Lam-Yuk-Tseung, S., “Nramp Metal Transporters: Insights into their structure, function, and subcellular targeting”, Doctoral Thesis, 2006.
NrampMetalTransporters:
Insightsintotheirstructure,function,and
subcellulartargeting
StevenLam-Yuk-Tseung
Department
ofBiochemistry
McGillUniversity
Montréal,Québec,Canada
June2006
AthesissubmittedtotheFaculty
ofGraduateStudiesandResearchinpartialfulfillment
oftherequirementsofthedegreeofDoctorofPhilosophy.
©StevenLam-Yuk-Tseung2006
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Abstract
ThisthesisexaminesthemolecularpropertiesofNrampproteinsbycenteringon
thetwomammalianorthologs.Nrampl
(Slcllal)isexpressedinphagocyticcellsand
restrictsreplication
ofintracellularpathogensbyremovingdivalentmetalsfromthe
phagolysosome.Nramp2
(DMTl,Slclla2)mediatesuptakeofdietaryironinthe
duodenumandaidsintheacquisitionoftransferrin-associatedironinmanycelltypes.
Thefirsthalf
ofthisthesisexploresstructure-functionrelationships.InChapter2,therole
ofchargedaminoacidswithinthemembrane-spanningsegmentsofNramp2was
examined
bysite-specifiemutagenesis.Thesestudiesidentifiedseveralinvariantcharged
residuesessentialformetaltransportandpHregulation
ofactivity.InChapters3and4,
theeffects
oftwoNRAMP2mutationsfoundinhumanpatientssufferingfromsevere
congenitalhypochromicmicrocyticanemiaandironoverloadwerecharacterizedinvitro.
ThefirstmutationwasanE399Dsubstitutioninaregionknownasthe”conserved
transport
motif’oftheprotein.ThesecondmutationwasanR416Csubstitutionatan
invariantresidueinTM9.Theeffects
ofbothmutationsonexpression,activity,and
subcellulartargetingwerecharacterized.Inbothcases,aquantitativereductionin
Nramp2expressionwasfoundtobethecause
ofmicrocyticanemiaandironoverloadin
thepatients.Thesecond
halfofthisthesisfocusesonthesubcellulartargetingofNramp1
and
2.InChapter5,cytoplasmicsignal(s)inNramp2responsibleforitssubcellular
targetinglintemalizationfromtheplasmamembranewerestudied.Thisworkledtothe
identification
ofatyrosine-basedmotifinthecarboxylterminusofNramp2(YLLNT555-
559)
criticalforthetransporter’sintemalizationfromthecellsurfaceanditsrecyc1ing
backtotheplasmamembrane.Chapter6exploreddifferencesintraffickingbetweentwo
splicingisoforms
ofNramp2andfoundthatoneisoform(isoform1)possessed
differencesinintemalizationlrecyc1ingwhichenabeledittobecomeenrichedatthe
plasmamembrane.InChapter
7,thesubcellulartraffickingpropertiesofNramp1,
inc1udingcytoplasmicsequencesresponsiblefortargetingtolysosomes,were
investigated
byusingchimericNrampl/Nramp2proteins.Thisworkledtothe
identification
ofatyrosine-basedmotif(YGSI15-18)intheaminoterminusofNramp1that
functionsasalysosomaltargetingsignal.
Abrégé
CettethèseexaminelespropriétésmoléculairesdesprotéinesNramp,enparticuliercelles
desdeuxorthologuesdemammifère.Nrampl(SIc1
laI)estexpriméparlescellules
phagocytairesetbloquelaréplicationintracellulaired’agentspathogènesendiminuantla
concentrationphagosomaledemétauxdivalent.Nramp2
(DMTI,SIcIla2)estresponsablede
l’absorptiondeferparleduodenumprovenantdel’alimentation
etaideàl’acquisitionferassocié
àlatransferrineparplusieurstypescellulaires.Lapremièremoitiédecettethèseexploreles
relationsstructure-fonction.
Auchapitre2,lerôledesacidesaminéschargésdessegments
transmembranairesdeNramp2aétéexaminé
parmutagenèsedesitesspécifiques.Cesétudesont
identifiéplusieursrésiduschargésconservéscommeétantessentielsautransportdemétauxet
à
l’activitéderégulationdupH.Auxchapitres3et4,leseffetsdedeuxmutationsdelaprotéinè
NRAMP2trouvéeschezdespatientssouffrantd’uneanémiehypochromiquemicrocytique
congénitalegrave
etd’unsurplusdeferontétécaractériséesinvitro.Lapremièremutation,la
substitutionE399D,sesituedansunerégiondelaprotéinenommée
“motifdetransport
conservé”.Lasecondemutation,lasubstitutionR416C,modifie
unrésiduinvariantdudomaine
transmembranaire
9.Leseffetsdesdeuxmutationssurl’expression,l’activitéetlalocalisation
intracellulaireontétécaractérisés.Danslesdeuxcas,uneréductionsignificativedel’expression
deNramp2
s’estavéréelacausedel’anémiemicrocytiqueetdelasurchargedeferobservées
chezlespatients.Lasecondemoitiédecettèthèseseconcentresurlalocalisationintracellulaire
desprotéinesNramplet2.Auchapitre
5,lessignauxcytoplasmiquesdelaprotéineNramp2
responsablesdesalocalisation
etdesoninternalisationàpartirdelamembraneplasmiqueontété
étudiés.Cetteétudeamenéàl’identification
d’unmotifàbasedetyrosinesituéencarboxy
terminaldeNramp2
(YLLNT555-559)essentielàl’internalisationdutransporteurdelasurface
cellulaireetàsonrecyclageverslamembraneplasmique.Lechapitre6explorelesdifférencesde
localisationentredeuxisoformesdeNramp2issuesd’épissagealternatif.
naététrouvéque
l’isoforme1présentedesdifférencesd’internalisationetderecyclage,cequiprovoqueson
enrichissement
àlamembraneplasmique.Auchapitre7,lespropriétésdelocalisation
intracellulairedelaprotéine
Nrampl,incluantlesséquencescytoplasmiquesresponsablesdesa
localisationauxlysosomes,ontétéexaminéesàl’aidedeprotéineschimèresNrampl/Nramp2.
Cetteétudeamené
àl’identificationd’unmotifàbasedetyrosine(YGSl1S-18
)danslaportion
aminoterminaldelaprotéineNramp1quifonctionneentantquesignaldelocalisationaux
lysosomes.
ii
Preface
TheworkdescribedinChapters2,3,4,5,6,and7ofthisthesisarepublishedasfollows:
Chapter2:Lam-Yuk-TseungS.,G.Govoni,J.Forbes,andP.Gros.2003.”Iron
transport
byNramp2/DMT1:pHregulationoftransportby2histidinesin
transmembranedomain6.”
Blood.101(9):3699-707.Reprintedwith
permission,©theAmericanSociety
ofHematology2003.
Chapter3:Lam-Yuk-TseungS.,M.Mathieu,andP.Gros.2005.”Functional
characterization
oftheE399DDMT1/NRAMP2/SLCIIA2protein
produced
byanexon12mutationinapatientwithmicrocyticanemiaand
ironoverload.”
BloodCells,MoleculesandDiseases.35(2):212-6.
Reprintedwithpermission,©Elsevier2005.
Chapter4:Lam-Yuk-TseungS.,C.Camaschella,A.Iolascon,andP.Gros.2006.”A
NovelR416CMutationinHumanDMTI(Slclla2)DisplaysPleiotropic
Effects
onFunctionandCausesMicrocyticAnemiaandHepaticIron
Overload.”
BloodCells,MoleculesandDiseases.Reprintedwith
permission,©Elsevier2005.
Chapter5:Lam-Yuk-TseungS.,N.Touret,S.Grinstein,andP.Gros.2005:
“Carboxyl-terminusdeterminants
oftheirontransporterDMTI/SLC11A2
isoformII(-IREIlB)mediateintemalizationfromtheplasmamembrane
intorecyclingendosomes.”
Biochemtstry.44(36):12149-59.Reprinted
withpermission,©AmericanChemicalSociety,2005.
Chapter6:Lam-Yuk-TseungS.andP.Gros.2006.”Distincttargetingandrecycling
properties
oftwoisoformsoftheirontransporterDMTl(Nramp2,
Slclla2).”Biochemistry.45(7):2294-301.Reprintedwithpermission,©
AmericanChemicalSociety,2006.
iii
Chapter7:Lam-Yuk-TseungS.,V.Picard,andP.Gros.2006.”Identificationofa
tyrosine-basedmotif(YGSI)intheaminotenninusofNrampl(Slcllal)
responsibleforlysosomaltargeting.”JournalofBiologicalChemistry.
(Submitted)
iv
ContributionofAuthors
Chapter2.Theworkinthischapterisessentiallymyown.GregoryGovoni,whobegan
theprojectpriorto
myarrivaI,created12ofthe21mutationsstudiedinthemanuscript
andprovidedmewithvaluableadviceatthestart
ofmygraduatestudies.1constructed
.theremainingmutants,expressedandfunctionallycharacterizedtheminyeastasweIlas
stablytransfectedChinesehamsterovarycells.AlIthefiguresthatappear
inChapter2
arebased
onexperiments1performed.JohnForbesperformedanadditionalradioisotopic
metaltransportexperimentthatwasrequested
byoneofourreviewersatthetimeof
publication.However,thedatafromthisexperimentwasnotinc1udedinthefinal
publishedmanuscript.
Chapter3.Inthischapter,allthefiguresarebasedonexperiments1performed.1
constructed,expressedandcharacterizedallthemutations
instablytransfectedLLC-PK1
cells.1optimizedtheconditionsfortheimmunofluorescencemicroscopyimagesandset
upandoptimizedanELISA-basedtechniquetoquantifythefraction
ofDMTl-HA
expressedatthecellsurface.Twoundergraduatestudents1supervised,LaurenHamlin
DouglasandRachelBeckerman,he1pedmecreatetheE399DandE399Qmutantsfor
theirundergraduateresearchprojects.1amalsoindebtedtoMelissaMathieuforher
he1p
inculturingandscreeningtheLLC-PKIcellsforDMTlexpression.
Chapter4.Allthefiguresinthischapterarebasedonexperiments1performed.Clara
CamaschellaandAchilleIolasconidentifiedtheoriginal
DMTlISLCIIA2human
mutationandapproachedustocharacterizetheeffectsonexpression,function,and
subcellularlocalization
oftheR416Chumanmutationinvitro.
Chapter5.Allthefiguresinthischapterarebasedonexperiments1performed.1created
themutants,stablyexpressedtheminLLC-PKIcells,andcharacterizedthemforproper
expression,activity,cellsurfacetargeting,subcellularlocalization,andfateupon
intemalization.1alsoset-upandoptimizedthecellsurfacebiotinylationtechniqueto
measuretherate
ofendocytosisofthevariantsfromtheplasmamembrane.Nicolas
v
Touretprovidedmewithvaluabletechnicaladviceandhelpedmeacquiretheimagesin
Figures3AandSA,which1perfonnedattheHospitalforSickChildren(Toronto)inthe
laboratory
ofSergioGrinstein.1amalsoindebtedtoDr.Grinsteinforhiscriticalreading
ofthemanuscriptandforhisscientificguidance.
Chapter6.Theworkdescribedinthischapterisentirelymyown.1expressedboth
DMTlisofonnsinstablytransfectedLLC-PK1cells,andcharacterizedthemfor
differencesinfunction,subcellularlocalization,endocytosiskinetics,andfateupon
intemalization.
Chapter7.Theworkdescribedinthischapterisessentiallymyown.VirginiePicard
createdtwo
oftheconstructs1usedinthestudy.1createdtheremainingconstructs,
expressed
aUthevariantsintransfectedLLC-PK1andstudiedthemforexpression,
stability,cellsurfacetargeting,transportactivity,andsubcellularlocalization.
Mysupervisor,Dr.PhilippeGros,providedsupervisionandadvicethroughoutaIlofthe
studies.
VI
TableofContents
Page
Abstract………………………………………………………………
…………………………………
Abrégé………………………………………………………………
…………………………………..
11
Preface………………………………………………………………
…………………………………..111
ContributionofAuthors………………………………………………………………
…………v
Table
ofContents…………………………………………………………………………………..VIl
ListofFigures………………………………………………………………
………………………..Xl
ListofTables…………………………………………………………………………………………xiv
Acknowledgements………………………………………………………………
……………….
,.xv
Objectives
ofthePresentedWork…………………………………………………………..XVI
Chapter1:IntroductionandLiteratureReview1
1.1TheNrampfamily……………………………………………………………………………….2
1.2Nramp
1:DiscoveryofthefirstNrampgene……………………………………………2
1.2.1TheBcg/lty/LshLocusandtheNramplgene……………………………….2
1.2.2Validation
ofNramplasacandidategene……………………………………4
1.2.3TheNramplprotein………………………………………………………………
….5
1.2.4Thefunction
ofNramp1atthephagosomalmembrane………………….7
1.2.5Pathogensunder
Nramplcontrol………………………………………………..9
1.2.6Effect
ofNramp1onintracellularMycobacterium………………………..Il
1.2.7EffectofNramp1onintracellularSalmonella………………………………13
1.2.8NRAMP1anditsroleinresistancetoinfectioninhumans……………..14
1.3Nramp2anditsroleinironhomeostasis…………………………………………………15
1.3.1CloningoftheNramp2gene………………………………………………………15
1.3.2Nramp2encodesadivalentmetaltransporter……………………………….15
1.3.3AmutationatNramp2causesirondeficiencyinrodents……………….16
1.3.4AltematesplicingofNramp2rnRNA………………………………………….18
1.3.5StructuralfeaturesoftheNramp2protein…………………………………….19
1.3.6Nramp2expression………………………………………………………………
……22
vii
1.3.7SubcellulartargetingofNramp2…………………………………………………26
1.3.8Nramp2andcellularironhomeostasis…………………………………………
28
1.3.8.1Mechanismsofcellularironabsorption…………………………….28
1.3.8.2Mechanismsofcellularironstorageandusage………………….31
1.3.8.3Mechanismsofironexport………………………………………………32
1.3.8.4Regulation
ofcellularironhomeostasis…………………………….32
1.3.9Nramp2andsystemicironhomeostasis……………………………………….34
1.3.9.1Systemicironregulators………………………………………………….34
1.3.9.2hnportanceofNramp2insystemicironhomeostasis………….36
1.3.9.3Diseasesassociatedwith
NRAMP2mutationsinhumans…….37
1.3.9.4Otheriron-overloaddisordersinhumans…………………………..38
Chapter2:IrontransportbyNramp2/DMTl:pHregulationoftransportby
twohistidinesintransmembranedomain6
42
Abstract……………………………………………………………………………………………..43
Introduction………………………………………………………………
………………………..44
MaterialsandMethods………………………………………………………………
…………46
Results………………………………………………………………
……………………………….
51
Discussion……………………………………………………..
…………………………………..64
Chapter3:FunctionalcharacterizationoftheE399D
DMTl/NRAMP2/SLCl1A2proteinproducedbyanexon12mutationina
patientwithmicrocyticanemiaandironoverload
69
Abstract………………………………………………………………
……………………………..70
Introduction………………………………………………………………
………………………..71
MaterialsandMethods………………………………………………………………
…………73
Results………………………………………………………………
……………………………….74
Discussion………………………………………………………………………………………….79
viii
Chapter4:AnovelR416CmutationinhumanDMT1(Slc11a2)displays
pleiotropiceffects
onfunctionandcausesmicrocyticanemiaandhepaticiron
overload80
Abstract………………………………………………………………
……………………………..81
Introduction……………………………………………………
…………………………………..82
MaterialsandMethods………………………………………………………………
…………84
Results………………………………………………………………
……………………………….87
Discussion………………………………………………………………………………………….
93
Chapter5:Carboxylterminusdeterminantsoftheirontransporter
DMTl/SLC11A2isoformII(IREIlB)mediateinternalizationfromthe
plasmamembraneintorecyclingendosomes96
Abstract………………………………………………………………
……………………………..97
Introduction………………………………………………………………
………………………..98
MaterialsandMethods………………………………………………………………
…………
101
Results………………………………………………………………
……………………………….107
Discussion………………………………………………………………
………………………….120
Chapter6:Distincttargetingandrecyclingpropertiesoftwoisoformsofthe
irontransporterDMT1(NRAMP2,
Slclla2)126
Abstract……………………………………………………………………………………………..127
Introduction………………………………………………………………
………………………..128
MaterialsandMethods………………………………………………………………
…………
131
Results………………………………………………………………
……………………………….135
Discussion………………………………………………………………
………………………….
144
Chapter7:Identificationofatyrosinebasedmotif(YGSI)intheamino
terminus
ofNramp1(Slclla1)responsibleforlysosomaltargeting151
Abstract………………………………………………………………
……………………………..152
Introduction………………………………………………………………………………………..153
IX
MaterialsandMethods………………………………………………………………
…………156
Results………………………………………………………………
……………………………….162
Discussion……………………………………………………..
…………………………………..174
Chapter8:SummaryandFuturePerspectives180
8.1Summary………………………………………………………………
……………………………181
8.2FuturePerspectives………………………………………………………………
……………..183
8.2.1Insightsintostructure/functionrelationshipsinNrampproteins……..183
8.2.2Evidenceforametalbindingsiteorpermeationpathway………………184
8.2.3AmolecularbasisforpHdependenceandproton-coupling……………186
8.2.4StructuraldeterminantsofNrampproteins…………………………………..
188
8.2.5SubcellulartargetingandtraffickingofNrampproteins………………..194
8.3FinalConclusions………………………………………………………………
………………..197
References………………………………………………………………
…………………………………..198
OriginalContributions
toKnowledge…………………………………………………………..230
Biohazardsafetycertification………………………………………………………………
………
231
x
Chapter1
Suppl.
Figure1
Figure1
Figure2
Figure3
ListofFigures
Page
Dendogram
ofsorneofthemembersoftheNrampfamilyofmetal
transporters……………………………………………………………….
…………..3
MetaltransportbyNrampproteinsattheinterface
ofhost-
pathogeninteraction……………………………………………………………….10
Theeffect
ofNramplonintracellularMycobacterium………………..12
AlternativesplicingofNramp2pre-mRNAgeneratesfour
theoreticalisoforms………………………………………………………………..20
Figure4Absorption
ofnon-hemeironattheintestinalbrushborderandin
reticulocytes……………………………………………………………….
………….
23
Chapter2
Figure1SchematicrepresentationofmouseNramp2
(DMTl)isoformII(-
IRE)andisoform1(+IRE)………………………………………………………52
Figure2Nramp2proteinexpryssioninsmfl/smf2mutantyeastand
functionalcomplementation
ofgrowthatalkalinepH…………………53
Figure3QuantitationofcomplementationoftheSmfllSmj2yeastmutant
byNramp2variants……………………………………………………………….
.54
Figure4ExpressionofNramp2variantsinstablytransfected
CHûcells…..56
Figure5RelativeironandcobalttransportactivityofNramp2mutations
affectingconservedchargedresiduesinTMdomains.
………………..58
Figure6RelativeironandcobalttransportactivityofNramp2mutations
affectingconservedhistidineresiduesinTM6……………………………
62
Figure7EffectofpHontransportactivityofNramp2mutantsatconserved
histidineresiduesinTM6.
……………………………………………………….63
Chapter3
Figure1Positionandconservation
ofE399residueinDMTl………………….75
Figure2StableexpressionandmetaltransportactivityofE399mutants……77
Figure3SubcellularandcellsurfaceexpressionofE399mutantsatsteady-
xi
state……………………………………………………………….
……………………..78
Chapter4
Figure1
Figure2The
positionandconservationofR416residueinDMTl……………
Stableexpressionandmetaltransportactivity
ofR416mutants…..
Figure3Doubleimmunofluorescencelabeling
ofDMTI-HAandthe86
88
recyclingendosomemarkerGFP-Syntaxin13……………………………
91
Figure4DoubleimmunofluorescencelabelingofDMTI-HAandthe
endoplasmicreticulummarkerGFP-Sec61……………………………….92
Chapter5
Figure1
Figure2Predicted
structuralfeaturesandmembranetopologyofDMTl…..
Expressionandmetaltransportactivity
ofDMTI-HAMutantsin
transfectedLLC-PKIcells……………………………………………………….
Figure3Subcellulardistribution
ofWTandmutantDMTI-HAin106
108
endomembranecompartments………………………………………………….110
Figure4Quantificationcellsurface
DMTI-HAexpressionatsteady-state…112
Figure5Fraction
oftherecyclingpoolexpressedatthecellsurfaceforeach
DMT1-HAvariant……………………………………………………………….
…114
Figure6Intemalization
ofDMT1-HAvariantsfromthecellsurface…………116
Figure7Subcellularlocalizationofintemalized
DMTI-HAmolecules……..119
Figure8Schematicmodelforendocytosis
ofcellsurface-expressedWT
andCterminusmutantDMT1molecules………………………………….122
Chapter6
Figure1ExpressionandfunctionalactivityofDMTlisoforms1andIIin
LLC-PKIcells……………………………………………………………….
………136
Figure2Subcellularlocalization
ofDMTlisoforms1andIIinLLC-PK1
cells……………………………………………………………….
……………………..138
Figure3Quantification
ofcellsurfaceexpressionofDMTlisoforms1and
II
inLLC-PK1cells……………………………………………………………….
..139
Figure4Quantification
oftherateofendocytosisofDMTlisoforms1and
XlI
IIinLLC-PK1cells……………………………………………………………….
..141
Figure5ThefateofintemalizedDMTisoform1andIImolecules……………143
Figure6AschematicmodelfordistincttraffickingofDMTlisoforms1
andII……………………………………………………………….
……………………148
Suppl.ThestabilityofDMTlisoforms1andIIproteins……………………….149
figure
Chapter7
Figure1PredictedtraffickingmotifsanddesignofNrampl/Nramp2
h·.t.c
Imencproelns………………………………………………………………
……161
Figure2ExpressionandstabilityofNramp112chimerasintransfected
LLC-PK
1cells………………………………………………………………
……….163
Figure3Cellsurfaceexpressionandmetaltransportactivityofthe
Nramp1/2chimeras………………………………………………………………
..
165
Figure4SubcellularlocalizationofNramp1/2chimeras………………………….167
Figure
5CharacterizationofNIN-HAtargetingmotifmutants…………………170
Figure6Subcellularlocalization
ofNIN-HAmutants…………………………….172
Figure7ModelforsubcellulartraffickingofNramplandNramp2(isoform
II)……………………………………………………………….
………………………..177
xiii
Chapter2
Table1
Chapter5
Table1
Table2
Chapter
7
Table1
Table2
Table3
ListofTables
Page
Primersusedformutagenesis……………………………………………………47
Oligonuc1eotidesusedformutagenesis………………………………………102
Summaryofsubcellularlocalization
ofWTandmutantDMT1-
HA……………………………………………………………….
……………………….102
Oligonuc1eotidesusedformutagenesis………………………………………157
Subcellularlocalization
ofNramp112chimeras…………………………..169
Subcellularlocalization
ofN1N-HAmutants……………………………..173
XIV
Acknowledgements
Foremost,1wouldliketothankPhilippeGrosforprovidingmewithmuchneeded
guidanceandsupport,especiallyduringthefirstfewyears
ofmygraduatestudies.He
providedclarityanddirectionwhen
1neededit,butatthesametimeallowedmeenough
roomtomakemistakesandgrowstrongerbothasapersonandasascientist-andfor
that,
1willalwaysbegrateful.1wouldliketothankSergioGrinsteinandothermembers
ofhisgroupfornotonlywelcomingmeintotheirlabontwoseparateoccasions,butalso
forprovidingatremendouslypositiveworkingenvironmentduring
mystay.1am
especiallyindebtedtoNicolasTouretforhelpingmewiththefluorescencemicroscopy
workforChapter
5.1wouldliketothankGregoryGovoni,fromwhom1tookoverthe
Nrampproject.Hewasagreathelpatthebeginning
ofmystudies,providingmewith
bothpersonalandprofessionaladvicethat
1valuestilltoday.1wouldalsoliketothank
severalformerpost-doctoralfellowsfromour
labo1amgratefultoFrançoisCanonne
Hergauxforhisadviceregardingtheuse
oftheanti-Nramppolycolonalantibodies,and
alsoforhisenthusiasmandwillingnessforscientificdiscussion.
1wouldliketothank
VirginiePicardforsetting-upandteachingmethefluorescencequenchingassayto
measureNrampmetaltransportactivity.
1alsothankJieCaiandJohnForbes,whose
advicehelpedguidemethroughthe”roughpatches”.
MythanksalsogoestoAnne
Fortier,whoisresponsiblefortheFrenchtranslation
oftheabstractandwhosefriendship
isgreatlyvalued.
1wouldalsoliketothankaIltheotherpastandpresentmembersofthe
Groslaboratorywhohaveprovidedapleasurableworkingenvironmentovertheyears.
1
wasfinanciallysupportedduringmystudiesbystudentshipsfromtheFondsdela
rechercheensantéQuebec(FRSQ),theCanadianInstitutes
ofHealthResearch(CIHR),
andtheMcGillUniversityFaculty
ofMedicine.1wouldliketothankmyfriendsfortheir
support,especially
myLoyolabrethren.Mostimportantly,1wouldliketothankmy
familyfortheirloveandsupport,withoutwhom1couldnothavefinishedthisdegreeand
withoutwhom,itwouldnotevenmatter.
xv
ObjectivesofthePresentedWork
ThefirstmemberoftheNrampfamily(Nrampl)wasidentifiedin1993asthe
generesponsiblefornaturalresistanceinmicetoinfection
bycertainintracellular
parasites
1.SinceNramp1wasisolatedbyapositionalc10ningapproachandrepresented
thefirstmember
ofanovelfamilyofproteins,itsmolecularfunctionwasunknownand
remaineddebatedforseveralyears.Thisdebatevirtuallyended
in1997whenasecond
member
oftheNrampfamily(Nramp2)wasshowntofunctionasapH-dependent
transporter
ofdivalentmetalsimportantforintestinalironabsorption2;3.Becauseofthe
highdegree
ofconservationofamongNrampfamilymembers,itwasbelievedthatall
Nramporthologssharedsimilarmetaltransportcharacteristics.
Atthestartofmythesis
work
inSeptember1999,themolecularbasisofproton-coupledmetaltransportby
Nrampproteinswasnotwellunderstood.Atthistime,structure-functionstudiesbecame
essentialtounderstandingthemolecularbasisforbothmetaltransportandpH
dependence
byNrampproteins.Alsounc1earwerethemolecularmechanismsof
subcellulartraffickingofmammalianNramp1andNramp2.Therefore,theobjectiveof
mythesisprojectwastousebiochemicalandmolecularbiologicaltechniquestoprovide
agreaterunderstanding
ofthestructure,function,andsubcellulartraffickingofNramp
proteins.
Toaccomplishthis,theworkdescribedinthefirstthreechapters
ofmythesis
focusontheimportance
ofconservedaminoacidresiduesinthefunctionofNramp2.
Chapter2addressestherole
ofchargedaminoacidswithinthemembrane-spanning
segments
ofNramp2andidentifiesseveralnegatively-chargedresiduesthatareessential
formetaltransportaswellastwoinvarianthistidinesnecessaryforthepHdependence
of
activity.Chapters3and4studytheimportanceofconservedresiduesinNramp2that
werefoundtobemutated
inhumanpatientssufferingfromseverecongenital’
hypochromicmicrocyticanemiaandironoverload.Thesestudieswereessentialto
understandingthecause
ofdiseaseinthepatientsandalsoprovidedinsightintotherole
ofNramp2inhumanironhomeostasis.
Thelastthreechapters
ofmythesiswereaimedatbetterunderstandingthe
subcellulardistribution
ofNramp1andNramp2.Chapter5addressestheimportanceof
xvi
cytoplasmicsequencemotifsinthesubcellulartargetingandtraffickingofNramp2.This
workledtotheidentification
ofanoveltyrosine-basedmotifinthecarboxylterminusof
Nramp2(isoformII)thatiscriticalforthetransporter’sintemalizationfromthecell
surfaceanditsrecyc1ingbacktotheplasmamembrane.Workdescribed
inChapter6was
aimedatcomparingthedistincttargetingandrecyclingproperties
oftwonaturally
occurringspliceisoformsofNramp2.Thisworkultimatelyshowedthataltematesplicing
ofNramp2criticallyregulatesthesubcellularlocalizationandsite
ofirontransport.
Finally,Chapter7
ofthisthesiswasaimedatbetterunderstandingthesubcellular
targeting
ofNramp1.ItwasknownforsometimethatNramp1isexpressedinthe
lysosomes
ofphagocyticcellshowever,specificsignaIsresponsiblefortargetingto
lysosomesremainedunc1ear.WorkdescribedinChapter7showedforthefirsttimethata
tyrosine-based
motifoftheformYXX<1>intheaminoterminusNramp1isresponsiblefor
thetransporter’sIysosomaitargeting.
XVll
Chapter1:
IntroductionandLiteratureReview
1
1.1TheNrampfamily
Nrampproteinsrepresentagrowingfamilyofmembranemetaltransportersthat
performavariety
offunctionsinorganismsrangingfrombacteriatohumans1.The
sequenceconservationamongmembers
oftheNrampfamilyisastoundinglyhigh,even
amongphylogenicallydistantspecies(supplementaryfigure1).Inmanyorganisms,
Nrampproteinsarecriticalfortheabsorption
ofdivalentmetalsandthemaintenanceof
normaldivalentmetalhomeostasis.MorespecializedrolesforNrampproteinshavealso
beenreported.In
Drosophila,Nramporthologsplayaroleintastediscrimination2andin
sornemammals,
Nrampliscriticalforhost-resistancetoinfectionbyintracellular
pathogens
3.Inhumans,polymorphismsinNRAMP1havebeenassociatedwithincreased
susceptibihtytoanumber
ofinfectiousandinflammatorydiseaseswhilemutationsat
NRAMP2disruptnormalironhomeostasis4-6,
TremendousadvanceshavebeenmadeinunderstandingNrampproteinfunction
sincethefirstmember
ofthisfamilywasclonedin19937.Muchofthecurrent
knowledge
onNrampproteinsoriginatesfromgeneticstudiesperformedinmouse
models
ofdiseases.Theusageofmousemodelsprovidesmanyadvantagesoverstudies
inhumans.Whereasgeneticandenvironmentalfactorsareheterogeneousandcomplexin
humans,thesefactorscan
becontrolledforininbredmousestrains.Furthermore,the
breeding
ofmiceisrelativelyfastandbiologicalsamplesareeasiertoobtainfrommice
thanfromhumanpatients.Finally,candidategenescan
beindividuallytargetedinmice
bygermhneinactivationthroughhomologousrecombinationandgain-of-functionalleles
can
bere-introducedbyBACclones.
1.2Nramp1:DiscoveryofthefirstNrampgene
1.2.1TheBcg/lty/LshLocusandtheNramplgene
Thirtyyearsago,therewerereportsofamouselocusthatpossiblycontrolledthe
intracellularreplication
ofanumberofdiverseintracellularpathogens.In1976,studies
performed
onseveralinbredmousestrainsshowedthatresistanceorsusceptibilityto
infection
bySalmonellatyphimuriumwascontrolledbyasinglelocus,whichwascalled
Ity8.Atthesametime,similarexperimentsdemonstratedthatanotherlocus,calledLsh,
2
1
1
1
1
i
—
….
Mycobacterium
ichiacoli
Candidaalbicans
YeastSmflp
Escher
YeastSmf2p
Yeas
Plant(A
tSmf3p
tNrampl)
C.Elegans
Xenopus)
Frog(
Chick
enNrampl
—-c
ouseNrampl
RatNrampl
–
Do
9Nrampl
HumanNrampl
Pig
Nrampl
r-SheepNrampl
~L[co
Bu
wNrampl
ffalo
re-~MOU
RatN
seNramp2
ramp2
CHU
Cowman
Nramp2
Nramp2
Nramp2
fish
Dog
Zebra
oosophila’r
Supplementaryfigure1.Dendogramofsomeofthemembersofthe
Nrampfamilyofmetaltransporters.Thesequenceconservationamong
members
oftheNrampfamilyisastoundinglyhigh,evenamongphylogenically
distantspecies.
controlledintracellularreplicationoftheprotozoanparasiteLeishmaniadonovaniand
that
LshwaseitheridenticalortightlylinkedtoIty9.Athirdlocus,Bcg,whichcontrolled
intracellularreplication
ofseveralmycobacteriawasalsomappedtothesameregion7.
TheBcglltylLshlocusthatgaveresistancetothesepathogensinadominantfashionwas
tracedbacktoaregionontheproximalportion
ofmousechromosome1.Subsequentin
vitroandinvivostudiesdemonstratedthatthecellsaffectedbyBcg/ltylLshweremature
macrophagesresidingespeciallyinthereticuloendothelialorgans(spleenandliver)
10-l2.
In1993,withoutthebenefitofaknowngeneproductorapracticalwayto
measuregenefunction,ourgroupusedapositionalc10ningapproachtoisolatethe
Bcgllty/Lshlocus7.Theminimalgeneticandphysicalintervalsforthegeneweredefined
andtranscriptionalunitswereisolatedfromtheinterval
byexonamplification.Ofthesix
genespresentintheregion,oneemergedasaIikelycandidatesinceitencodedrnRNA
thatwashighlyexpressedinthereticuloendothelialorgansandaimostexc1usivelyin
macrophagesandneutrophils.Thecandidategenewasnamed
Naturalresistance
associatedmacrophageprotein1
orNrampl(OMIM#600266,nowre-c1assifiedas
SlcllalbutwillbereferredtoasNramplinthisthesis).
1.2.2ValidationofNramplasacandidategene
FollowingtheidentificationofNramplasacandidategeneforBcg/lty/Lsh,
severalsubsequentexperimentsconfirmedNramp1asthegeneresponsibleforhost
resistancetoinfections.First,thesequencing
ofNramplmRNAfrom27inbredmouse
strainsshowedthatsusceptibilitytoinfectioncouldbetracedbacktoasinglenuc1eotide
pointmutation.Theresult
ofthismutationwasanon-conservativeglycinetoaspartate
substitution(GI69D)
inthefourthmembrane-spanningsegment(TM4)oftheNrampl
protein7.ThismutationwasIatershowntocauseacompletelossofNramplprotein
expressioninmacrophages
7;13andresultinaproteinthatismisfoldedandretainedinthe
endoplasmicreticulumforeventualdegradation
14.Second,anullmutationatNrampl
(Nrampr/)introducedintoembryonicstemcellsbyhomologousrecombinationcaused
normallyresistantI29svmicetobesusceptibletoinfection
byMycobacterium,
Salmonella,
andLeishmania15.Finally,introducingatransgenecontainingtheNrampl
4
resistancealle1e(0169)intoanaturallysusceptiblemousestrain(DI69)granted
resistancetoinfection
byMycobacteriumandSalmonella16.ThebroadeffectofNrampl
mutationsonsusceptibilitytoinfectionwithantigenicallydiversepathogenssuggesteda
criticalroleforthisproteinininnateimmunity.
Inmice,theNramplgeneismadeupof15exonsthatspanan11.5kbregionand
istranscribedfromonemajorandseveralminortranscriptionstartsites.Usingprimer
extensionand
SInuc1easemappingexperiments,Oovoniandcolleaguesshowedthatthe
upstreamregion
ofNramplcontainsaTATAbox-Iesspromoter,withconsensusSPI
bindingsitesandinitiatorsequenceslinkedtoRNApolymeraseIItranscription
17.Alsoin
thepromoterregionareconsensusbindingsitesforseveralubiquitousandtissue-specifie
transcriptionfactors.AmongthemaresitesforamacrophageandB-cellspecifie
transcriptionfactor(PU.1),generaltranscriptionfactors(AP-1,AP-2,AP-3,andSPI),as
wellasresponseelementsforlipopolysaccharide(NF-IL6)andinterferon-y
17.Nrampl
mRNAisfoundinprimarymacrophagesandgranulocytes7;18anditsexpressionis
increasedinresponsetoinfectionwith
mycobacterium,exposuretoLPS,interferon-y,or
byotherinflammatorystimuli17-23.Inaddition,studiesbyLafuseandcolleaguessuggest
that
NramplmRNAlevelscanbecontrolledatthelevelofstabilitybyMAPkinase
signalingcascadesinactivatedmacrophages
24;25.BycomparingRAW264.7cellsstably
transfectedwitheitherresistant
(0169)orsusceptible(D169)Nramp1,theyshowedthat
inhibition
ofERK1,2andp38MAPkinaseactivitiesdecreasesNramp1mRNAstability
incellsinfectedwith
Mavium.Furthermore,phosphorylationofERK1,2andp38MAP
kinaseswashigherininfectedcellsexpressingresistantNramp1
Gl69comparedto
susceptibleNramp1
D16924.
1.2.3TheNramplprotein
In1996,Vidalandcolleaguesgeneratedapolyc1onalanti-Nramp1antiseraand
usedittodemonstratethat
Nramplencodesa90-100kDaintegralmembraneproteinthat
ishighlyglycosylated(upto50%
ofitsmass)andphosphorylatedinperitoneal
macrophagesobtainedafterthioglycolatestimulation
13.Theyalsoshowedthat
susceptible
(NramplD169)macrophagesfailtoexpressmatureNramp1protein.The
5
topologyofNramp1inthemembranewasinitiallypredictedfromhydropathyplotsand
hasbeenpartiallyconfirmed
byepitopeaccessibilitystudiesinintactcells1;26;27.Nramp1
contains
12putativetransmembranedomainsandalargeextracellularloopflankedby
TM7/8,whichisheavilyglycosylatedattwoneighboringN-linkedglycosylationsignaIs
1.ComplexglycosylationofNramp1seemstobetheendresultofastableandproperly
foldedtransporterbutglycosylationitselfdoesnotappearto
becriticalforsubcellular
targeting
14.Nramplcontainsanumberofhighlyconservedyetthermodynamically
disfavoredchargedaminoacidswithinitstransmembranedomains.Thesecharged
residueshavebeenshowntobecriticalfortheproperstructure/function
ofNramp
proteins28;29.Nramp1alsopossessesseveralphosphorylationsitesincludingputative
sitesforproteinkinaseCphosphorylationandaSrchomology3domain(SH3)binding
region
intheaminoterminusoftheprotein1;30.Littleisknownabouttheroleof
phosphorylationorthebindingofSH3domain-containingproteinsinthefunctionof
Nrampl,however,thefactthattheseregionsarenotconservedinNramporthologs
suggeststheirlack
offunctionalimportance.Altematively,theseregionsmaybecritical
fortheuniqueregulation
ofNrampl.Finally,thefourthintra-cytoplasmicloopof
Nramp1flankedbyTM8/9isaregionofextremelyhighconservationthatcontainsa
sequence
motifknownasthe”binding-protein-dependenttransportsysteminner
membranecomponentsignature”orsimply”conservedtransport
motif'(CTM).This
motifwasoriginallydiscoveredonthecytoplasmicfaceofthemembraneanchorsubunits
ofbacterialperiplasmicpermeases,whereitisbelievedtoparticipateintheinteraction
of
membranecomponentswithperipheralATPbindingsubunitsofthesetransporters31;32.
ThismotifinNramp1alsoshowssimilaritytoasequencemotifidentifiedintheK+
channelsuperfamilyandotherionchannelsandtransportersasakeystructural
determinant
oftheionpermeationpathway33.TheexactfunctionoftheCTMinthe
context
ofNrampproteinsisnotcurrentlyknown,however,mutationsatkeyresidues
withinthisregionareknowntoabrogatefunction
34.
Atsteady-state,Nramp1isnotexpressedattheplasmamembranebutisalmost
exclusivelyfound
inthemembranesofLampl-positivelateendosomes/lysosomesof
macrophagesandotherphagocyticcells35.Thesortingmachineryandspecifieproteins
6
involvedinthesubcellulartraffickingofNramp1arecurrentlynotwellunderstood.In
Chapter7
ofthisthesis,experimentsperformedonNramp1/Nramp2chimericproteins
expressed
intransfectedLLC-PK1kidneycellsdemonstratedthatatyrosine-basedmotif
oftheformYXXintheaminoterminalregionofNramp1(YGSII5
-18)canfunctionasa
lysosomaltargetingsignal.Usingsubcellularfractionationandimmunofluorescence
microscopy,GruenheidandcolleaguesdemonstratedthatNramp1isrecruitedtothe
membrane
ofthephagosomeandremainsassociatedwiththisstructureduringits
maturationtophagolysosome.Afterphagocytosis,Nramp1isacquired
bythephagosomal
membranewithkineticssimilartoLamp1
35whereitcontrolsthereplicationof
intracellularparasitesbyalteringtheluminalenvironmentofthemicrobe-containing
phagosome.Inadditiontomacrophages,Nramp1isexpressed
inpolymorphonuc1ear
(PMN)leukocytessuchasneutrophilsandothergranulocytes.Usingsubcellular
fractionation
ofgranulepopulationstogetherwithimmunoblottingwithgranule-specifie
markersaswellasimmunogoldelectronmicroscopy,Canonne-Hergaux
etal
demonstratedthatNramp1isexpressedprimarilyinthetertiarygelatinase-positive
granulesofneutrophils
36.TheyalsoshowedthatNramplisrecruitedfromtertiary
granulestothemembrane
ofCandidaalbicans-containingphagosomesinhuman
neutrophils
36.
1.2.4ThefunctionofNramplatthephagosomalmembrane
Nramp1israpidlyrecruitedtothephagosomalmembraneuponphagocytosisof
livebacteria(Salmonella,Leishmania,Mycobacterium,Yersinia)orinertpartic1es(latex
orzymosanbeads)whereitisproposedtoexerciseitsantimicrobialactivity
37-39.The
molecularbasisforNramp1
‘sfunctionremainedunclearforseveralyearsafterits
c1oning.However,thepolypeptidesequenceofNramp1showedstructuralfeaturesthat
suggesteditwasamembranetransporter
7.In1997,theidentificationand
characterization
ofasecondNrampproteininmammals,Nramp2,providedstrong
evidencethatNrampproteinswerepH-dependentdivalentmetaltransporters.Gunshin
andcolleaguesidentified
Nramp2(DMT1/S1c11a2,OMIM#600523)asanovel
intestinalirontransporterinascreenforduodenalgenesup-regulatedinratsfedalow-
7
irondiet40.TheyshowedevidencethatNramp2behavedasaproton-coupleddivalent
metaltransporterwithanunusuallybroadsubstratespecify,inc1udingFe
2+andMn2+.
Thec10ningandcharacterizationofNramp2isdescribedindetailinChapter1.3ofthis
thesis.Indeed,theidentificationandcharacterization
ofnumerousNramporthologsin
speciesrangingfrombacteriatohumanssuggestedapotentialfunctionforNramp1atthe
phagosomalmembraneasapH-dependentdivalentmetaltransporter.Byanalogytothe
knownsubstrates,membraneorganization,anddirection
oftransportestablishedfor
Nramp2,weandothersproposedamodelwherebyNramp1wouldactasaneffluxpump
atthemembrane
ofpathogen-containingphagosomes,restrictingmicrobialaccessto
essentialmetalssuchas
Mn2
+andFe2
+41;42.However,earlystudiessuggestedthat
Nramp1wouldactasametalinfluxpumpatthephagosomalmembranetofacilitatethe
production
ofoxygenradicalsviatheHaber-Weissreaction43;44.Thesestudiesreported
anNrampl-dependantincreaseintheaccumulationlbindingofradiolabeledFe
2+into
isolatedphagosomescontainingeitherLatexbeads
orMyocobacteriumavium43-45.The
phagosomalFe
2+accumulationwasblockedbytheadditionofanti-Nramp1antibodies
suggestingthat
NramplmighttransportcytoplasmicFe2+intophagosomes.However,a
potentiallimitation
ofthesestudieswasthelackofdistinctionbetweenphagosomalmetal
binding(whetherNrampl-dependent
orindependent)andactualmetaltransportacross
thephagosomalmembrane.Alaterstudyusing
NramplmRNAinjectedintoXenopus
laevis
oocytesfoundanincreaseinZn2+-dependentinwardcurrentswhenthecellswere
exposedtoalkalineextracellularpH,whichsuggestedmetaluptakeagainstthe
experimentalprotongradient
46.BasedonadditionalpH-dependenttransportstudies
usingradioabeled
Zn2+,theseauthorsconcludedthatNramplmighttransportmetalsfrom
thecytoplasmintophagosomes
byaprotonldivalent-metalantiportmechanism.They
proposedthatincreasedphagosomalFe
2+couldstimulateoxygenradicalproductionvia
theFentonreactionforincreasedbactericidalactivity
43-47.However,forNramp1to
functionasprotonldivalent-metalantiporteratthephagosomalmembraneitwoulddictate
atransportmechanismdistinctfromotherNramporthologsasweIlasNramp2,bothwith
respecttothedirection
oftransportandutilizationofthetransmembranepHgradient.
8
Thiswasveryunlikelyconsideringthehighaminoacidconservationinthepolypeptide
sequences
ofNramp1andNramp2(64%identifyand78%similarity)1.
RecentexperimentshaveprovidedstrongevidencethatNramp1indeedfunctions
asapH-dependentdivalentmetaleffluxpumpatthephagosomalmembrane,consistent
withthebiochemicaltransportdatareportedinstudies
ofNramporthologs26;27;40;48-51.
First,ametal-sensitivefluorescentdyewascovalentlycoupledtozymosanpartic1es,
whichwerethenfedtoperitonealmacrophagesisolatedfromNrampl-expressing(+/+)
andNrampI-deficient(-/-)mice.Thetransport
ofdivalentmetalsacrossthephagosomal
membranewasmonitored
bytrackingfluorescencequenching.Nramp1+1+phagosomes
showedreducedaccumulationandincreasedefflux
ofMn2+comparedtoNramprl
–
phagosomes.Thisdifferencewaslostwhenphagosomalacidificationwasblockedwith
theV-typeATPaseinhibitorbafilomycin,suggestingthatdivalentmetaltransport
by
NramplwasH+-dependent,likeNramp252.Second,anotherconsequenceofthe
functionalsimilaritybetweenNramp1andNramp2isthatshouldNramp1
beexpressedat
theplasmamembrane,itwouldfunctioncomparablytoNramp2andtransportdivalent
metalsintothecellinapH-dependentmanner.Suchan
Nramplvariantwascreatedby
insertingahemaglutininepitopetaginthefourthpredictedextracytoplasmicloopofthe
proteindelineated
byTM7andTM827.Atthecellsurface,theNramp1variantindeed
causedpH-dependentuptake
ofFe2+andMn2+,asmeasuredbybothfluorescence
quenchingandradioisotopicmetals
27.Interestingly,Nramp1wasfoundtobeamore
efficienttransporterfor
Mn2+thanFe2+,suggestingthatMn2+isthenaturalsubstratefor
Nramp1atthephagosomalmembrane
27.Theseresultsgivestrongcredencetothemodel
thatNramp1controlstheintracellularreplication
ofmycobacteriaandotherpathogensby
actingasaneffluxpumpatthephagosomalmembrane,restrictingmicrobialaccessto
essentialmetals(Figure1).
1.2.5PathogensunderNramplcontrol
InadditiontoSalmonellatyphimurium,Leishmaniadonovani,andseveralspecies
ofMycobacteria
(Mbovis53,Mavium54,Mlepraemurium55,Mintracellulare56),
thereareanumberofotherpathogenstowhichdifferentleve1sofresistanceor
9
Figure1.MetaltransportbyNrampproteinsattheinterfaceofhost
pathogeninteraction.Inthetwodiagrams,Nramp1(green),Iysosomal
associatedmembraneproteinLamp(yellow),vacuolarW-ATPase(red),and
metalions(blue)arecolourcoded(Ieft)andidentified(right).
Inmacrophages,
bacterium-containingphagosomesmaturebyfusiontovacuolarW-ATPase
positivevesicles,andLamp-1/Nramp1positivelysosomes.ATP-dependent
acidification
ofthephagosomeisrequiredforNramp1-mediatedeffluxof
divalentmetals(Fe2+,Mn2+,Zn2+).Atthephagosomalmembrane(rightpanel),
thevacuolarW-ATPasecreatesaprotongradient([H+]versus
[H+),whichis
necessaryforNramp1-mediatedmetaleffluxfromthephagosomallumen.
Bacteriapresent
inthephagosomeexpressNramphomologues(e.g.MntH)
thatmayimportdivalentmetalsfromthephagosomalspacebyasimilarpH
dependentmechanism.Themembranetopology
ofNrampproteinsisshown
withrespecttotheamine(N)andcarboxyl
(C)terminus.(From:Lam-Yuk
Tseung,
S.andGros,P.”Geneticcontrolofsusceptibilitytobacterial
infectionsinmousemodels”.CellularMicrobiology.2003May;5(5):299-313.)
susceptibilityhavebeenassociatedwithpolymorphicvariationsinNrampl.These
inc1udeavariety
ofovine57andavian58strainsofSalmonella,aswellasBrucella
abortus
59,Pasteurellapneumotropica60,Candidaalbicans47,Toxoplasmagondii61;62,
andFrancisellatularensis63;64.Surprisingly,Nramplisnotcriticalforresistanceinmice
to
invivoinfectionwithH37rvMycobacteriumtuberculosis,thecauseofpulmonary
tuberculosisinhumans65-67.Thesereportsareincontrasttothesituationinhumans
wherepolymorphicvariantsat
NRAMP1havebeenassociatedwithtuberculosis68-74.
However,thisapparentparadoxmaybeexplainedbydifferencesbetweenthe
experimentalmodel
oftuberculosisinthemouse(loworhighdoses,i.v.),andthenatural
humansituation(lowdoses,aerosol).Thenextsections
ofthisthesiswilldescribethe
effects
ofNramp1oncontrollingtheintracellularreplicationofMycobacteriumand
Salmonella,sincetheroleofNramplininfectionbytheseparasiteshavebeenrelatively
wellcharacterized.
1.2.6EffectofNramplonintracellularMycobacterium
NrampI-mediatedmetaleffluxfromthephagosomehaspleiotropiceffectsonthe
engulfedpathogen.
Ithaslongbeenestablishedthatmycobacteriasurvivewithin
macrophages
bypreventingthematurationofphagosomesintofullybactericidal
phagolysosomes
75-81.MycobacteriapresentinNrampI-deficientphagosomesareableto
inhibitluminalacidification
byblockingrecruitmentofvacuolarH+-ATPasepumpstothe
phagosomalmembrane,aswellasreducingtheacquisition
oflysosomalmarkerssuchas
Lamp1andCathepsinD
78-81.Rather,thesephagosomesretainsortinglearlyendosome
markerssuchastransferrinreceptor(animportantsource
ofTf-iron)andRab5(required
forearlyendosomefusion).Thisinhibition
ofphagosomalmaturationappearstobean
activeprocessfromthelivepathogensinceitdoesnotoccur
whenmacrophagesengulf
latexbeads
ordeadmycobacteria.InthepresenceofNrampl(Nrampl+/+),mycobacteria
areunabletoblockphagosomalmaturation,resultinginincreasedluminalacidification,
increasedfusionwithlysosomes,enhancedbacterialdamage,anddiminished
mycobacterialreplicationcomparedto
Nrampr/-phagosomes82;83(Figure2).Nrampl+/+
macrophagesalsoproducemorenitricoxide(NO)inresponsetomycobacterialinfection
11
Nramp+/+
Macophage
•Nramp1/Slc11a1
oRab5,TfR
Nramp-/-
LE/Lysosome
Macophage
=Lamp1,H-ATPase,CathepsinD
1Livemycobacterium
eKilledmycobacterium
Figure2.TheeffectofNramp1onintracellularMycobacterium.Inthepresenceof
Nramp1(Nramp1+/+,leftpanel),mycobacteriaareunabletoblockphagosomalmatura
tion,resultinginincreasedluminalacidification,increasedfusionwithlysosomes,
enhancedbacterialdamage,anddiminishedmycobacterialreplication.Mycobacteria
survivewithinmacrophagesbypreventingthematuration
ofphagosomesintofully
bactericidalphagolysosomes.MycobacteriapresentinNramp1-deficientphagosomes
(rightpanel)inhibit
luminaiacidificationbyblockingrecruitmentofV-ATPasepumpsto
thephagosomalmembrane,asweilasreducingtheacquisition
ofIysosomalmarkers
suchasLamp1andCathepsin
D.Rather,thesephagosomesretainsorting/earlyendo
somemarkerssuchastransferrinreceptorandRab5.
and/orinterferon-ystimulationthanNramprl-controIs,possiblycontributingto
bacteriostasis84-86.
Themechanismbywhichmycobacteriacaninhibitphagosomalmaturationis
poorlyunderstood.However,Nramp1c1earlyplaysaroleinantagonizingthisprocess.
Onepossibleexplanationisthatinhibition
ofphagosomalmaturationbymycobacteriais
ametal-dependantprocessthatcanbecountered
byNrampI-mediatedmetalefflux.
Indeed,anumber
ofstudieshavedemonstratedtheimportanceofironinmycobacteria
mediatedinhibition
ofphagosomalmaturationaswellasinthevirulenceofthe
intracellularpathogens
87-90.Inaddition,microarraygeneexpressionanalysisofM
tuberculosisisolatedfromactivatedmacrophages,showedup-regulationofmycobacterial
genesinvolvedinmetaluptake,accountingforover
halfoftheactivatedgenes89.Iron
promotesmycobacterialgrowthin
Nramp1+/+miceandpromotesthedevelopmentof
activetuberculosisinhumans90;91.Likewise,extracellularironcanstimulate
intracellulargrowth
ofMaviuminbothNramp1+/+andNrampr/-macrophages92,
suggestingthatexcesscellularironmayoverwhelmthefunctionofNramp190;92.
Collectively,theseresultssuggestthatironisrequiredbymycobacteriatoarrest
phagosomalmaturationandsurviveintracellularly.
1.2.7EffectofNramplonintracellularSalmonella
Unlikemycobacteria,Salmonelladonotsurviveintracellularlybyinhibiting
phagosomalacidification.Rather,theysequesterthemselvesinsidespecialized
Salmonella-containingvacuoles(SCVs)thatbecomeacidifiedandareabletofusewith
LampI-positivelysosomes
93.InpermissiveNrampr/-macrophages,Salmonellasurvive
byblockingrecruitmentofmannose-6-phosphatereceptor(M6PR)positivevesiclesand
isolatethemselvesfromtheendosomalpathway
37.Innon-permissiveNrampl-expressing
macrophages,SCVsfusewithM6PR-positivevesic1esandmaintainaccesstothe
endosomalpathway,expressingendosomalmarkerssuchasEEAIandfluidphaseFITC
dextran
37.TheseeffectscouldbemimickedinNrampI-negativemacrophages
13
byusingmembrane-permeantironchelators94.Theseresultssuggestamode!inwhich
Nramp1counteractstheability
ofSalmonellatosequesteritselffromthedegradative
phagosomalpathway
byrestrictingitsaccesstodivalentmetals.
Divalentmetals,suchasFe2
+andMn2+,have
beenshowntobeessentialin
Salmonellaforvirulenceinvivoandreplicationinculturedmacrophages91;95.Salmonella
possessseveralhighandlowaffinityironandmanganesetransporters96-99andseveralof
thesetransportershavebeenshowntocontributetovirulenceinvivo98;100-102.Infact,
mutationsatanumber
ofthesetransporters(suchasfeoB,sitA-D,orMntH)ordivalent
metalchelationhavebeenshowntoreduce
orabrogatevirulenceofSalmonella101.Thus,
NrampI-mediateddepletion
ofmetalsfromtheintra-phagosomalspaceappearstohavea
majorimpact
onintracellularsurvivalandreplicationofSalmonella.Thepathogen,in
tum,reactstothemetaldepletion
byupregulatingexpressionofmetaltransporterssuch
as
MntHandsitA103andincreasingexpressionoftheirPathogenicityIsland2(SPI2)
virulencegenes104.
1.2.8NRAMPIanditsroleinresistancetoinfectioninhumans
Inhumans,NRAMP1hasbeenmappedtochromosomalregion2q35andconsists
of15exonsthatspana13kbregion105;106.Twotranscriptionalstartsiteshavebeen
mappedandregulatorymotifs
intheNRAMP1promoterregionhavebeenidentified.
Theseinc1udeaTATAboxelement,interferon-yresponseelements,andbindingsitesfor
thetranscriptionalfactorsNFKB,
AP-l,andPU.II05-108.Resultsobtainedinmurine
modelshaveraisedthepossibilitythat
NRAMPgenescouldbeimportantdeterminantsof
susceptibilitytocommonhumandiseases.Anumberofstudiesconductedonethnically
andgeographicallydiversepopulationsareingeneralagreementthat
NRAMP1allelesare
riskfactorsfortuberculosis
74;109-116.Indeed,anumberofgenomicDNApolymorphisms
havebeenidentifiedin
ornearNRAMP1thathavebeenassociatedwithsevera!infectious
andinflammatorydiseasessuchasleprosy
117-119,non-tuberculousmycobacterial(NTM)
lungdisease
120,humanimmunodeficiencyvirus(HIV)121,rheumatoidarthritis122-125,
inflammatoryboweldiseases126;127,multiplesc1erosis128,andtype1diabetes129;130.The
numerousillnessesaffected
byNRAMP1polymorphismsreflectthepleiotropiceffectsof
14
Nramp1functionandtheimportanceofdivalentmetalsindiseaseandinfection.
Recently,Kissler
etalusedlentiviraltransgenesisandRNAinterferencetoknock-down
expression
ofNramp1inanonobesediabetic(NOD)mousemodel,andshowedthat
Nramp1-silencingreducedthefrequency
oftype1diabetesintheseanimaIs130.Their
resultsprovidedstrongevidencethattheinsulin-dependentdiabeteslocus5.2
(idd5.2),
whichhasbeenimplicatedintheNODmousemodel,isindeedNrampl.
1.3Nramp2anditsroleinironhomeostasis
1.3.1Cloning
ortheNramp2gene
TwoyearsafterthepositionalcloningofNrampl,asecondmouseNrampgene
wasidentified
bycDNAcross-hybridizationstudiesbasedonitshighsequencehomology
with
Nrampl131.ThenovelgenewasnamedNramp2,andwasmappedtothedistalpart
ofmousechromosome15.ThemurineNramp2geneconsistsof18exonsspreadover
morethana30kbregion.Analysis
ofthenucleotideandpredictedaminoacidsequence
ofNramp2,indicatedthatitwasanovelproteincloselyhomologoustoNramplandthat
thetwoproteinsdefinedanewfamily
ofproteins1.ThetwoNrampproteinsidentified
shareahigh63%sequenceidentityand78%similarity
1;131.However,incontrasttothe
macrophage-specifie
Nrampl,Nramp2rnRNAexpressionisdetectedalmostubiquitously
40;131
1.3.2Nramp2encodesadivalentmetaltransporter
AlthoughthepolypeptidesequencesofNramp1andNramp2suggestedthatboth
proteinsfunctionedasmembranetransporters,thesubstrateandmechanism
oftransport
ofNrampproteinsremainedelusive.PerhapsthefirstindicationofthefunctionofNramp
proteinsoccurredin1996whenSupekandcolleaguesreportedtheidentificationanovel
Saccharomycescerevisiaemutantthatwasunabletogrowinthepresenceofthemetal
chelatorEGTA
132.Themutantcou1dbesuppressedbyoverexpressionoftheyeastgene
SMF1,ageneoriginallyclonedasamulticopysuppressorofatemperature-sensitive
mutant
(mifl-l)defectiveinthefunctionofmitochondrialprocessingpeptidase133.
However,theabilityofSmflptocomplementgrowthinthepresenceofEGTAsuggested
15
thatSMFlpIayedadirectroleinmetalhomeostasisinyeast.Consistentwiththis
hypothesis,Supek
etal.showedthatasm/1nullmutantexhibitedreducedMn2
+uptake.
Interestingly,
Smflpshowedsignificantresemblance(46%similarity)tobothNramp1
andNramp2proteins
1.
FurtherinsightintothefunctionofNrampproteinsoccurredin1997,whentwo
simultaneousindependentstudiesshowed
bydifferentmethodsthatNramp2functionsas
atransporter
ofdivalentcations,includingiron.Inthefirststudy,Nramp2wasisolatedby
expressioncloningfromaratcDNAlibraryinXenopusoocytesbyGunshinand
colleaguesinasearchforirontransportproteins
40.Thegoalofthisstudywastoidentify
theintestinaltransporterresponsibleforabsorption
ofnon-hemedietaryiron.To
accomplishthis,theypreparedacDNAlibraryusingduodenalrnRNAfromratsthatwere
kept
onalow-irondiet.Theproximalduodenumhadbeenpreviouslyidentifiedasthe
mainsite
ofnon-hemeironabsorptionbyphysiologicalironuptakemeasurements134;135.
AsinglecDNAclonecauseda200-foldincreaseinironuptakecomparedtocontrol
uninjectedoocytes.Theclonewasfoundtocontainrat
Nramp2,althoughitwasre-named
DCT1(Diva/entCationTransporter1)inthisreport,andwaslaterre-namedagainto
DMT1forDiva/entMetalTransporter1.AlthoughDMT1isthemostcommonlyused
termfor
Nramp2today,ithasbeenrecentlyre-namedonceagaintoS1c11a2(solute
carrierfamily
Ilmember2).Forclarity,1willusetheNramp2/DMT1nomenclaturein
theremainderofthisthesis.
Expression
ofNramp2inoocyteswasdemonstratedtomediateuptakeofabroad
range
ofdivalentmetalsincludingFe2+,Mn2+,Zn2+,C02+,Cu2+,Ni2+,Pb2+,and
Cd2
+but
not
Ca2+orMg2
+40.MetaltransportwasshowntobedependentonextracellularpHand
thecellmembranepotential.Furthermore,mRNAexpressionstudiesconfirmedthe
ubiquitousexpression
ofNramp2butalsodemonstratedastrikingtissue-specifie
upregulation
ofNramp2rnRNAuponchronicirondepletion.Thisupregulationwas
strongest
intheproximalintestinebutwasalsoseentoalowerextentinthekidney,liver,
brain,heart,lung,andtestis
40.
16
1.3.3AmutationatNramp2causesirondeficiencyinratsandmice
InaparallelstudytoGunshinetal.40,Flemingandcolleaguesusedapositional
c10ningapproachtoidentifyNramp2asthecausativemutationinmicewithmicrocytic
anemia(mk)
136.Homozygousmklmkmicesufferfromseverehypochromic,microcytic
anemiaduetoimpairedintestinalironabsorptionanddefectiveerythroidironutilization
137-140.ThemkmutationarosespontaneouslyinthebreedingstocksoftheJackson
Laboratoryoverthirtyyearsagoandisinheritedasanautosomalrecessivetraitwithfull
penetrance
137.Newbommicehomozygousforthemutation(mklmk)werereadily
distinguishableatbirth
bytheirsmallsizeandpalecoloration,andweresubsequently
maintained
bybreedingontoseveralgeneticbackgrounds137.ThestudybyFlemingetal.
tracedthe
mkphenotypetoamutationinNramp2thatresultsinanon-conservative
glycinetoargininesubstitution(G185R)
inthepredictedTM4oftheprotein136.
Strikingly,itwaslaterdiscoveredthattheidenticalmutationinNramp2(G185R)wasthe
cause
ofdiseaseinaradiation-inducedratmutantthatexhibitedanmk-likephenotype,
theBelgrade(b)rat
141.Likemk,bisinheritedasanautosomalrecessivetraitthatresults
inhypochromic,microcyticanemiaassociatedwithimpairedreticulocyteironuptakeand
gastrointestinalironabsorption
142-145.Together,theseresultsprovidedstrongevidence
thatNramp2functionsasapH-dependentirontransporteressentialforbothnormal
intestinalironabsorptionanderythroidironusage.
InvitrostudieshaveshownthattheNramp2
Gl85Rproteinexpressedinbothmk
miceandBelgraderatsdisplaysmultiplebiosyntheticandfunctionaldefectsthatcombine
tocauseirondeficiencyinrodents.SuandcolleagueshaveshownthattheG185R
mutationreducestheability
ofNramptotransportironintransfectedHEK293kidney
cells
146.Incontrast,proteinexpressionstudiesinwild-typeandmutantanimaIsled
Canonne-Hergaux
etaltoconc1udethatNramp2Gl85Risinadequatelytargetedorretained
bytheplasmamembraneofenterocytesandreticulocytesfrommkmice147.Experiments
performed
byTouretetalintransfectedLLC-PK1epithelialcellsshowedthat
Nramp2
Gl85Risfunctionallyimpaired,lessstable,andinefficientlyprocessedresultingin
defectiveglycosylationandreducedplasmamembraneexpression
148.Consequently,
17
Nramp2G185Rwasaisoshowntoaccumulateintheendoplasmicreticulumwhereitis
rapidlydegraded
byaproteosome-dependentmechanism148.
ThefactthattheG185Rmutationhasoccurredspontaneouslyontwooccasionsin
mkmice136andonceinBelgrade141ratsisstrikingandsuggeststhateitherthecodon
containingG185ishypermutableorthatG185Rrepresentsagain
offunctionthatconfers
sornesurvivaladvantagetotheorganism.Work
byXuandcolleaguessupportthelatter
hypothesis.TheyshowedthatNramp2
G185R
,despitepresentingmultiplebiosyntheticand
functiondeficiencies,
confersanovelCa2+-selectivepermeabilitypathway149.Theyargue
thattheinflux
ofCa2+mightpotentiatetheresidualNramp2iron-transportactivity.The
transferrincycleisessentialforironuptake
byerythroidprecursorcells150andNramp2
mediatestransfer
ofironfromtransferrincycleendosomestothecytoplasm42;51;141.
ElevatedintracellularCa2+hasbeenreportedtoaccelerateironuptakethroughthe
transferrincycle,apparentlythroughactivation
ofproteinkinaseC151.
Recently,mutationsinNRAMP2havebeenidentifiedinthreehumanpatients
sufferingfromseveremicrocyticanemiaandhepaticironoverload
4-6.Theeffectsof
thesemutationsatboththephysiologicalandmolecularlevelsarediscussedfurtherin
section1.3.9.3
ofthischapter.
1.3.4AlternatesplicingofNramp2rnRNA
TheamineacidsequencepredictedfromtheratcDNAisolatedbyGunshinetal.
wasaimostidenticaitothesequencereportedformouseNramp2,exceptattheextremeC
terminus,wherethetwosequencesdiverged
40;131.However,theratNramp2sequence
washomologoustothepublishedC-terminaisequence
ofhumanNRAMP2152.The
discrepancy
intheC-terminaisequenceswasexplainedinsubsequentstudiesreporting
theexistence
oftwoisoformsofNramp2,generatedbyalterativesplicingattwo3’exons
inmice,ratsandhumans40;131;136;152;153.ThisaltematesplicinggeneratestwoNramp2
proteinswithdifferentC-terminaisegments.Interestingly,one
ofthetwoNramp2splice
isoforms,namedisoform1or
+IRE,containsanironresponsiveelement(IRE)inits3’
untranslatedregion(UTR)andproducesa561aminoacidproteininratsandmice.The
otherspliceisoform,namedisoformIlor
-IRE,lacksanIREinits3’UTRandproduces
18
a568aminoacidprotein.IREsareknowntoplayregulatoryfoIesincellulariron
metabolismthroughinteractionswithironregulatoryproteins(IRPs).TheroleIRPsin
regulatingcellularironhomeostasisisdescribedfurtherinsection1.3.8.4
ofthisChapter.
Recently,a
novel5’upstreamexonhasbeenidentifiedinthehurnan,mouse,andrat
Nramp2genes
154.Thisnovelexon(exonlA)isgeneratedbytheusageofanaltemate
transcriptioninitiationsite,andispredictedtoproduceanNramp2proteinbearingan
additional29-31aminoacids(exon
lA)upstreamofthepreviouslyidentifiedstartcodon
ofNramp2isoforms1andII(exonlB).TheusageofexonlAalsoappearstobehighly
tissue-specifie,withenhancedusageinthekidneyandduodenum
154.Thus,atheoretical
total
offourNramp2mRNAsandproteinscanbeproducedbythegene,dependingon
altemate
5′(exonlAvs.lB)and3′(I/+IREvs.ill-IRE)splicing,givingrisetofour
Nrampisoforms:I(A),I(B),II(A),andII(B)(Figure3).However,most
invitrostudieson
Nramp2,inc1udingaIlthestudiesdescribedinthisthesis,havebeenperformedusingthe
originallyidentifiedisoformsI(B)andII(B).Littleiscurrentlyknownaboutthe
importance
ofexonlAortheextra29-31N-terminalresiduesintheregulation,function,
ortargetingofNramp2.
1.3.5StructuralfeaturesoftheNramp2protein
LikeNramp1,Nramp2isexpressedasa90-100kDaintegralmembrane
phosphoglycoproteinintransfectedCHOorLLC-PK1cellsthatispredictedtohave
twelvemembrane-spanningdomains.Thepredictedtopology
ofNramp2inthe
membranehasbeensupported
byseveralepitopeaccessibilitystudiesinintactcells
26;27;51.PicardandcolleaguesusedimmunofluorecenceexperimentsonintactCHOcells
expressinganepitope-taggedNramp2varianttoshowthattheregionconnectingTM7
and8isindeedexofacial.SimilarexperimentswithNramp1andNramp2variants
possessingN
orCterminalepitopetagshavedemonstratedthattheaminoandcarboxyl
terminalsegments
ofbothtransportersfacethecytosol26;27.GlycosylationinNramp2is
extensive,withthetransporterowing
~50%ofitsmolecularmasstosugarmoietiesadded
attwoconsensusN-linkedglycosylationsignaIspresentinthefourthextra-cytoplasmic
loop
42.ExtensiveglycosylationofNramp2mayhelpprotecttheproteinagainst
19
Isoform1(A)
Nram2•
IsoformIl(A)
Nramp2I<;'IRE"'~:I
IsoformIl(8)
Isoform
1(8)
,Nramp2
Figure3.AlternativesplicingofNramp2pre-mRNAgeneratesfourtheoretical
isoforms.Theuseofalternatetranscriptioninitiationsitesatthe5'exon(exon.1Avs.
1
B),generatesdiversityatthe5'endofthemessage.Alternatesplicingatthe3'terminal
exongeneratesfurtherdiversity,causingtheinclusion(+IRE)ornot(-IRE)aniron
resonpiveelement(IRE)
inthe3'untranslatedregion.
degradationormaybeimportantforpropermaturationandtargetingofthetransporterto
itsite
ofaction.TabuchiandcolleagueshaveshownthatdisruptionoftheN-linked
glycosylationsitesinNramp2affectthetransporter'spolarizeddistributiontotheapical
membrane
ofconfluentMDCKkidneycells155.AsinNramp1,thefourthintra
cytoplasmicloop
ofNramp2containsaconservedtransportmotif(CTM)ofunknown
funetion1;131.
ConsistentwithothermembersoftheNrampfamily,Nramp2possessesnine
highlyconserved,yetthermodynamicallydisfavored,ehargedaminoaeidswithinitsTM
domains
1.AisopresentaretwohighlyeonservedhistidineresiduesinTM6ofthe
protein.Beeause
oftheirhighconservation,theseehargedresidueslikelyplayaeritieal
roleinthestructureand/orfunctionofNramp2.TheworkdeseribedinChapter2
ofthis
thesisidentifiesthreenegativelyehargedresidues(D86,D192,E299)within
transmembranedomains
1,4,and7ofNramp2thatareessentialformetaltransport
activity
28.Thesenegativeresiduesarethoughttoformpartofametalbindingsiteorline
ahydrophilicporeortransportpath.Theseresultshavebeensupportedbysimilar
subsequentmutagenesisstudies
ofthechargedTMresiduesinthebacterialNramp
ortholog,
MntH156.SeveralstudieshavebeenaimedatbetterunderstandingthepH
dependence
ofmetaltransportbyNrampproteins.Sacherandcolleagueshaveprovided
evidencefromelectrophysiologicalmeasurementsthatNramp2,andotherNramp
proteins,mayexhibitaprotonclutchmeehanism,allowingmultipleprotonsto"slip"
throughthetransporteratacidic
pH50.Theadvantageofsuchaprotonslippage
mechanismisnotcurrentlyknown,however,ithasbeenpostulatedtoprotectthe
organismagainsttheoverloading
ofmetalsinthepresenceofexeessprotons.Atthe
molecularlevel,experimentsdescribedinChapter2identifytwoconservedhistidines
(H267,H272)inTM6
ofNramp2thatareeritiealforregulatingthepHdependeneeof
metaltransport28.SubsequentstudiesonratNramp2expressedinXenopusooctyesusing
electrophysiologiealandradioisotopictechniqueshaveshownthatH267andH272are
criticalfortheH+-couplingrequiredformetaltransport
157.Finally,electrophysiological
studieshaveshownthatasingleaminoacidsubstitution(F227DinTM4
ofNramp2can
increasetheratio
ofmetalstoprotonstransportedby14-fold158.
21
StudiesinvolvingtheyeastNramporthologs(Smfl-3)havealsoprovidedvaluable
insightintothemolecularstructureandfunction
ofNramp2.Amutantyeaststrainwith
two
ofitsNramporthologsinactivated(SmflLYSmj2L1)isunabletogrowinthepresence
ofthemetalchelatorEGTA34;132.ExpressionofNramp2inSm/lLYSmj2L1yeastisknown
tosuppressthisphenotypeandrestoregrowthinthepresence
ofEGTA34.Pinneretal
tookadvantageofthistodemonstratethatmutations(Q384E,G394V)athighly
conservedresidueswithintheCTM
ofNramp2abrogatethefunctionoftheprotein34.
Cohenandcolleaguesusedcomplementationassaysinyeastinconjunctionwith
e1ectrophysiologicalmeasurementsinoocytestoanalyzethestructuralandfunctional
importance
ofthefirstextra-cytoplasmicloop(ECI)ofNramp2159.Theyshowedthat
criticalsinglemutations
inECI,Gl19AandQ126D,resultedinacompletelossofmetal
uptakeactivitybyNramp2.However,thenon-functionalQl26Dmutantcouldbe
partiallyrestoredbytheintroductionofasecondmutationattwoaminoacidsupstreamof
Ql26(D124A).ThedoublemutantD124NQl26Dalsoappearedtoalterthemetalion
specificity
ofthetransporterinfavorofFe2+whileatriplemutant
(Gl19ND124NQ126D)displayednotransportactivityyetpossessedalteredpre-steady
statecurrents
159.Theseresultssuggestthatthefirstextra-cytoplasmicloopofNramp2
maybeinvolvedinmetalionbindingandproton-couplingoftransport.
1.3.6Nramp2expression
Nramp2expressionhasbeendetectedathighlevelsinepithelialcellsaswellas
peripheraltissues.Ingeneral,isoform1
ofNramp2appearstobepredominantlyexpressed
inepithelialcellswhileisoformIIappearsto
beexpressedmostlyinnon-epithelialcells.
However,preferentialexpression
ofNramp2isoforms1andIIisnotnecessarilymutually
exclusive:simultaneousexpression
ofbothisoformsatthemRNAslevelhasbeen
observedinseveraltissuesincludingkidney,thymus,andliver
40;155;160.
Isoform1isexpressedattheapicalmembraneofduodenalbrushborder
enterocytes
161,whereitisresponsiblefortheuptakeofdietaryinorganiciron(Figure4).
Immunostainingexperiments
oftissuesectionsshowthatNramp2expressionislimitedto
thevilliandabsent
inthecryptscells161-165.Nramp2expressionappearstobestrongest
22
2+••Fe.••F3+•e
~~e
01
l
MNramp2/DMT1
•Ferroportin
'"Ferrireductase
...Hephaestin
-:Iron
Figure4.Absorptionofnon-hemeironattheintestinalbrushborderandin
reticulocytes.DMT1/Nramp2isoform1isexpressedattheapicalmembraneof
intestinalenteracytes,whereitmediatestheabsorptionofnon-hemedivalentiranthat
hasfirstbeenreduced
byaputativeferrireductase(1).Atthebasolateralmembrane,
Ferroportin(Fpn)isresponsiblefortheexport
ofiranfromtheenterocyteintocircula
tion(2).FolrowingoxidationatthebasolateralmembranebytheferroxidaseHephaes
tin,ironisrapidlyboundtotransferrin(3).Non-epithelialcellssuchaserythroid
precursorsuptakeironprimarilythroughinternalization
ofthetransferrin-transferrin
receptorcomplex(4).Endosomalacidificationfacilitatestherelease
ofironfrom
transferrinandcreatestheprotongradientrequiredforirontransportbyNramp2
isoformIlacrasstheendosomalmembraneandintothecytosol(5).Ironisthen
transportedintothemitochondriafor
hemesynthesisorstoredboundtoferritin.
Apotransferrin-transferrinreceptorandNramp2isoform
Ilaresubsequentlyrecycled
backtothe
ceUsurface.
intheapicaltwo-thirdsofthevilliwhereitislimitedtoenterocytesthatmakeupthe
columnarabsorptiveepithelium
ofthemucosa.Inthesecells,Nramp2localizesprimarily
totheapicalplasmamembraneknownasthebrushborder.Thispattern
ofexpressionwas
alsoobserved
inthehumanintestinalcelllineCaco-2163;166-168.Intheintestine,Nramp2
isoform
1expressionisup-regulatedinresponsetodietaryirondepravationandseems
repressedunderironoverloadconditions161.lmmunohistochemicalstudies
byCanonne
Hergauxandconeaguesshowedthatin
mk/mkenterocytes,Nramp2expressionis
upregulated
butisnotproperlytargetedtothebrushborder147.
Incontrast,isoformIIofNramp2ispredominantlyexpressedinperipheralornon
epithelialcellssuchasinerythroidceIls.StudiesinprimaryasweIlastransfectedceIl
lineshaveshownthatisoformIIisnotonlyexpressedattheplasmamembrane
ofthese
censbutalsointransferrinreceptorpositiverecyclingendosomes42;51;146.Inmice,
reticulocytesandreticulocyteprecursorsceIls(whichgiverisetomatureoxygen-carrying
redbloodcens)arethemajorphysiologicalsite
ofisoformIIexpression,wherethe
transporterisdetectedasa70
kDaspeciesandhasbeenshowntocolocalizewith
transferrinreceptor
169.Duringerythropoiesis,precursorredbloodceIlshavealarge
requirementforironforhemebiosynthesisandtheproductionofhemoglobin.Thehigh
level
ofexpressionofisoformIIinthesecenspermitsthecoordinateduptakeofironinto
recyclingendosomesviathetransferrincycleandthetransport
ofironacrossthe
endosomalmembraneintothecytosol
byNramp2(Figure4).AlthoughNramp2
expressioninreticulocytesdoesnotseemto
beregulatedbybodyironstatus,treatmentof
micewithphenylhydrazineorerythropoietin(toboosterythropoiesis)inducesthe
productionofNramp2-expressingreticulocytes
170.However,reticulocytesfrommk/mk
miceexpressnodetectableNramp2protein.
Macrophagesareanothersource
ofsignificantNramp2isoformIIexpression42.
Thesephagocyticcellsplayacriticalroleintherecyclingofhemeironthrough
phagocytosis
ofsenescentredbloodcens.Nramp2mayindeedtransportphagosomaliron
intothecytosolafterhemoglobindegradation.Immunoblottingexperimentshaveshown
thatNramp2isexpressedasaglycosylated70-90
kDaproteininmacrophages42.
ImmunofluorescenceexperimentsrevealedthatNramp2islocalizedprimarilytotheearly
24
endosomecompartmentwithsornepresenceinLampI-positivelate
endosomes/lysosomesintransfected
RAW264.7macropagesandtwomurineSerolicell
lines
ofthetestis(TM4and15P-l)171.Immunofluorescencestudieshavealsoshownthat
Nramp2can
beassociatedwithphagosomalmembranesduringphagocytosis42.More
recently,Jabado
etalshowedthatNramp2canassociatewitherythrocyte-containing
phagosomes
intransfectedRAW264.7macrophages171.
Inthekidney,Nramp2proteinhasbeendetectedinmicrosomalmembrane
fractionsasa70-75
kDamembraneprotein161.Inmousekidneys,Canonne-Hergauxand
colleaguesusedimmunohistochemicalstainingwithanaffinitypurifiedanti-Nramp2
antibodytodetectNramp2inthecortexbutnotthemedulIa,andatthebrushborderand
apicalpoles
ofepithelialcellsoftheproximaltubule172•Inratkidneys,Fergusonand
colleaguesdetectedNramp2expressionintheintracellularvesicles
ofproximaltubule
cells
ofS3segment,collectingducts,thickascendinglimbsofHenle'sloopand,more
intensely,attheapicalmembrane
ofdistalconvolutedtubules173.Recently,Abouhamed
etalreportedthedetectionofNramp2inLampl-positivelateendosomesandlysosomes
withinratproximaltubulecells
174.Becausethesestudiesusedpolyclonalantibodies
recognizingtheNterminus
ofNramp2,theexactisoform(1orII)expressedinthekidney
couldnot
bedetermined.AlthoughthefactCanonne-Hergauxetalcouldnotdetectany
significantexpressioninthekidneyusinganantibodyspecificallyrecognizingisoform
il
stronglysuggeststhatthemajorityofNramp2presentinthekidneyisindeedisoform1
172.Insharpcontrasttotheintestine,Nramp2mRNAandproteinexpressionappearsto
beonlymodestlyaffectedbydietaryirondeprevationandisnotaffectedbystimulationof
erythropoiesis172;175.Furthermore,whileanemicmkandBelgrademutantsshowstrong
upregulation
ofNramp2intheintestine,theyshowverylowNramp2expressioninthe
kidney
172;176.
Inthebrain,workbyGunshinandcolleaguesshowedNramp2mRNAis
expressedinmostneuronsatlowlevelsbutnotinglial
orependymalceUs40.Stronger
Nramp2expressionwasdetectedindenselypackedcellgroups,suchasthehippocampal
pyramidalandgranulecelIs,cerebellargranulecelIs,thepreopticnucleusandpyramidal
cells
ofthepiriformcortex40.StrongNramp2mRNAexpressionwasalsoseeninthe
25
ventralportionoftheanteriorolfactorynucleusandintheepithelialcellsofthechoroid
plexus
40.Attheproteinlevel,BurdoandcolleaguesdetectedNramp2inneuronsof
striatum,cerebellum,thalamusandintheependymalandvascularcellsthroughouttherat
brain
177.ImmunohistochemicalstudiesbyMoosandMorganinratsconfirmedthat
Nramp2proteinisexpressedinneuronsandthechoroidplexus,
butnotinbraincapillary
endothelialcells
orinmacro-ormicroglialcells178.Sornestudieshavesuggestedthat
Nramp2expressioninneuronalcellsisregulated
bythehypoxiaregulator.Recentstudies
byLisandcolleagueshaveshownthathypoxiaselectivelyincreasesexpressionof
Nramp2,particularlytheexonlAcontainingisoformsofthetransporter,inrat
pheochromocytoma
(PCI2)cells179.
Intheliver,themainsiteofbodyironstorage,Nramp2mRNAexpressionhas
beendetectedinanumber
ofcellstypes.Usinginsituhybridizationandquantitativereal
timePCR,ZhangandcolleaguesdetectedNramp2
mRNAexpressioninratliver
hepatocytes,Kupffercells,sinusoidalendothelialcells(SECs),andhepaticstellatecells
(RSCs)
180.Attheproteinlevel,immunohistochemicalstudiesbyTrinderandcolleagues
detectedNramp2
ontheplasmamembranesofrathepatocytesandthisexpression
appearedtoincreaseanddecreaseinresponsetoironoverload
orirondeficiency,
respectively
162.TrinderetalsuggestedthatupregulationofNramp2intheliverduring
ironoverloadcouldreducetherisk
ofincreasedironintakeandcelldamageelsewherein
thebody.
Nramp2hasalsobeendetectedinseveralothertissues,althoughthedistribution
ofthetransporteratthesesiteshasbeenlessextensivelycharacterized.Inthethymus,
Nramp2
mRNAwasdetectedinthecortical,butnotmedullary,thymocytes40.Inthe
testis,Nramp2
mRNA40andprotein171expressionhasbeendetectedintheSertolicells
ofseminiferoustubules.Nramp2proteinhasalsobeendetectedintheplacenta,bothin
thecytoplasmatthejunction
offetalmembraneandfetalvessels181.Inthisstudy,
GeorgieffandcolleaguessuggestedthatNramp2
maytransportendosomalferrousiron
intothecytoplasm
ofthehumansyncytiotrophoblasttosupplythefetus181.
26
1.3.7SubcellulartargetingofNramp2
AlthoughbothNramp2isoforms1andIIareexpressedattheplasmamembrane,
immunolocalizationstudiesintransfectedcellshaveshownthatisoforms
1andIIexhibit
differentsubcellularendosomaltargetingatsteady-state.
IntransfectedLLC-PKI,CHO
and
RAW264.7cells,isoformIIismostlyexpressedinearlyandrecyclingendosomes
42;51.Studiesusinganexofacially-taggedNramp2moleculehaveshownthatisoformII
moleculespresentatthecellsurfaceandinrecyclingendosomesareindynamic
equilibrium,withsurfacetransportersbeingcontinuouslyinternalizedviaaclathrinand
dynamin-dependentprocess
51.Incontrast,isoform1hasbeendetectedinLampl-positive
lateendosomesandlysosomesintransfectedHEp-2andLLC-PK
Icells155;182.Lysosomal
expression
ofisoform1inphagocyticcellssuchasmacrophagesmayberequiredforthe
recycling
ofironfromsenescentredbloodcells.WorkdescribedinChapter6ofthis
thesiscomparesNramp2isoforms
1andIIwithrespecttofunction,subcellular
localization,endocytosiskinetics,andfateuponinternalizationintransfectedLLC-PK
I
cells.Thisworkshowsthatisoform1isexpressedathigherlevelsatthecellsurface
comparedtoisoformIIduetoaslowerrate
ofinternalizedfromtheplasmamembrane.In
addition,isoform1isnotefficientlyrecycleduponendocytosisandistargetedtolate
endosomesandlysosomes
182.Highersurfaceexpressionisadvantageousforisoform1-
expressingepithelialcells,whichmustabsorbFe2+and
possiblyotherdivalentmetals
acrosstheirplasmamembranes.Thus,alternativesplicing
ofNramp2criticallyregulates
thesubcellularlocalizationandsite
ofmetaltransport.
Cytoplasmicmotifssuchastyrosine-based(NPXY,
YXX
haveanadditionalbutlessdeterminateeffect
onsubcellulartargetingofthetransporter
184
1.3.8Nramp2andcellularironhomeostasis
Ironisanessentialelementforlife,playingavarietyofrolesinorganismsranging
fromthetransportation
ofoxygentothegenerationofanti-microbialreactiveoxygen
species.One
ofthekeypropertiesofironisitsabilitytomediateoxidation-reduction
reactions
byalteringbetweenitsFe3+andFe2+oxidativestates.However,itisthishighly
reactivenaturethatmakesanexcess
of”free”ironverytoxictoorganismsand
necessitatesstrictmechanisms
ofregulation.
1.3.8.1Mechanismsofcellularironabsorption
Highlyreactiveironinitsionicformisnotnormallyfoundathighlevelsin
normalorganisms.Rather,themajority
ofcirculatingiron(Fe3+)existsinaninertform
boundtotheabundantplasmaglycoproteintransferrin(Tf).Mostcellsgainaccesstoiron
byintemalizingtheentirediferric-Tfcomplexthroughtheubiquitously-expressed
transferrinreceptor
1(TfRl),whichresidesinc1athrin-coatedpitsonthesurfaceofcells.
Diferric
TfbindsTfRlattheplasmamembraneandentirecomplexisintemalizedby
clathrin-dependentreceptor-mediatedendocytosisintoearlyendosomes.Endosomal
acidification(pH5.5-6.0)
byvacuolarW-ATPasestrengthenstheinteractionbetweenTf
andTfRlbutweakensthebindingofirontoTf,causingthereleaseofFe3+inthe
endosomallumen.Arecentlyidentifiedferrireductase,
Steap3,reducesFe3+toFe2+inthe
endosomallumen
185;186.Theacidicenvironmentintheendosomealsoprovidesthe
protongradientforNramp2totransportFe
2+acrosstheendosomalmembraneintothe
CytOSOI141;169.Apo-TflTfRlaswellasNramp2arethenrecyc1edbacktothecellsurface,
28
wheretheycanbeusedinfurthercyclesofironuptake51(Figure4).Althoughmostcells
arethoughttouptakeironviatheTf-
TfRlcycle,thisprocessisofparticularimportance
fordevelopingerythrocyteswhichhaveanenormousneed
ofironforhemoglobin
synthesis.Ahomolog
ofTfRl,transferrinreceptor2(TfR2),isexpressedexclusivelyin
erythroidcells,hepatocytes,andduodenalcryptcells
187.TfR2alsofunctionsinthe
uptake
ofTf-iron,althoughitbindsTfwithlessaffinitycomparedtoTiRlandisnot
regulated
bytheIRP/IREregulatorysystem.TfR2clearlyplaysacriticalroleiniron
homeostasisasmutationsatthehuman
TjR2generesultinsystemicironoverload188.
PolarizedepithelialcellsofthekidneyareabletouptakeTf-ironbyamegalin-dependent
mechanismusingtheTf-bindingendocyticreceptorcubilin
189.
ThereareseveralTf-independentcellularmechanismsofironuptake.Brush
borderintestinalenterocytesuptakedietarynon-hemeirondirectlyacrosstheirapical
membranesthroughtheaction
ofNramp2(Figure4).Non-hemeironisfoundin
vegetablesandgrainproductsandexistsinitsinsolubleFe3+form,whichmustfirstbe
convertedtoFe
2+beforeitcanbetransportedbyNramp2.Thisisthoughttooccur
throughtheaction
ofacytochromeb-likeferrireductasecalledDcytb(CybrdJ),whichis
expressedattheapicalenterocytemembrane
190.Otherredundantferrireductasesmay
alsoexistsincestudiesinvolvingtargeteddisruption
oftheCybrdlgeneinmicesuggest
Dcytbisnotessentialforintestinalironabsorptioninmicefedanormalirondiet
191.
Gastricacidsecretionbythestomachpromotesthesolubilityofironcomplexesandalso
providestheprotongradientrequiredforNramp2-mediatedFe
2+uptake.
Ironisrequiredtomaintainproperneuronalcellfunction,however,anexcess
of
ironcausesneurodegeneration.EarlyimmunohistochemicalstudiesbyMoosclearly
showedadiverseexpressionoftransferrinreceptorthroughoutthecentralnervoussystem
(CNS)
192.Morerecently,severalindependentgroupshaveshownthatNramp2isalso
expressedinneuronalcells
40;177;193,suggestingthatTiRandNramp2workin
conjunctioninthesecellstouptaketransferrin-boundiron.Irontransportedthroughthe
blood-brainbarriermayexistinthebraininterstitialfluidinalowmolecularweight
form,suchasiron-citrate.Thepresence
ofnontransferrin-boundironinbrain
29
extracellularfluidssuggeststhatneuronscanalsotakeupironinatransferrin-freeform
194-197.ThismaybemediatedbyNramp2expressionattheneuronalplasmamembrane.
Ironre-absorptioninthekidneyisnotcompletelyunderstoodyetstudies
by
Canonne-Hergauxetal172andFergusonetal173suggestthatapicallyexpressedNramp2
mayfunctionaspart
ofanironre-absorptionsysteminthekidney,possiblytopreventthe
loss
ofpreciousironintheurine.ThisideaissupportedbytheobservationthatNramp2
expressioninthekidneyincreasesapproximatelytwo-foldinmicefedalow-irondiet
172.
Furthermore,Wareingandcolleagueshaveshownusingmicroinjectionand
microperfusiontechniquesthatincreasedNramp2expressioninratkidneywas
accompanied
byadecreaseinurinaryironexcretionrateandviceversa198.Interestingly,
mk/mkmiceandb/bratssufferingfromseveremycrocyticanemiashowalmosta
completeloss
ofNramp2expressioninthekidney172;176.However,anotherstudyby
Fergusonetalshowedthatalthoughb/bratsexhibithigherserumironlevelsthan+/b
rats,bothanimaIsdisplaysimilarurinaryironexcretionrates,suggestingthatNramp2is
notplayacriticalroleinironre-absorption
bythekidney176.
Dietaryhemeironisanimportantnutritionalsourceofironincarnivoresand
omnivoresthatismorereadilyabsorbedthannon-hemeiron.Ironisreleasedfromheme
throughthebreakdownofhemoglobinandmyoglobinpresent
inredmeat.Inmammals,it
hasbeenknownforsometimethatproximalduodenalenterocytesandhepatocytesare
themajorsites
ofhemetransport199;200.Themechanismofhemetransportacrossthe
plasmamembraneremainedelusiveuntilShayeghiandcolleaguesrecentlyidentifieda
duodenalmembraneprotein,hemecarrierprotein1(HCPI),thatmediatescellularheme
uptake
201.HCPIshowssequencesimilaritytoseveralbacterialmetal-tetracyclin
transporters,isupregulatedisresponsetohypoxia,andappearstoberecruitedtothe
plasmamembranefromthecytoplasmuponirondeficiency
201.Upontranslocationacross
theplasmamembrane,hemeappearstolocalizeinmembraneboundvesic1eswithin
thecytoplasm
199;200;202;203;203beforeitisdegradedbytheenzymehemeoxygenase(HO-l
andHO-2)toyieldferrousiron
204.
Smallpeptidesiderophoresaresecretedbymicro-organismstotrapandimport
ironacrossthecellmembrane.Likethisbacterialiron-uptakesystem,mammalianneural
30
gelatinase-associatedlipocalin(NGAL/24p3)hasbeenreportedtocomplexwithironand
beintemalizedandtraffickedtolateendosomes205.RecombinantNGAL/24p3loaded
withironhasbeenshowntodeliverthemetalintoculturedcellsandaffecttheexpression
ofiron-regulatedgenes.ThisNGAL/24p3deliverypathwayappearstobeimportantfor
differentiatingepithelialcellsduringearlyembryonicdevelopment
205;206.
Finally,tissuemacrophagesabsorbironindirectlythroughthephagocytosisofold
ordamagederythrocytes.Engulfedredcellsarelysed,hemoglobinisdegraded(withthe
help
ofhemeoxygenase),andthereleasedironiseitherstoredboundtoferritinor
exportedtoreloadcirculatingapo-Tf.Therecycling
ofironbymacrophagesisahighly
efficientprocessthatrepresentsthemajorsource
ofironforcellularprocessessuchas
erythropoesis.
1.3.8.2Mechanismsofcellularironstorageandusage
Onceimportedintothecell,excessironthatwillnotbeimmediatelyusedis
storedboundtotheubiquitousproteinferritin
207.Ferritinisahighlyconserved
multimericproteinthatisabletosequesterinorganicFe
2+byfirstconvertingittoFe3+
beforestoringitasthechemicallylessreactiveferrihydrite208.Mammalianferritin
consists
of24light(L)andheavy(H)chainsubunitsthatcanaccommodateupto4500
atoms
ofiron209.Whencellsexperienceagreaterrequirementforiron,ferritinis
degradedandironisreleased,howeverthisprocessisnotcurrentlywellunderstood.
Most
oftheironwithincellsisdirectedtothemitochondriaforeitherheme
biosynthesis
ormaturationofiron-sulfur(Fe-S)clusters.Herneissynthesizedbyan
enzymecalledferrochelatase,whichcatalyzestheinsertion
offerrousionsinto
protoporphyrinIX(reviewedin
210).Hernesynthesisisofparticularimportancein
oxygen-carryingerythroidcells,howeverthemechanism
bywhichironisdeliveredto
mitochondriaisnotcurrentlyknown.Recently,Zhangandcolleaguesproposedthata
transientinteractionbetweeniron-richendosomesandmitochondriaisresponsibleforthe
transfer
ofironacquiredfromtransferrintoferrochelatase211.Newlysynthesizedhemeis
rapidlyexportedoutsidethemitochondriaintothecytosolandendoplasmicreticulum,
andassociateswithapo-hemoproteins
212;213.Fe-Sclustersareubiquitousandparticipate
31
inanumberofproccsscsincludingclcctrontransfer,substratebindinglactivation,and
ironlsulfurstorage
214.Themitochondrialmatrixproteinfrataxinisrequiredfor
biogenesis
ofFe-Sproteinsandisthoughttoplayrolesinmitochondrialironexportand
storage
215-217.Mutationsatthefrataxingenecausetheneurodegenerativedisorder
Friedreich’sataxia.PatientswithFriedreich’sataxiapossessadeficiency
inFe-S
containingproteinsanddisplaymitochondrialironoverload
218.
1.3.8.3Mechanismsofironexport
Becauseironissuchapreciouselement,thecurrentdogmaisthatthereisno
excretionmechanismforiron,withlossfromthebodynormallyresultingfrom
desquamation
orthelossofbiologicalfluids(especiallyblood).Atthecellularlevel,in
contrasttothenumerousmechanismsidentifiedforcellularironimport,muchlessis
knownaboutthemechanismsunderlyingcellulariron
eXPOrt’Infact,theonlyputative
mammalianironexporteridentifiedtodateisferroportin(MTP,Ireg
1)219-221.Ferroportin
isexpressedatthebasolateralmembrane
ofduodenalenterocyteswhereitmediatesiron
(Fe2
+)eXPOrtinto
thebloodstreaminconjunctionwiththeferroxidasehephaestin,which
convertsFe2
+to
Fe3+before
itcanbeboundbycirculatingapo_Tf222.Mutationof
hephaestininsex-linkedanemia(Sla)miceleadstoironaccumulationintheepitheliaand
resultsin
Slamicehavingasystemicirondeficiency222.Innon-intestinalcells,iron
exportrequiresoxidation
bytheplasmaproteinceruloplasmin223.Ferroportinisalso
highlyexpressed
inintracellularvesic1esoftissuemacrophages,whereitisthoughtto
playaroleinthe
eXPOrtofironrecyc1edfromengulfedredbloodcells.Whenferroportin
expressionisupregulatedthroughirontreatment
orerythropagocytosis,ferroportin
expressionisstronglyenhancedattheplasmamembrane
ofmacrophages224.Incontrast,
treatmentwiththeironregulatoryproteinhepcidin(seebelow)causesrapid
intemalizationanddegradation
ofthemacrophageironexporter224;225.
1.3.8.4Regulationofcellularironhomeostasis
Proteinsinvolvedinironuptake,usage,storageandexportrequiretight
regulation.Thebestcharacterizedform
ofiron-dependentregulationisatthelevelof
32
mRNAstability.Ironresponsiveelements(IREs)areconservedhairpinstructures
approximately30nuc1eotidesinlengthfoundineitherthe
5’or3’untranslatedregions
(UTRs)
ofmRNAsencodingproteinsinvolvedinironhomeostasissuchasNramp2,TfR,
ferroportin,andferritin.Specializedironregulatoryproteins(IRP
l,IRP2)interactwith
theIREsbasedoncellularlabileironpoollevels.Ironlevelsregulatethebinding
ofIRP1
andIRP2toIREs
bydistinctmechanisms226.Whencellularironlevelsarehigh,iron(as
Fe-Sclusters)bindsIRPIandinhibitsitsbindingtoIREs
byconvertingittoanaconitase,
whichinteraconvertscitrateandisocitrate.Whencellularironstoresarelow,IRPIisable
tofreelybindIREs.IRP/IREinteractionsarealsocontrolled
byotherfactorsincludingthe
presence
ofreactiveoxygenspecies,nitricoxide,andhypoxia.Themechanismbywhich
ironregulatesIRP2islesswellunderstood.UnlikeIRPI,IRP2doesnotbindFe-S
clustersbuthasbeenshowntoaccumulateiniron-starvedcellsandistargetedfor
degradationwhenironlevelsarehigh
227.IRP2isalsosensitivetodegradationinthe
presenceofnitricoxide(NO),whereasIRPIisactivated
byNO228;229.Recentstudies
havesuggestedthatIRP2isthepredominantregulator
ofironhomeostasisinmammalian
cells.Indeed,micedeficientinIRPIappearnormalyetmicedeficientinIRP2show
pronouncedmis-regulation
ofironmetabolismandnervedamage230;231.
SingleIREsarefoundinthe5’UTRsofmRNAsencodingferritin,erythroid
5-aminolevulinicacidsynthase(requiredforhemebiosynthesis),mitochondrialaconitase
(acitratecycleenzyme),andferroportin
220;221;227;232.TheformationofIRP/IRE
complexesonthe
5’UTRofthesetranscriptsinhibitstheirtranslation,leadingtodown
regulation
oftheprotein233.TheoppositeresultoccurswhenIREsarelocatedinthe3′
UTRoftranscripts.,MultipleIREsarepresentinthe3’UTRofmRNAencodingTfRl
andbinding
ofIRPservestostabilizethetranscript,leadingtoincreasedtranslationand
up-regulation
oftheprotein227,The3’UTRofmRNAencodingNramp2isoform1
containsasingleIREthathasbeenshowntobindIRPI
invitro234,However,thisIRE
hasbeenshowntoonlymarginallyregulateNramp2expressioninculturedcells
234.The
recentlyidentified
5’promoter/exonlAregionofNramp2appearstoworkinconjunction
withthe
3’UTRIREmodulateNramp2expressioninresponsetothecellularand
systemicironlevels
154.
33
Genesinvolvedinironhomeostasisarealsoregulatedatthetranscriptionallevel.
Forexample,cytokineproductionhasbeenreportedtomodulateexpressionofvarious
iron-relatedgenes.Interferon-y
(INF-y),tumornecrosisfactor-a,interleukin-l(IL-l),and
IL-6haveallbeenreportedtostimulateferritinexpressionbutdown-regulate
TfRl207.
INF-yandlipopolysaccharide(LPS)havebeenreportedtoinduceNramp2expressionbut
inhibitferroportinexpressioninactivatedmonocytes
235;236.Theresponseofferroportin
toLPSappearstorequiresignalingthroughtheLPSreceptor,Toll-likereceptor4(TLR-
4)
236,andmaycontributetotheironsequestrationbyphagocyticcellsobservedduring
inflammation.
1.3.9Nramp2andsystemicironhomeostasis
Over65yearsago,studiesonironbalanceinhumansshowedthatvirtuallyno
ironisexcretedandthatstableironlevelsaremaintained
bymodulatingabsorptionof
ironfromthegut.Insufficientorexcessiveaccumulationofironcanbeproblematic,
especiallycoupledwiththefactthatmammalslackanaturalphysiologicalexcretion
systemforiron.Therefore,organismshavedevelopedsophisticatedmechanismstosense
andregulatebodyironlevelsinresponsetothedemandingneeds
ofsystemicprocesses
suchaserythropoesis.Themaintenanceofhealthyironhomeostasisrequiresefficient
communicationbetweenthecellsthatusethemostiron(erythroidprecursors)andthe
cellsthatobtainandstoreiron(duodenalenterocytes,tissuemacrophages,hepatocytes).
1.3.9.1Systemicironregulators
Theprimaryironstoragesitesaretissuemacrophagesandhepatocytes.Ironis
storedboundtoferritininthesecellsandthelevel
ofthesestoresismonitoredand
maintained
byaso-called”storesregulator”237;238.Thestoresregulatorcan,inpart,alter
intestinalironabsorptioninresponsetobodystores.Erythroidcellsarebyfarthemajor
consumers
ofironandtheamounttheironpresentinthebonemarrow,erythroid
precursors,andcirculatingredbloodcellsisnormallygreaterthantheamount
ofiron
presentinthestores.Whensystemicirondemandforerythropoeisisexceedswhatis
availableinthestoragepools,an”erythroidregulator”up-regulatesintestinalironuptake
34
tocompensateandreplenishthestores238.A”hypoxiaregulator”isaIsebelievedteexistl
alteringironhomeostasisinresponsetoinsufficientbloodandtissueoxygenlevels.
Meanwhile,an”inflammatoryregulator”isthoughttomediatetheretention
ofironwithin
tissuemacrophagesasweIlasthereduction
ofintestinalironabsorption,asahost
responsetoinfection
orinflammation.Thisactionisbelievedtopromotehostresistance
byrestrictingtheamountofironavailabletopathogens.
Itisclearthatahierarchyexistsamongtheregulators.ThisisespeciaUyevidentin
diseaseswhereerythropoesisisiron-deficientyetthe
bodyironstoresareoverfilled.
Hypotransferrinemicmice
(Tr!PX),whichpossessamutationdisruptingasplicedonor
siteinthe
transferringene,haveaneartotaldeficiencyincirculatingserumtransferrin
239.Erythroidprecursorsuptakethemajorityoftheirironasdiferric-TfviaTfR!and
thereforeerythropoesisisseverelyimpaired
inTr!pxmice150.However,sinceanumber
ofnon-erythroidceUsandtissuesreadilyuptakenon-transferrinboundiron,ironoverload
developsinvariousorganssuchastheliverandpancreas.Inthiscase,signaIsfromthe
erythroidregulatorseemtooverridesignaIsfromthestoresregulator,up-regulatingthe
absorption
ofdietaryironintheduodenum.Thebodyappearstoprioritize(andrightfully
so)ironneededforerythropoesisovertheneedtoavoidironoverload.
Ananalogous
scenariooccurs
intwoothermousemodelsofironoverloadgeneratedbytargeted
disruption
oftheHfeandf3-2microglobulingenes240;241.
Thesearchforregulatoryeffectorsthatcanmodulateintestinalironabsorption,
ironrecycling
bymacrophages,andironstoragebyhepatocyteshasbeenextensive.This
searchledtotherecentidentification
ofhepcidin(HAMP,LEAP-l),asmaUcysteine-rich
peptidehormonethatisproduced
byhepatocytesandexcretedthroughthekidneys242-244.
Producedfromthecleavageofalargerprecursorprotein,hepcidinshowssequence
similaritytopeptides(defensins)involved
ininnateimmunityandpossessesitselfanti
microbialproperties
242;243.HepcidincontrolsextraceUularironlevelsbyregulatingits
intestinalabsorption,placentaltransport,recyc1ing
bymacrophages,andreleasefrom
stores.Hepcidin-deficient
(USF2-1-)micedevelopelevatedbodyironstoreswhile
transgenicmiceconstitutively-expressinghepcidineventuallydiefromsevereiron
deficiencyanemia
245;246.Hepcidinalsoappearstobeanacutephaseresponsepeptide
35
induceddirectlybyvariousinflammatorystimuliincludingbacteriallipopolysaccharide
247-250.Severalstudieshavelinkedhepcidintotheerythroid,stores,hypoxia,and
inflammatoryregulators(reviewedin
251).Althoughthesignalingpathwaysofthese
mechanismsarenotcompletelyunderstood,moststudiesare
inagreementthatwheniron
isscarce,decreasedserumhepcidinexpressionisassociatedwithenhancedironrelease
fromintestinalcellsandmacrophages
248;252;253.Conversely,inastateofironoverload,
increasedserumhepcidinexpressionisassociatedwithdecreasedironreleasefrom
intestinalcellsandmacrophages
244;254;255.Recently,hepcidinwasfoundtoinhibit
cellularironefflux
bybindingtotheironexporterferroportinandinducingits
internalizationfromthecellsurfaceanddegradation
224;256.Mutagenesisstudieshave
revealedthattheNterminus
ofhepcidinisresponsibleforitsinteractionwithferroportin
257
1.3.9.2ImportanceofNramp2insystemicironhomeostasis
Becauseofitsdualroleinintestinalironabsorptionaswellasinacquisitionof
transferrin-iron,mutationsinNramp2severelyaffectsystemicironhomeostasis.Bothmk
miceandBelgraderats,whichpossesstheidenticalmutation(G185R)inNramp2,suffer
fromseveremicrocyticanemia
136;141.Theneedforincreasederythropoesisinthese
rodentspromptstheerythroidregulatortoincreaseintestinalironabsorption
byup
regulatingNramp2expressionattheduodenalbrushborder
147.However,becausethe
G185RmutationattenuatesNramp2function,theanemiapersists
146;148.
RecentworkbyGunshinandcolleagueshasfurtheredourunderstandingofthe
roleofNramp2invivo.TheyshowedthatglobalinactivationofNramp2inmiceresults
inasimilarbutmoreseverephenotypethanthatseenforanimaIshomozygousforthe
G185Rmutation
258.AlthoughNramp2-1
-micewerebornalivewithnoapparent
anatomicalabnormalities,theywerenoticeablypaleratbirthandlaterdisplayed
progressivepost-natalgrowthretardation,withnomicesurvivingmorethan7days
258.
TherelativelynormaldevelopmentoftheNramp2-1
-miceinuterosuggestedthatfetal
Nramp2isnotessentialformaterno-fetalirontransfer.Transplantinghematopoeticstem
cellsfrom
Nramp2-1
-miceintoirradiatedWTmiceresultedinabnormalerythrocyte
36
morphologyanddecreasedhemoglobinlevels,confirmingtheimportanceofNramp2in
erythroidironutilization
258.Furthermore,studiesinvolvingselectiveinactivationof
Nramp2intheintestineconfirmedtheimportanceofNramp2intheabsorptionofdietary
non-hemeiron
258.
1.3.9.3DiseasesassociatedwithNRAMP2mutationsinhumans
Currently,therearethreereportedcasesofmutationsinhumanNRAMP2
associatedwithiron-relateddisorders.Thefirstcasewasidentifiedin2004,when
Priwitzerovaandcolleaguesreporteda20-year-oldfemalepatient
ofCzechorigin,the
product
ofaconsanguineousunion,whosufferedfromliverhemosiderosisandsevere
congenitalhypochromicmicrocyticanemiaduetodefectiveerythroidironuse
259.Ayear
later,Mims
etalreportedthatthispatientwasinfacthomozygousforaGtoC
substitutionatthelastnuc1eotide
ofexon12ofNRAMP2(NRAMP2G1285C)4.This
mutationhadadualeffect,impairingnormalsplicing
ofexon12byanestimated90%
andintroducinganaminoacidsubstitution(E399D)intheremaining
(-10%)properly
splicedtranscriptfoundinthepatient.Theimpairedsplicingisthoughttoresultfrom
alteration
oftheconsensussequenceforbindingoftheUlsnRNPattheexon12/intron12
boundaryandtranslatesintoatruncated,non-functionalNramp2protein
4;260;261.
AlthoughE399Drepresentsaconservativemutation,E399residesinthehighly
conservedfourthintra-cytoplasmicloop
ofNramp2definedastheconservedtransport
motif.WorkdescribedinChapter3
ofthisthesisexaminedtheeffectsoftheE399D
mutationaswellasothermutationsatthatpositionintransfectedLLC-PK
1cells.These
resultsshowedthattheE399Dmutationdoesnotinitselfaffectexpression,function,or
targeting
oftheNramp2protein,andthatthereducedNramp2functioninthepatientis
likelycaused
byaquantitativereductioninNRAMP2mRNAlevelsduetoimproper
splicing
262.Thisresultwasconfirmedbystudiesfromothergroups260;261.
Inearly2006,Iolasconandcolleaguesreportedasecondhumanpatientwhois
compoundheterozygousfortwonovelmutationsin
NRAMP2263.Thepatientisa5-year
oldmale
ofltalianoriginsufferingfromseverehypochromicmicrocyticanemiawith
hepaticironoverload.Thepatientpossessedtwonovelmutations
inNRAMP2:a3base
37
pairdeletion(deICTT)inintron4andaCtoTtransition(NRAMP2c/246T)inexon13
resultinginanArgtoCyssubstitutionatposition416(R416C)ofNramp2.TheCTT
deletionwasshowntodisruptnonnalsplicing
ofNRAMP2pre-mRNA,causingapartial
(30-35%)skipping
ofexon5263.Thisskippingresultsinatruncated,non-functional
Nramp2proteinthatisinheritedinafullyrecessivemanner.R416representsahighly
conservedresiduelocatedinTM9.WorkdescribedinChapter4
ofthisthesisexamined
theeffects
ofconservativeandnon-conservativemutationsatR416.Theseresultsshowed
thatnon-conservativesubstitutionsatR416(C,A,E)causemultiplefunctional
deficienciesincludingdefectiveproteinprocessing,loss
oftransportactivity,impaired
cellsurfacetargetingandrecyclingthroughendosomes,concomitantwithretention
ofthe
transporterintheendoplasmicreticulum
264.ThesefindingsdemonstratedthattheR416C
mutationrepresentsacompleteloss-of-functionandthataquantitativereductionin
Nramp2expressionisthecause
ofthemicrocyticanemiaandironoverloadinthepatient.
Recently,Beaumontandcolleaguesreportedathirdhumanpatientcompound
heterozygotefortwonovel
NRAMP2mutations,associatedwithmicrocyticanemiaand
progressiveliverironoverload
265.Inthefirstmutation,aGTGdeletioninexon5causes
thein-framedeletion
ofV114inTM2.Thesecondmutation,aG>Ttransitioninexon8,
causesaG212VsubstitutioninTM5.Theeffectsoftheserecentlyidentifiedmutationson
Nramp2functionarenotcurrentlyknownandneedtobestudied.AlthoughV114isnota
highlyconservedamongNramp2orthologs,itsin-framedeletionmaystillaffectNramp2
function.Ontheotherhand,G212inTM5issignificantlyconservedanditislikelythat
thenon-conservativeG212VsubstitutionaffectsNramp2function.
Together,thesethreereportedcasesdefineanewsyndrome
ofcongenital
microcytichypochromicanemiawithliverironoverload.
Inthefirsttworeports,a
quantitativereductioninNramp2expressionwasthecause
ofdisease,indicatingthe
presence
ofaminimumthresholdofNramp2expressionrequiredfornonnal
physiologicalfunction.Thisminimumthresholdappearsto
bebetweenthelowlevelof
activityobservedinbothNRAMP2mutantpatientsandthe50%activityretainedintheir
phenotypicallynonnalheterozygoterelatives
4;263.ResultsfromstudieswithNramp2-/
micesuggestthatcompletelossofNramp2activitymaynotbecompatiblewithlife258.
38
1.3.9.4Otheriron-overloaddisordersinhumans
Hereditaryhemochromatosis(HH)isanautosomalrecessiveinheritediron
overloaddisordercharacterized
byincreasedintestinalironabsorptionandiron
accumulationinvitalorgans,eventuallyleadingtoorganfailure(reviewedin
266).When
identifiedbeforesevereorgandamagehasoccurred,hemochromatosiscanbetreated
by
phlebotomy(bleeding)toremoveiron-richredbloodcells.Mutationsinfourgeneshave
beenlinkedtoHH:HFE,TFR2,hemojuvelin,andhepcidin.
ThefirstgeneshowntobeassociatedwithHHwasHFE,anatypicalmajor
histocompatibilityclass1proteinthatheterodimerizeswith
(3-2microglobulinbutdoes
notbindasmallpeptide
267;268.Mostpatients(80%)withHFE-linkedhemochromatosis
arehomozygousforamissensemutation(C282Y)thatpartiallydisruptsHFEfunction
267.TheC282YmutationisparticularlycommoninindividualsofNorthemEuropean
descent.Othermutationsat
HFEarerareandtheircontributionstoclinicaldiseasehave
notbeenwellestablished.Onlyafraction
ofpatientswithahemochromatosisgenotype
developclinicalironoverloadsymptomsandenvironmentalfactorsplayasignificantrole
indiseasepenetrance.Despitevastefforts,theprecisemolecularfunction
oftheHFE
proteinhasremainedelusiveandexperimentsexpressingHFE
intransfectedcellshas
yieldedconflictingresults
268-271.However,itisclearthatHFEislinkedtothetransferrin
cycleandcanformahigh-affinitycomplexwith
TfR!,competingwithtransferrintobind
thereceptor
268;272;273.RecentstudiesinHfe-/-micehavelinkedHFEtohepcidinlevels
andsuggestedthetwoproteinsarepart
ofthesamesignalingpathway274-276.
AlthoughlesscommonthanHFEmutations,homozygousmutationsinTfR2have
alsobeenidentifiedinpatientswithhemochromatosis
188;277-282.Theclinicalsymptomsof
TFR2hemochromatosisaresimilarbutmoreseverethantheHFE-relateddisease283.A
definingcharacteristic
ofTFR2hemochromatosisisthepersistenceofhightransferrin
saturation,evenfollowingphlebotomy.
Sornepatientswithjuvenilehemochromatosisarehomozygousformutationsat
HAMP,thegeneencodinghepcidin284.Mutationsidentifiedthusfarcauseeithera
completeinactivation
oftheprotein285orsubstituteoneoftheinvariantcysteinesofthe
39
peptide286-288.However,mostjuvenilehemochromatosispatientspossessmutationsin
thegenehemojuvelin(HFE2orHJV)289.Hemojuvelinencodesa
glycosylphosphatidylinositol(GPI)-linkedprotein,characterized
byanRGDmotifanda
vonWillebrandtypeDdomainthatishighlyexpressedintheliver,skeletalmuscle,and
heart.Over30differentmutationshavebeenidentifiedinthe
HJVgeneinpatientswith
juvenilehemochromatosis(reviewedin
290).HJVhemochromatosissharesnumerous
featureswith
HFEhemochromatosis,butaUtheclinicalmanifestationsdevelopearlier
duetogreaterintestinalironabsorptionandfasterrate
ofironaccumulation.Thefactthat
patientswithcriticalmutationsin
HJVdisplayloworunmeasurablehepcidinlevels289
suggestthattheproteinisacomponentofthehepcidinregulatorypathway.
Adistincttype
ofironoverloadinheritedinanautosomaldominantfashionis
foundinpatientswithcriticalmutations
inferroportin291;292.Thesepatientspresent
variableclinicalphenotypesdependingonthespecifiemutationtheyharbor.
Ferroportin
mutationsessentiaUyfaUintotwomainclasses:(1)mutationsthatdisruptceUsurface
expressionandinhibitironexport
293;and(2)mutationsthatlocalizetotheceUsurface
butareunabletorespondtheregulatorypeptidehepcidin
294.Patientswiththefirsttype
ofmutationshowtypicalferroportindiseasewithlowtransferrinsaturationandiron
accumulationinmacrophages.Patientswiththesecondtype
ofmutationshowhigh
transferrinsaturationandearlyhepatocyteironloadingsimilartoclassichereditary
hemochromatosis.Thedominantinheritance
offerroportin-linkedhemochromatosismay
beexplained
byinvitroexperimentsthatsuggestferroportinfunctionsasamultimerand
thatmutantsactasdominantnegatives,affectingthebehavior
ofthewildtypeprotein295.
However,thisstillremainscontroversialsinceotherstudieshaveshownthatWTand
mutantferroporinsdonotformoligomersintransfectedHEK293ceUs
293;296.
40
PrefacetoChapter2
TheidentificationofNramp2asatransporterofdivalentmetalsin1997provided
newinsightintothefunction
ofNrampproteinsinvivo40;136,Electrophysiological
measurementsindirectlyidentifiedFe
2+,Mn2+,C02+,Cd2+,Cu2+,Ni2+,Pb2+,andZn2+as
substratesforNramp2andshowedthattransportoccurred
byapH-dependentmechanism
40,ThedemonstrationofdivalentmetaltransportbynumerousdistantNramporthologs
highlightedfunctionalconservationintheNrampsuper-family
1;2;297;298,Atthistime,
structure-functionstudiesbecameessentialtounderstandingthemolecularbasisforboth
metaltransportandpH-dependence
byNrampproteins,
Chapter2
ofthisthesisdescribesoneofthefirstsystematicattemptstoexplore
structure-functionrelationshipsinNrampproteins
bysite-specifiemutagenesis.Through
multiplesequencealignments
ofvariousprokaryoticandeukaryotieNrampsequences,
weidentifiedanumber
ofhighlyconservedyetthermodynamicallydisfavoredcharged
aminoacidsresidingwithinthehydrophobicTMdomainsofNramp2.Ourmutagenesis
studiesrevealedseveralchargedresiduesthatareessentialforNramp2-mediatedmetal
transport,aswellastwoinvarianthistidines(H267andH272)thatarecriticalforpH
dependence.Later,similarmutagenesisstudiesperformedinabaeterialNramportholog,
MntH,confirmedtheimportance
ofTMchargedresiduesinmetaltransportbyNramp
proteins
299,Furthermore,ouridentificationofH267andH272ascriticalforpH
dependeneewaslatersupportedbyelectrophysiologicalexperimentsinXenopusoocytes
300
41
Chapter2:
IrontransportbyNramp2/DMT1:pHregulation
oftransportbytwohistidinesintransmembrane
domain6
42
ABSTRACT
MutationsatNramplimpairphagocytefunctionandcausesusceptibilityto
infectionswhilemutationsat
Nramp2(DMTl)affectironhomeostasisandcausesevere
microcyticanemia.Structure-functionrelationshipsintheNrampsuperfamilywere
studied
bymutagenesis,followedbyfunctionalcharacterizationinyeastandin
mammaliancells.Thesestudiesidentifythreenegativelychargedandhighlyconserved
residuesintransmembranedomains(TM)1,4,and7asessentialforcationtransport
by
Nramp2/DMTl.Theintroductionofachargedresidue(G185R)inTM4foundinthe
naturally-occurringmicrocyticanemia
mk(mouse)andBe/grade(rat)mutantsisshownto
causeapartialorcompleteloss
offunctioninmammalianandyeastcells,respectively.A
pair
ofmutation-sensitiveandhighlyconservedhistidines(H267,H272)wasidentifiedin
TM6.Surprisingly,inactiveH267andH272mutantscould
berescuedbyloweringthe
pHofthetransportassay.ThisindicatesthatH2671H272arenotdirectlyinvolvedin
metalbindingbutrathertheyplayanimportantrolein
pHregulationofmetaltransportby
Nrampproteins.
43
INTRODUCTION
Nramp2(DMTl,DCTl)isessentialfornutritionaliron(Fe2+)uptakebythe
duodenumbrushborder
40;136;141andforirontransportacrosstheendosomalmembranein
peripheraltissues
42;141;301,Nramp2isanintegralmembraneproteincomposedof12
predictedtransmembrane(TM)domains
1.TheNramp2geneproduces2rnRNAsby
alternativesplicingoftheterminal3’exonthatshowdifferent3’untranslatedregions
containing(isoform
I,+IRE)ornot(isoformII,-IRE)anironresponseelement(IRE),as
wellasdistinctC-terminalproteinsequences
153;302,Innormaltissues,Nramp2protein
(isoform
1)isexpressedatthebrushborderoftheproximalportionoftheduodenum,
whereitisregulated
bydietaryirondeprivation161.Nramp2(isoformII)isalsopresentin
the
Tfreceptorpositiverecyc1ingendosomecompartmentoferythroidprecursorsthatcan
berecruitedinvivobytreatmentwithphenylhydrazineorerythropoietin169.Nramp2
mRNAexpressionwasalsodetectedinthekidney(IsoformsIandII)175andNramp2
proteinexpression(isoform
1)wasdetectedatthebrushborderofthekidneyproximal
tubule
172,DirecttransportstudiesinXenopuslaevisoocytessuggestthatNramp2isa
pH-dependentdivalentmetaltransporterwithbroadsubstratespecificityinc1udingFe
2+,
Mn2+,C02+,Cd2+,Cu2+,Ni2+,Pb2+,andpossiblyZn2+andthatmayfunctionbyaH+co
transportmechanism
40.Parallelstudieswithmetal-sensitivedyeshaveshownsimilar
transportpropertiesforNramp2expressedattheplasmamembrane
26;168,An
independentlyarisingnaturalmutationinTM4ofNramp2(G185R)isresponsiblefor
microcyticanemia
ofthemkmouse136andtheBelgraderat141,bothassociatedwitha
severedefectinintestinalironabsorptionandimpaireduse
byerythroidcells138,
TransportexperimentsinvitrointransfectedHEK293cells,asweIlassubcellular
localizationstudiesinvivointargettissuesfrommk/mkmicehaveshownthatthe
Nramp2G185Rmutationcausesaseverelossoffunctioncharacterizedbyreducedactivity
andpossiblyimpairedmaturation/targeting
146;147.Together,thesestudiessuggestadual
roleforNramp2astheTf-independentironacquisitionsystem
oftheduodenum,andas
transportingreducedTf-ironacrosstheendosomalmembrane
251303.
TheNramp2homolog,Nrampl1;7isexpressedinthelysosomalcompartmentof
macrophagesandneutrophils18andisrecruitedtothemembraneofpathogencontaining
44
phagosomesfonnedintheseceUs35;304,whereitmayfunctionasaMn2+effluxpump52.
Anaturally-occurringmutationinpredictedTM4ofNrampl(G169D)impairsmaturation
andmembranetargeting
oftheprotein,andcausessusceptibilitytoinfectionwith
unrelatedintracellularpathogens
7;15.NramplandNramp2definealargesuper-familyof
membranetransportershighlyconservedfrombacteriatohumans1;41;305.Demonstration
ofdivalentcationtransportbydistantNramphomologsfrombacteria(MntH,Mramp),
yeast(Sm!1,Smj2,Smf3),fly(M/v),andplant(AtNramp)hashighlightedfunctional
conservationinthisfamily
1;2;297;298,Inaddition,expressionofmouseNramp2proteinin
adoublesmfl/smj2mutantcanrestoretheability
ofthismutanttogrowatalkalinepH
andonmediumcontainingmetalchelators
34;306,Likewise,expressionofhuman
NRAMP1intheflymutantma/voUacorrectsthetastediscriminationdefectofthismutant
2,inamannersimilartothatproducedbyincreasingdietaryFe2+orMn2+307.
Inthepresentstudy,wehaveusedmultiplesequencealignmentstoidentifyhighly
conservedresiduesintheNrampsuper-family.Wehavestudiedthefunctionalrole
of
conservedchargedTMdomainresiduesinsubstrateselectivityandpHregulationof
Nramp2.
45
MATERIALSANDMETHODS
Materials
Calceinacetoxymethylester(calcein-AM;500/lMstocksolutionpreparedin
DMSO)wasobtainedfromMolecularProbes(Eugene,OR).StockFe
2+(2mM)aqueous
solutions
offerrousammoniumsulfate(FAS;Sigma)werealwayspreparedfreshin
degassed,de-ionizedwater.CoClz(Sigma)waspreparedasastocksolution(2mM)in
water.Themembrane-permeableironchelatorsalicylaldehydeisocotinoylhydrazone
(Sm,25mMstocksolution)waspreparedinDMSO,andthemembrane-impermeable
ironchelatorHES-DFO(6desferroxamine/M
r50,000starchmolecule;38mMstock)was
preparedinwaterandstoredat-20°C.
smandHES-DFOweregenerousgiftsfromDr.
P.Ponka(McGillUniversity,LadyDavisInstitute,Montreal,Canada).
PlasmidsandConstructs
AfulliengthmouseNramp2(DMTl)cDNAIsoformIIlackingtheIRE(GenBank™
AccessionNumberL33415)wasmodified
bythein-frameadditionof2antigeniccMyc
epitopetagsatthecarboxyterminus
oftheprotein(N2-2Myc)34.Mutationsatspecific
aminoacidpositionswerecreated
bysite-directedmutagenesisusingarecombinantPCR
protocol308,andusingoligonucleotideprimerslistedinTable1.Themutantswerecloned
intothemammalianexpressionvectorpCB6.Forcomplementationstudies
inyeast,
mutantswereintroduced
intheyeastexpressionvectorpVT.
YeastTransformationandsmfl/smj2ComplementationAssays
TheS.cerevisiaesmf1/smf2doublemutant,isamutantinwhichtheSMF1and
SMF2geneshavebeeninsertionallyinactivated(MATaura3-521eu2-3-112gal2
SMFl::LEU2,SMF2::LEU2)
34.Thismutantcannotgrowonalkalinemediumoron
mediumcontainingmetalchelators
34;306.YeastcellsweretransformedwithpVT/Nramp2
constructs309andura+transformantsweregrownasmasspopulations,andcrude
membranefractions
310werepreparedforNramp2proteinexpressionbyimmunoblotting
usingthemouseanti-cMycmonoclonalantibody9E10(Babco,Berkeley,CA)
161.Totest
46
Table1.Primersusedformutagenesis.
PrimerNucleotidesequence(5’to3′)PositioninNramp2
pCB6Faatgggcggtaggcgtgta848-866ofpCB6
pCB6Raggccaggagaggcactgg1006-1034ofpCB6
N2
D86AFcctacctagCcccaggaaac248-267
N2R119AF
ctgctgctccagGCccttgcag346-367
N2
R146AFcaaggtcccaGCgatcatcctgtg426-449
N2
E154AFctgatggtg9Qgttggcaatca451-472
N2D161AF
cattggttctgCcatgcaggaagtc471-495
N2G184DF
ccctgtgggAcggagtcctca542-563
N2G185RF
gggtccccctgtggggcaGagtcctcatcaccatc536-570
N2
D192AFcaccatcgcagCcacttttgtg564-585
N2
E225AFgtttggatat9Qgtacattac663-684
N2H267AF
atcatgccgGCcaacatgtacct790-825
N2H267CF
atcatgccgTGcaacatgtacct790-825
N2H267RF
atcatgccgcGGaacatgtacct790-825
N2
H272AFaacatgtacctgGCttctgcctta802-825
N2H272CF
aacatgtacctgTGttctgcctta802-825
N2H272RF
atgtacctgcGttctgcctta805-825
N2H267/272AF
atcatgccgGCcaacatgtacctgGCttctgcgtta790-825
N2H267/272CF
atcatgccgTGcaacatgtacctgTGttctgcgtta790-825
N2H267/272RF
ctgtgatcatgccgcGGaacatgtacctgcGttctgc785-828
cttagtc
N2E299AFacttcttcatc9Qgtcctgcatcg883-907
N2
R416AFgtgatcctgaccGCgtctatcgc1234-1256
Findicatesforward;
R,reverse.Codonsofmutatedaminoacidsareunderlined.Uppercaseletiersin
nucleotidesequencesindicatebasesthatdifferfromtheNramp2sequence.
47
forpossiblecomplementationofthegrowthdefectsofthesmJllsmJ2mutantbyNramp2
variants,duplicatealiquots(1mL)ofura+transfonnantswereresuspendedineither
YPDmediumorinalkalineYPDmedium(pH7.9,
OD595=0.02)in96-wellplates(100
~Llwell).Growthwasmeasuredafter24hincubationat30°CusinganELISAmicroplate
reader(Bio-Radmodel450).SensitivitytoalkalinepHwasmeasuredasrelativegrowth
ofeachtransfonnant(expressedasa%)inalkalineYPD7.9comparedtogrowthofthe
sametransfonnantinnonnalYPDmedium(pH5.5-6.0).Forcomplementationstudieson
alkalineagar,cellsgrowntosaturationinnonnalYPDweredilutedtoOD
S95=0.2,0.02,
0.002,0.0002,and0.00002inYPD7.9,and20
~Lofeachdilutionwasspottedon
alkalineYPDagarplatesandgrownfor36to48hoursat30°Cbeforephotography.
CellCultureandTransfection
LR-73ChineseHamsterOvary(CHO)cellswereroutinelygrownina-minimum
essentialmedium(MEM)supplementedwith10%fetalbovineserum,50units/mL
penicillin,and50
~glmLstreptomycin(Invitrogen).AlIpCB6(neo)plasmidconstructs
weretransfectedintocellsascalciumphosphateco-precipitates,accordingtoaprocedure
wehavepreviouslydescribed
311.Clonesofstabletransfectantswereselectedinmedium
containinggeneticin(G418;770
~glmL;Invitrogen)andwerepickedafter8-13daysof
selection.
CrudeMembranePreparationfromCHOTransfectants
Cellpelletswereresuspendedin250
~LofTNEBuffer(100mMNaCI;10mM
Tris-Cl,pH7.0;10mMEDTA)containingproteaseinhibitors(1mMPMSF,1
~M
pepstatin,0.3~Maprotinin,1~Mleupeptin).Cellswerehomogenizedby20passages
througha
25gaugex5/8inneedle,followedbycentrifugation(4°C,2000xg,10
minutes)toeliminatenuc1eiandunbrokencells.Membraneswerethenpelletedfromthe
supematant
byultracentrifugation(75000rpm,TLA-I00rotor(Beckman),4°C),and
wereresuspendedinTNEcontaining30%glycerolandproteaseinhibitors.Recombinant
Nramp2proteinvariantsweredetectedusingthemousemonoclonalanti-c-Mycantibody
9ElO(1:1000,Babco)aspreviouslydescribed
26.
48
CalceinLoadingoftheCellsandDivalentMetalTransportAssay
CHONramp2transfectantswereloadedwiththemetalsensitivefluorescentdye
calcein-AM,aswehavepreviouslydescribed
26.Briefly,CHOtransfectants(lx106cells)
wereincubatedwith0.250
J.LMcalcein-AMfor10minutesat37°Cin1mLofloading
medium(a-minimumessentialmedium,1mg/mLBSA,20mMHEPES,pH7.4).The
cellswerewashedtwice
inandresuspendedin500J.LLoftransportbuffer(150mMNaCI,
20
mMMES,pH5.0-6.5).Thecellsuspensionwastransferredtoastirredthermostated
(37°C)semi-microcuvette,andfluorescencewasrecordedusinganLS-50Bfluorescence
spectrometer(PerkinElmer,Inc.;excitation,488nm;emission,517nm;excitationand
emissionbandpass,5nm;responsetime,6
s;datainterval,0.5s).Divalentmetals(20J.LM
finalconcentrationofFe2+orC02+)wereaddedtothecellsuspensionafterallowing
fluorescencetostabilizefor60
s.Forassaysusingiron,acombinationofmembrane
permeable
(Slli)andmembrane-impermeable(HES-DFO)ironchelatorswereusedat
varioustimepoints(Fig.SA)todistinguishintracellularfromcell-associatedquenchable
fluorescence:Afterfluorescencewasallowedtostabilizefor60s,20
J.LMFe2+wasadded
tothecellsuspension(Fig.SA,
arrow1).After240s,themembrane-impermeableiron
chelatorHES-DFOwasadded(Fig.SA,
arrow2)toreleasemetal-inducedquenchingof
extracellularcell-associatedcomplexedcalcein.At300s,amembrane-permeableiron
chelator
Slliwasadded(Fig.SA,arrow3),toreleasemetal-inducedquenchingof
intracellularcalceinfluorescence.Initialrateswerecalculatedfromquenchingcurves,
andthesize
oftheintracellularlabileironpoolwasextractedfromdataobtainedwiththe
2metalchelators.
CellSurfaceProteinBiotinylation
CHONramp2transfectantswerewashedthoroughlywithcoldPBS
(supplementedwith1
mMMgCh,0.1mMCaCh)andthenwithcoIdboratebuffer(10
mMboricacid,154mMNaCI,7.2mMKCI,1.8mMCaCh,pH9.0).Cellswerelabeled
for
15minonicewith0.5mg/mLSulfo-NHS-SS-Biotin(Pierce)incoIdboratebuffer.
AfterwashingthreetimeswithcoIdquenchingbuffer(PBS,200
mMglycine),cellswere
49
scraped,collectedandresuspendedin1mLlysisbuffer(1%TritonX-100,150mM
NaCI,2mMEDTA,10mMTris-ClpH7.4,30%glycerol)plusproteaseinhibitors.
Lysateswereincubated
onicefor20minandpelleted(10min,10OOOxg,4°C).
Supematantswerecollectedandproteinlevelswerequantified
byBradfordassay.500Ilg
oftotalproteinlysatewereincubatedovemightat4°Cwith50ilLofimmobilized
streptavidinbeads(Pierce)inafinalvolume
of500ilL(withlysisbufferandprotease
inhibitors).Streptavidinbeadswerewashedthreetimeswithlysisbufferthenoncewith
PBS.Labeledcellsurfaceproteinswereelutedwith50ilL1xSDS-PAGEloadingbuffer
andseparated
bySDS-PAGE.
50
RESULTS
MutagenesisStrategy
Alignmentof28eukaryoticandprokaryoticNrampsequencesidentifyacommon
andconservedhydrophobiecore
of10TMdomains(~30%identitybetweenbacteriaand
man)
1thatcontainfourabsolutelyinvariant(D86,E154,E299,R416)and5highly
conserved
(RI19,R146,D161,D192,E225)chargedresidues(6negative,3positive)in
TMdomains(Fig.1;darkblueresidues).AnotheruniquefeatureoftheNrampfamilyis
thepresence
oftwoinvariantHistidineresidues(H267,H272)inthepredictedTM
domain6(Fig.1;redresidues).Inmice,naturallyoccurringmutationsatadjacent
residuesinTM4
ofNramplandNramp2causesusceptibilitytoinfectionandmicrocytic
anemia,respectively,andtheloss-of-functionphenotype
ofthemicrocyticanemia
Nramp2variant(GI85R)wasstudied.MutantcDNAswereexpressedinyeastS.
cerevisiae,andtestedfortheirabilitytocomplement2phenotypesofasmfllsmj2mutant:
(a)impairedgrowthonmetalchelators
306and(b)impairedgrowthatalkalinepH.34
Mutantsshowingpartialorcompleteloss-of-functionweresubsequentlyexpressedin
CHûceIls,andtestedforFe2+andCo2+transportattheplasmamembrane.26
ConservedChargedResiduesinMembraneDomains
ImmunoblottingindicatedthataIlmutantscouldbestablyexpressedas60-65kDa
immunoreactiveproteins
inyeastmembranefractions(Fig.2A,leftpanel).Theseresults
suggestthatnone
ofthemutationshadamajoreffectonproteinexpressionorstabilityin
yeast.Theability
ofeachmutanttocomplementthenullsmfl/smj2yeastmutantwas
testedinparaIlel
byplatingseriaIdilutionsofeachtransformantonYPDagarpH7.9
(Fig.2B,
leftpanel),andusingagrowthinhibitionassayinliquidYPD(pH7.9).
Routinely,yeast
WTNramp2transformantsshoweda9-foldstimulationforgrowthon
alkalinemediumovernegativecontrols(pVT,
smf-/-;Fig.3,leftpanel).TwoNramp2
mutantspreviouslyshowntoeitherabrogate(G394V)ortohavenoeffect(Q395E)on
Nramp2function
inyeast34wereusedasadditionalcontrols(Fig.3,leftpanel).
Inbothassays,mutantsR119A,R146A,D161A,E225Ashowedfull
complementation
ofthesmfl/smj2growthdefect,whilemutantsD86A,E154A,D192A,
51
314-358
Isoforml(+IRE)
Figure1.SchematicrepresentationofmouseNramp2(DMT1)isoform”(-IRE)and
isoform
1(+IRE).Thetwelvetransmembranedomainsarepredictedfromhydropathyprofiling,
calculations
ofhydrophobiemomentandothercomputer-assistedanalyses6andfromdirect
epitopemappingstudies.13Individualpredictedintraeellularandextracel/ularsegmentsare
identifiedandtheirpositionwithintheprimarysequenceisshown.Aminoacidresiduesdefining
sequencelandmarksandsignaturemotifsaredepictedindifferentcolors,includingnegatively
andpositivelychargedresidueswithinpredictedTMdomains(darkblue),conservedhistidine
residuesinTM6(red),glycineresiduesinTM4alteredinanemicmkiBelgrademutants(G185R),
andmutated(inNramp1)
inmicesusceptibletoinfections(G185D)(yellow).Aisoidentifiedare
Asn-linkedglyçosylationsignaIsintheTM7-TM8extracytoplasmicloop(black),predicted
membranetargeting/sortingmotifs(tyrosine-based,anddi-Ieucine)(green),andconsensus
transportsignaturecommontoNramporthologsandpresentinthecytoplasmicface
ofmem
braneanchors
ofbacterialperiplasmicpermeases(orange).The2differentC-terminiofthe
proteingeneratedbyalternativemRNAsplicingcontaining
ornotaniron-responseelement
(isoforml,+IRE;isoformIl,-IRE)
inthe3’untranslatedregionareidentified,withcorresponding
numbering.Final/y,thepolarity
oftheproteinandmembranedomainswithrespecttothe
membrane(Iightblue)isindicated(in,out,lumen).
A<:u0::NNN
N"-"-"-0-<:<:<:00::<:
<:<:u0::<:u0::NNN<:.....................E<:0 ~.-~L.I"lN "-"-"-NNN"-"-"-
~ro ~L.I"l 00000 "-"-"- ....00NNNNNNNNN0-Z0J:
J:J:
J:J:J:
J:J:J:
.'.
B<:u0::NNN
N"-"-"-10-<:<:<:<:0::<:<:<:~LJ.J<:<:u0::<:u0::NNNj-.....................E<:0 ~.-L.I"lNL.I"l0L.I"l "-"-"-NNN"-"-"-
~....ro .-~L.I"l 000N0000.- "-"-"- E....00.-.-
.-.-.-.-NNMM~NNN
NN
NN
NN0-VIZ00::0::LJ.J0.:)0LJ.JLJ.J.:).:)J:J:
J:J:J:J:J:J:J:
Figure
2.Nramp2proteinexpressioninsmf1/smf2mutantyeastandfunctionalcomple
mentation
ofgrowthatalkalinepH.(A).Crudemembranefractionsfromsmf1/smf2yeastcells
(pVT)expressingeitherwildtype(Nramp2)
orindividualmutantvariantsofNramp2(indicatedon
top)wereseparated
bySDS-polyacrylamidegelelectrophoresis.Immunoblottingwasperformed
usinganaffinitypurifiedrabbitanti-mousepolyclonalanti-Nramp2antibody.Apparentelectropho
reticmobility
oftheimmunoreactivespeciesisconsistentwithamolecularmassof60-65kDa.
(8).Functionalcomplementation
ofthegrowthdefectofthesmf1/smf2mutantwastestedonsolid
YPDagaradjustedatalkalinepH(pH7.9).Seriaiten-folddilutions
ofculturescorrespondingto
individualNramp2mutants(identified)werespotted(fromtoptobottom)onYPD-agarplates(pH
7.9),followedbyincubationfor48hat30?Candphotography.
140%.------------------------------,------------------~
120%
.!:100%
~-80%(9
~60%+='~
&40%
Figure3.QuantitationofcomplementationoftheSmf1/Smf2yeastmutantby
Nramp2variants.IndividualNramp2mutants(identified),togetherwithpositive
(Nramp2)andnegative(pVT)controlsweregrownto
s~turationanddilutedto00595=
0.02ineitherYPOorYPOpH7.9.CeUswereseededin96-wellplatesandgrowthw~s
measuredafter24hincubationat30CbymeasuringOD595.Resultsrepresentrelative
growth(expressedasapercentage)
ofindividualmutantgrownatpH7.9vs.normal
YPO.Errer'barsrepresentstandarddeviations
ofthemeans.
E299A,R416Afailedtodoso.Inaddition,testingtheseIlmutantsfortheirabilityto
complementsusceptibility
ofsmfl/smj2mutanttometalchelator(growthinEGTA
containingliquidmedium)showedrelativeactivitiessimilartothatseenforgrowthat
alkalinepH(datanotshown).Theseresultsidentify5
ofthe9chargedresiduesinTM
domainsasessentialforNramp2functionand
sm/1/smj2complementationinyeast.
TheD86A,E154A,D161A,D192A,E299A,R416Amutantswereanalyzedfor
metaltransportaftertransfectionin
CHûcells(Fig.4A,leftpanel).Transfectantswere
screenedbyimmunoblottingforexpression
ofthecorrespondingNramp2variantsatthe
cellmembrane.MembranesfromcellstransfectedwiththepCB6emptyvectorwereused
asnegativecontrols,whiletwopreviouslycharacterizedtransfectants
26expressingeither
low(N2GG)
orhigh(N2.3)levelsofNramp2proteinwereusedaspositivecontrols.
CHûcellclonesstablyexpressingmutantsEl54AandR416A,couldnotbeobtainedin4
independenttransfections(141clonesscreened)forE154Aand3independent
transfections(75clonesscreened)forR416A,suggestingpossibleeffect
ofthese
mutationsonproteinfoldinglprocessingand/ortoxicityforthecells.StableCHû
transfectantsexpressingD86A,D192AandE299Acouldbereadilyisolated.Inthese
clonesthelevel
ofexpressionvariedbutwasintherangeoflevelsseeninpositive
controlsexpressingWT(N2.3,N2GG)orD161Aproteins(Fig.4A,
leftpanel).The
variabilityinexpressioncanbeattributedtoacombination
ofthesiteofintegrationofthe
vectorandtheeffectoftheintroducedmutation.Inaddition,biotinlabelingexperiments
inintactcellsidentifiedcellsurfacereactivity
ofallexpressedmutants(D86A,D161A,
D192A,E299A),withlevelsapproximatelyproportionatetototalexpressionlevels
detectedinmembranefractions
byimmunoblotting(Fig.4B,leftpanel).Theseresults
indicatethatmutationsatD86,D161,D192,andE299donothaveamajoreffecton
Nramp2proteinmaturation
ormembranetargeting.
Transportproperties
ofthemutantswereinvestigatedinintactcellsusinga
calceinquenchingassay
26.MetaltransportbyNramp2usingthecalceinquenchingassay
andtransportofisotopic
55Fehas
beenshowntobecomparableintheidenticalcontrol
Nramp2
CHûtransfectants26aswellassorneNramp2mutantvariants(datanotshown).
55
A(9«0:::«
0
…. ……..
….0I..!:)0
Figure5.RelativeironandcobalttransportactivityofNramp2mutationsaffectingconserved
chargedresiduesinTMdomains.(A)ControlCHOcells(pCB6),asweilasCHOtransfectantsexpressing
wildtypeNramp2(N2.3)wereloadedwiththemetalsensitivefluorescentdyecalcein.Cellswerewashed,
resuspendedtransportbuffer(pH6.0),andfluorescencewasrecorded
withafluorescencespectrometer.
Whenfluorescencestabilized,divalentmetals(20
/lMfinalconcentrationofFe2+orCo2+)wereaddedto
thecellsuspension(arrow1),andfluorescencewascontinuouslymonitoredforanadditional3minutes.
Forirontransportstudies(rightpanel),
themembrane-impermeableironchelatorHES-DFO(200/lM)was
addedat4
min(arrow2)torevealcellassociatedextracellularFe-calceincomplexes.At5min,the
membrane-permeantironchelatorSIH(250/lM)wasadded(arrow3),andrevealedintracellularFe-calcein
complexes(seeMaterialsandMethods).Thetransportactivity
ofNramp2wasmeasuredasaninitialrate
(siope)duringtheearlyportionofthecalceinquenchingcurve,andisshownasashadedarea(siope
interval).
(B).Relativetransportactivitiesofwildtype(N2.3,N2GG)andmutantNramp2variants
(identified)werecalculatedfrom
theinitialslopeofcalceinquenchingcurvesasshownin(A),andare
expressed
asarelativetransportactivitywithtransportinthepositivecontrolN2.3setat100%.Errorbars
representstandarderrorson
themeansof3ormoreindependentexperiments.Asterisks(*)marktrans
portactivitiessignificantlydifferent(Le.morethan2standarddeviations)fromthosedetectedinthe
negativecontrol,vector-transfected(pCB6)cells.
clearlysuggestthattheG185Rmutationonlyattenuatesbutdoesnoteliminatetransport
function
ofNramp2proteininmammaliancells.
ConservedHistidinePairinTM6.
Histidinepairscanformmetalbindingsitesinsoluble312orinmembraneproteins
313;314.ThehighlyconservedHistidinepairfoundinTM6(H267,H272)oftheNramp
super-familywasinitiallystudiedinNramp2
bymutagenesistothesmallneutralresidue
AlanineinmutantsH267A,H272AandinthedoublemutantH267A/H272A.Metal
binding
byHisresiduesinvolvesdonatingimidazolenitrogenlone-pairelectronstothe
unfilledorbitaIs
ofthemetal,andCysteinescansubstituteforHisinthisprocess312;315-
317.Thus,mutantsH267C,H272CandH267CIH272Cwerecreated.Theimidazole
proton
ofHishasapKaof6.6andcanmediatepH-dependenteffectsinproteins.Thus,a
lastset
ofmutantswerecreatedinwhichHiswasreplacedbythepositivelychargedArg
(pKa~12)whichmayfunctionallymimmictheprotonatedHis,albeitwithamuchlarger
bulk.Mutantsweretransformedinyeastcells,andimmunoblottinganalysesshowthatall
9singleanddoublemutantscouldbestablyexpressedinthemembranefraction
ofthe
smfl/smf2mutant(Fig.2A,rightpanel).Complementationstudiesforgrowthatalkaline
pH(Fig.3)andinthepresenceofmetalchelators(datanotshown)indicatethatH267is
highlymutation-sensitivewithreplacementstoAla,CysandArgcausingsevereor
completeloss-of-function.H272waslessmutationsensitivethanH267,withH272Aand
H272CretainingnearWTactivity,andonlysubstitutiontothebulkierArg(H272R)
abrogatedcomplementation.Finally,thethreedoublemutants(H267A/H272A;
H267C1H272C;H267R1H272R)werecompletelyinactiveinyeast.
Immunoblottinganalysis(Fig.4A,
rightpanel)revealedthatallmutantscouldbe
stablyexpressedaftertransfectionin
CHûcells,withtheexceptionofthedouble
H2671H272Cmutant(3independenttransfections,119clonesscreened).Theother8
Nramp2mutantswereexpressedatvaryinglevels,butthatgenerallyfellbetweenthose
seeninthepositivecontrolsexpressinglow(N2GG)andhigh(N2.3)amounts
of
Nramp2.Biotinlabelingexperimentsinintactcellsidentifiedcellsurfacereactivityofall
expressedmutants,withlevelsapproximatelyproportionatetototalexpressionlevels
59
detectedinmembranefractionsbyimmunoblotting(Fig.4B,rightpanel).However,
sornereducedtrafficking
ofproteintotheplasmamembranemayhaveoccurredin
H267CandH272A.Metaltransportactivity
ofthemutantswastestedinthecalcein
quenchingassayforFe
2+andC02+atpH6.0.Mutantsbearingeithersinglemutationsto
Argateitherposition(H267R,H272R),
ordoublemutations(H267NH272A,
H267R1H272R)werecompletelyinactivefortransport(Fig.6).Likewise,mutantH272A
wasalsoinactive
intheseconditions,andmutantH267Conlyretainedlowtransport
activitybutforC0
2+only(Fig.6).InfactonlyH267AandH272Cretainedmodestbut
significanttransportactivitytowardsbothmetalcationsanalyzed
intheassay,with
H267Apossiblyhavingahigherselectivityforcobalt(seebelow).Theseresultsindicate
thatH267andH272playanimportantroleinNramp2metaltransport,bothinyeastand
mammaliancells.
Theapparentchangeissubstrateselectivity(cobaltoveriron)
intheH267A
mutantwasinvestigatedfurtherthroughradioisotopic
55Fetransportmeasurements.
Competitionexperimentswithcoldironandcobaltfailedtorevealasignificant
preferenceforcobaltinH267Acomparedtothewild-typeNramp2.Therefore,thechange
insubstrateselectivityinH267Aobservedinthecalceinquenchingassay,while
statisticallysignificant,couldnotbeconfirmed
byradioactivityandremainsunclear.
Possible
pHeffectsontransportactivityofthemutantswereinvestigated(Fig.7).
Fe
2+(Fig.7A,toprow)andC02+(datanotshown)transportbyNramp2inclonesN2.3
andN2GGapproachedmaximumat
pH~6.0,anddecreasingthepHto5.0hadlittle
effectontransport.Likewise,backgrounduptake
ofmetalinnegativecontrolswasnot
affected
byloweringpH.However,thelowleveloftransportactivitydetectedatpH6.0
inmutantsH267A,H267C,andH272C(Fig.6)couldbesignificantlyenhanced
by
progressivelyloweringpH(Fig.7A,7B).Thisincreaseoccurredgraduallyover
background,pH-insensitivelevelsdetectedincontrolpCB6cells.Surprisingly,mutants
272Aand267A/272Awhichshowedcompleteloss-of-functionat
pH6.0(Fig.6)could
berescued
byloweringthepH.Interestinglyhowever,loweringthepHofthe
extracellularmediumhadnoeffectonC02+(notshown)orFe2+transport(Fig.7B)ofany
ofthehistidinetoargininemutants(H267R,H272R,H267/272R).ThepHeffectseenin
60
theCys/AlamutantswasspecifiefortheconservedHispairofTM6andwasnot
observedinany
oftheotherinactivatingmutationsstudied(e.g.D86A,E299Aor
G185R)(datanotshown).Therefore,mutationsatthetwoconservedHisinTMD6
clearlyaffect
pHsensitivityofNramp2transport,andsuggestthattheseresiduesmaybe
involved
inpHregulationoftransport.
61
120
—:::R0……..**>-.100+-‘.:;:
”;::;80uro+-‘~0600.*VIc:ro40~+-‘CI)
.~20+-‘roCI)a:0 coU0.
(V)19N19ZNZ
‘*
«ua:«u……………………NN ……..
……..NNN
NN::r:
::r:::r:::r:::r:
a:N……..N::r:
•Cobalt
Oiron
«a:NN…………….NN………………………….. N
N::r:::r:
Figure
6.RelativeironandcobalttransportactivityofNramp2mutationsaffectingconserved
histidineresiduesinTM6.
TransportstudieswereconductedatpH6.5.Resultsarepresentedfor
different
mutantsatH267andH272,asdescribedinthelegendtoFigure5.
A
1.6)
.
1—.
B·1.6
Figure7.
Effect
of
pH
on
transport
activity
of
Nramp2
mutants
at
conserved
histi-
0.8
~
m~
~
dine
residues
in
TM6.(A)Iron
0.6
transportinthevariousnegative
0.4
(pCB6),andpositivecontrols
0.2
pCB6
WT(low)
0.2
(N2.3,N2GG)asweilasin
0
0
CHOclonesexpressing
0
1
2
3
4
5
60
1
2
3
4
5
60
1
2
3
4
56
0
1
2
3
4
5
6
Nramp2mutants
at
theplasma
1.6
1.6
1.41.4
membrane(H267A,H272A,
1.2
1.2
H267/272A,H267C,H272C,
1.0
1.0
H267R,H272R,H267/272R)
~
0.8
~
0.8
wasstudiedbythecalcein
c
0.6
c
quenchingassayasdescribed
Q)
Q)
0.6
inthelegendtoFigure5A,with
u
0.4
~
0.4
11’1
thefollowingmodifications.
~
02
H267A
H272AH267/272A
~
0.2
o
0
0
0
Afterloadingwithcalcein-AMat
::J
0
,
2
3
4
560
12
3
4
5
60
,
2
34
5
6
::J
0
1
2
3
4
5
6
pH
7.4,
ceUs
werewashedand
ü:
ü:
Q)
1.6
Q)
.1.6
resuspendedintransportbuffer
>
.~
1.4
at
differentpH(indicated).
”;::::;
pH
5.0
……
ra
ra
Fluorescencewasallowedto
v
v
stabilizefor1min,followedby
0:
pH
5.5
0:
0.8
addition
of
20
JlM
Fe2+.(B)
0.6
pH
6.0
Summary
of
transportresults
0.4
pH
6.5
0.4
obtainedforthehistidine
0.2
H267C
H272C’
0.2
mutants
at
pH5.0.
0
001
2
3
4
5601
2
34
56
0
1
2
3
4
5
6
1.6
l’L
1.4
-pCB6
1.4
-H267R
1.2
-H272R
1.0
-H267/272R
0.8
0.8
0.60.6
0.40.4
0.2
H267RH272RH267/272R
0.2
00
012
3
4
5
60
,
2
34
56012
34
5
6
0
1
2
3
4
56
Time
(min)
Time(min)
DISCUSSION
WeaimedtostudythefunctionalroleofhighlyconservedchargedresiduesintheTM
domains
ofNrampproteinsonsubstratetransportandpHregulation.Suchresidueswere
mutatedinthebackbone
ofNramp2andcorrespondingmutantsweretestedinyeastand
mammaliancens.Withfewexceptions,therewasgoodgeneralagreementbetween
sm/l/smj2complementationresultsinyeastandtransportdatainmammaliancens.
Interestingly,although
anmutantscouldbeexpressedinthemembranefractionsofyeast
cens(Fig.2A),severalcouldnotbeexpressedinCHOcellmembranes.Theseresults
suggestthatsuch
TMmutations1)affectednormalproteinfolding,maturationand
processing,possiblyleadingtoproteininstabilityand/ordegradationor2)that
overexpression
ofthecorrespondingmutantproteinwassomehowtoxicforthecells.
Theseresultshighlightpossibledifferencesinproteinsorting,maturationandtargeting
mechanisms
inbothcellsystems,butalsostresstheimportanceofmaturationand
membranetargetingforproperNramp2function.
Gly185isinvariantinallNramporthologsfrombacteriatoman.Strikingly,its
mutationtoArgaroseindependentlyin2rodentmodels
ofirondeficiency,themicrocytic
anemiamouse
mk136,andtheanemicBelgrade(h)rat141.TheG185Rmutationshoweda
complexphenotype.Inyeast,G185Rcouldbeexpressedinyeastmembranes,butcould
notcomplementthe
smfl/smj2mutant,suggestingalossoftransportactivity,in
agreementwithresultsfromSuetal
146,andWorthingtonetal318whoreporteda95%
and85%reductionin
55Fe2+transportactivityofG185RinHEK293censandCOS-7
cells,respectively.G185Rcouldalsobeexpressed
inthemembranefractionofCHO
cells(Fig.4),whereitretainedsignificanttransportactivityforbothC0
2+(50%ofWT)
andFe2+(35%ofWT).SimilartransportactivityofG185Rhasalsobeenobservedin
stabletransfectantsintheLLC-PKIporcinekidneycells(datanotshown).This
significanttransportactivitysuggeststhatloss
oftransportfunctionmaynotbethesole
defectresponsibleforthe
mkphenotypeinvivo.Rather,themutationmayaffect
membranetargetinginacell-specificfashion,perhapsinc1udingtargetingtoatransport
incompetentcompartment
inyeastcells.Recentstudiesinvivosupportsuchatissueor
cell-specificeffect.Indeed,immunoblottingstudiesshowrobustexpression
oftheG185R
64
isoforminduodenurnmembranefractionsofmk/mkmice.butimmunohistochemistry
studiesrevealedabsenceofproteintargetingtothebrushborder,thesiteofactive
transport
147.Likewise,mklmkmiceshowastrongreductionofNramp2(GI85R)protein
expressioninthekidney
172,whilemklmkreticulocytesarecompletelydevoidofNramp2
expression169.ParallelanalysisofthediseasesusceptibilityG169DmutationinTM4of
Nrampl(reconstructedinNramp2;datanotshown)associatedwithsusceptibilityto
infectionsalsoindicatedabsence
ofsmfl/smj2complementationinyeast.AIso,wecould
notisolate
CHûclonesstablyexpressingthismutant,asituationsimilartothatseenin
vivoinmacrophagesfromNramplG169Dmousestrains,wherenomatureproteinis
detected
13.Together,theseresultssuggestanimportantroleforthisresidueandTM
segmentfortransportactivity,butalsosuggestthatitsintegrityisrequiredforproper
maturation,foldingand/ortargeting
oftheNrampproteins.
Helicalwheelprojectionsintheconservedhydrophobiccore
ofNrampproteinsreveal
strongamphipathiccharacterforseveralTMdomains,inc1uding
TM3,5and91
.
Sequenceconservationexpressedasavariabilitymomentl,indicatesperiodicitywith
strongconservation
ofthepolarface,withtheapolar”lipid-accessible”faceofthehelix
beingheavilysubstituted.Severalhighlychargedresiduesmaptothepolarside
ofTM
helices.Suchanarrangementischaracteristic
offamilesofiontransportersandion
channels
1.Mutationsatthe9conservedchargesinTMdomainshadeithernoeffect
(Rl19,R146,D161,E225),orcausedpartial(D192)orcompleteloss-of-function(D86,
E154,E299,R416).D86,E154,E299,andR416arethemosthighlyconservedbeing
invariantinmultiplesequencealignmentsperformed(appended).
Itisstrikingthat3of
themhavenegativelychargedsidechains,raisingthepossibilitythattheymaymediate
interactionwiththepositivelychargeddivalentcationsubstrates
ofNramptransporters.
Alternatively,suchresiduesmaybeinvolvedinhydrogenbonding,saltbridgeformation
(dipole),orotherinteractions
intheformationofawater-filledporeortransportpath.
Theabsoluteconservation
oftheHistidinepairH2671H272inTM6ineukaryoticand
prokaryoticNrampsequencessuggestsanimportantrole.Inaddition,studies
byus(data
notshown)andothers
318showthatNramp2-mediatedtransportintransfectedcellsis
sensitivetotheaction
ofthehistidine-specificreagentdiethylpyrocarbonate(DEPC).
65
Here,weshowthatbothresiduesaremutation-sensitive(inparticularH267),with
independentsubstitutionsateitherorbothresiduescausingloss-of-function.Strikingly,
several
poodyactive(H267A,H267C,H272C)orcompletelyinactiveHismutants
(H272A,H272C,H267
AlH272A)atpH6.0couldberescuedbyloweringthepHofthe
transportassay(Fig.7).TheobservedpHeffectwasincremental,withmaximaltransport
attainedatorbelow
pH5.0.However,complete1yinactivehistidinetoargininemutants
(H267R,H272R,H267/272R)couldnotberescued
byloweringthepHofthetransport
assay.This
maybearesultofincreasedsterichindranceassociatedwithreplacementof
histidinetobulkyarginine.Thus,mutationsateitherHisresiduesinTM6shiftedthepH
requiredtoachievemaximaltransporttoamoreacidicvalue.
Severalexplanationscanbeputforwardtoaccountfortheuniqueeffect
ofpHon
transportproperties
oftheNramp2Hismutants.Firstly,H267andH272maybeinvolved
indirectbinding
ofthemetalsubstrateinapH-dependentfashion.MetalbindingbyHis
pairshasbeendocumentedinsolubleproteinssuchastranscriptionfactors
312,andhas
alsobeenused
inmembraneproteinswheretheyhavebeenengineeredtostudyproximity
relationshipsbetweenindividual
TMdomainsbyelectroparamagneticresonance314.
AlthoughH267andH272mayindeedformpartofabindingsiteformetalsinthe
membraneportion
ofNramp2,itappearsunlikely.Indeed,anNramp2mutantlacking
bothHistidines(H267
AlH272A)isstilltransportactiveatpH5.0(Fig.7B).Asecond
possibilityisthatH267,H272orbothparticipate
inH+movementacrossthemembrane
inaH+co-transportmechanismpossiblybyaprotonrelaysystem40.Sucharelaysystem
hasbeendescribedforthelactosepermease
ofE.coli,andinvolvesTMresidues
R3021H322/E325319.SuchaprotonrelaysystemmayexistinNramp2andmayinvolve
conservedresiduessuchas
H2671H272aswellasothernegativelyandpositivelycharged
residuesin
TMdomains.Partialorcompleteinactivationofthissystemwouldbe
predictedtohaveamajoreffectonthepHdependenceoftransport.Athirdexplanation
fortheobserved
pHeffectontransportpropertiesofsingleordoubleHismutants,isthat
H2671H272maybeimplicatedinpHregulationofthetransporterthroughgainorlossof
theimidazoleproton(pK5.5to6.5).Inthisfavoredmodel,protonationofH2671H272
wouldberequiredtomaintaintheprotein
inafunctional,transport-competent
66
conformation.Thiseffectcouldbeeithergeneralandinvolveadditionalresidues,
resultinginapH-dependentglobalconformationalchangefromaninactivestate(neutral
pH)toanactivestate(acidicpH).
Inthismodel,lossofthekeyHisresidueswouldshift
the
pHformaximaltransporttoamoreacidicvalue,requiringprotonationofother
groupsorsidechainstocreatethesameoverallconformationalchange.Altematively,
either
orbothH2671H272couldplayamorespecifieroleincreatingapH-dependent
transportpath
inthetransporter.Forexample,aninteractionwithadjacentandhighly
conservednegative1ychargedresiduesinother
TMdomainscouldbenecessarytoopen
aniontransportpath.LossofH2671H272wou
Idrequireformationofcompensatory
interactions
ofconservednegativelychargedresidueswithotherprotonatedsidechains
and/orwatermoleculesinthetransportpath.Althoughhighlyspeculative,sucha
mechanismappearstoaccountforthepH-dependence
ofaniontransportbytheBand3
transporter
320;321.
ACKNOWLEDGMENTS
TheauthorsareindebtedtoDrs.S.Grinstein(UniversityofToronto,Toronto)and
H.R.Kaback(University
ofCalifomia,LosAngeles)forhelpfuldiscussionsand
suggestionsduringthiswork,andDr.P.Ponka(McGillUniversity,Montreal)forthe
generouspreparationandgift
ofironchelators.
67
PrefacetoChapters3and4
Followingitsinitialfunctionalc10ningin1997,mountingevidencemadeit
increasinglyc1earthatNramp2playedadualroleinironhomeostasis
40;136.Wenow
knowthatNramp2isthetransporterresponsiblefornotonlytheintestinalabsorption
of
nutritionalnon-hemeironbutisalsotheproteinresponsibleforthetransferoftransferrin
ironfromendosomestothecytosol.Before2005,much
ofwhatwasknownaboutthe
role
ofNramp2insystemicironhomeostasiscamefromstudiesinrodentmodelsofiron
deficiency.BothmkmiceandBelgraderatspossessidenticalmutationsinNramp2
(G185R)leadingtoseverehypochromicmicrocyticanemia136;141.Criticalmutationsin
human
NRAMP2werethoughttocauseasimilarphenotypeinpeople.
In2004,Priwitzerovaandcolleaguesreportedafirsthumanpatientsufferingfrom
aniron-relateddisorderlinkedtoamutationin
NRAMP2.Thepatient,ayoungfemaleof
Czechorigin,sufferedfromseverecongenitalhypochromicmicrocyticanemiaandiron
overload
4;259.Thephenotypewassimilartothatofthemk/Belgraderodentmodels
exceptfortheelevatedhepaticironstoresthatwerespecifictothehumanpatient.
Recently,asecondhumanpatientcompoundheterozygotefortwonovelmutationsin
NRAMP2wasidentified263.Thepatient,a5-year-oldmaleofItalianorigin,sufferedfrom
severehypochromicmicrocyticanemiaandhepaticironoverloadsimilartothefirst
patient.
Inbothhumancases,mutationsinNRAMP2resultedinaminoacidsubstitution
mutationsatconservedresiduesintheNramp2protein.Theeffects
ofthesemutationson
theexpression,function,andsubcellulartargeting
ofNramp2wereunc1earandneededto
bestudiedinordertoexplainthephenotypes
ofthepatientsatthemolecularlevel.The
followingtwochapters
ofthisthesis(Chapters3and4)describethefunctionalproperties
andpossiblecontributiontodisease
ofthetwohumanmutationsexpressedintransfected
LLC-PKIkidneycells.
68
Chapter3:
FunctionalcharacterizationoftheE399D
DMT1/NRAMP2/SLC11A2proteinproducedby
anexon12mutationinapatientwithmicrocytic
anemiaand
itonoverload
69
ABSTRACT
DMTl(Nramp2,Slclla2)mediatesironuptakeattheintestinalbrushborderand
acrossthemembrane
ofacidifiedendosomes.Asinglepatientwithseveremicrocytic
anemiaandironoverloadwasrecentlyreportedtocarryamutationinexon12
ofDMTl
(1285G>C).Themutationhastwoeffects:itseverelyimpairssplicingcausingskipping
ofexon12,andintroducesanaminoacidpolymorphism(E399D)intheproteinencoded
bytheremainingproperlysplicedtranscriptfoundinthepatient.Thefunctional
propertiesandpossiblecontributiontodisease
oftheDMTlE399Dmutationare
unknownandhavebeenstudiedinindependentmutantsatthatposition(E399D,E399Q,
E399A)expressedinLLC-PK
1kidneycells.The3mutantsareshowntobefully
functionalwithrespecttostability,targetingandtraffickingtothemembrane,andare
transportcompetent.Thisindicatesthat
DMT1Gl285Cisnotacompletelossoffunctionbut
ratherthatamodestamount
ofactiveDMTlisproducedinthispatient.Thisactivitymay
explainthedistinguishingironoverloadseeninthispatientinaddition
tomicrocytic
anemia,thatisabsentinparallelrodentmodels
ofDMTldeficiency.
70
INTRODUCTIONStudiesinmousemodels
ofdisordersinironmetabolismhaveidentifiedsorneof
thekeytransporters(DMTlINRAMP2/SLCllA2andferroportin),enzymes(hephaestin),
andregulatorypeptides(hepcidin)thatplayacriticalroleinironhomeostasis251.
DMTlINRAMP2/SLCllA2(hereinreferredtoasDMT1)isexpressedatthebrushborder
oftheduodenumwhereitmediatesuptakeofdietaryiron161,butisalsoexpressedatthe
plasmamembraneandinrecyclingendosomes
ofperipheraltissuesandcells,including
erythroidprecursors,whereitisrequiredfortransport
oftransferrin-associatedironinto
thecytoplasm
169.DMTltransportsseveraldivalentcations(Fe2+,Mn2+,C0
2+,Zn2+)in
a
pH-dependentfashion,anddownaprotongradient
40.Amutation(GI85R)inDMTl
causesmicrocyticanemiaandirondeficiencyinthemkmouseandintheBelgraderat
136;141.ThisG185Rmutationmapsintransmembranedomain4(TM-4)ofDMTland
causesmultiplebiosyntheticandfunctionaldefects,affectingstability,maturation,
targetingandtransport
148.Recently,Priwitzerovaetalidentifiedafirsthumanpatient
homozygousforamutationin
DMTl259.Thepatient,aproductofconsanguineousunion,
presentedseverecongenitalhypochromicmicrocyticanemia,highserumironandserum
transferrinreceptor(TfR)levels,andrequiredseveralbloodtransfusionsafterbirthand
duringinfancy.Shealsoshowserythroidhyperplasiawithdefectivehemoglobinization,
anddevelopedliverhemosiderosis
bytheageof19.Erythroidprogenitorsisolatedfrom
thepatientweresmall,poorlyhemoglobinized,andgrewlessefficientlyinthepresence
oferythropoietincomparedtohealthycontrols.Erythroidgrowth,morphologyand
hemoglobinizationwererescuedinthispatient
bytreatmentwithFe-SIR,anirondelivery
strategythatby-passesthe
DMTl/TfRironpathway259.Intriguingly,anddespiteobvious
similarityinphenotypes,theliverironoverloadnotedinthispatientisnotseeninDMT1-
deficient(G185R)
mkmiceorinBelgraderats.
Thepatienthasintact
FerroportinandTransferrinReceptor(Tfri)genesbutis
homozygousforaGtoCsubstitutionatthelastnucleotide
ofexon12ofDMTl
(DMT1G1285C)4.DMT1G1285CdisruptsnormalsplicingofDMTlpre-mRNAleadingto
skipping
ofexon12,andconcomitantproductionofatruncatedDMTlproteinwhich
lackspredictedTM-8,andisunlikelytobefunctional.Thistruncated
DMTlvariant
71
(~TM-8)appearstorepresentaloss-offunctionandisunlikelytoactasadominant
negativesincethedefectinironmetabolismcaused
byDMT1Gl285Cisinheritedinafully
recessivemanner
4.Althoughskippingofexon12normallyoccursatlowlevels(~10%of
mRNA)incertaintissuesofhealthyindividuals,theDMT1Gl285Cmutationgreatiy
exaggeratesthisprocess
(~90%rnRNA)4.Conversely,asmallamountoffulliength,
properlyspliced,andexon12-containing
DMTlRNAisexpressedinthepatient.Inthis
fulliengthtranscript,however,the
DMT1Gl285CmutationcausesaGlutoAspsubstitution
atposition399(E399D)inthe4
thpredictedintracellularloopofDMTl.
TheconsequenceoftheE399Dmutationonproteinfunction,inc1udingapossible
contributiontotheironmetabolismdisorderseeninthispatient(aloneorincombination
withexon12skipping)remainunknown.Here,wehavecreatedindependentmutationsat
E399andanalyzedtheeffectonproteinstability,targetingandfunctionintransfected
LLC-PKIcells.
72
MATERIALSANDMETHODS
ConstructionandExpressionofDMTlMutants
AmouseDMTl(Nramp2,Slclla2)isoformII(non-IRE)cDNAmodifiedby
insertionofahemagglutininepitope(HA)taginpredictedTM7-TM8extracytoplasmic
loopwasusedformutagenesis
26,usingarecombinantpolymerasechainreaction
strategl8andprimers5′-gtttgtcatggacggattcctga-3′(E399D),5′-tcaggaatccctgcatgacaaac-
3′(E399Q),and5′-gtttgtcatggcgggattcctga-3′(E399A).Mutantswereintroducedin
plasmidvectorpCB6as
SacI/EcoR1fragmentsfoUowedbytransfectionandexpression
inporcineLLC-PKIkidney
ceUsaspreviouslydescribed?8DMT1transportofFe2+and
Co
2+wasmeasuredusingacalceinfluorescencequenchingassay(pH5.0,20~Mfinal
metalconcentration)
26-28.Initialratesofmetaltransportwerecalculatedfromthe
fluorescencequenchingcurves.
Immunojluorescence
LLC-PKIceUsgrownoncoverslipsweretransfectedwithGFP-Syntaxin1351,
fixedwith4%paraformaldehyde,permeabilized(5%non-fatmilk,0.2%saponinin
PBS),andincubated(16hrs,4°C)withanti-Nramp2NTpolyc1onalantibod
y
42(1:300).
Coverslipswerethenwashed,incubatedwithgoatanti-rabbit-Cy3(1:2000)for1h,and
mounted
onglassslides.
CellsurfaceQuantification
CeUsweregrowntoconfluencyin48-weUplatesandfixedin4%paraformaldehyde
(30′).Cellswerewashed(PBScontaining1
mMMgClz,0.1mMCaClz),blocked(5%nonfat
milkinPBS),andincubatedfirstwithanti-HAAb(1:500,90min)andthenwithdonkeyanti
mouse-HRPsecondaryantibody(1:4000,1h).Fortotal
DMTI-HAexpression,cellswere
permeabilized(5%nonfatmilk,0.2%saponininPBS)priortoincubationwithanti-HAantibody.
PeroxidaseactivitywasmeasuredwithanHRPsubstrate(0.4mg/mLo-phenylenediamine
dihydrochloride),accordingtoconditionsfromthecommercialsupplier(Sigma).Background
absorbancereadingsfromnon-specifiebinding
ofsecondaryantibody,andnon-specifiebinding
ofprimaryantibodytountransfectedcellsweresubtractedforeachsample.Cellsurfacereadings
werenormalizedtototal
DMTI-HAvaluesforeachcloneexpressedasapercentage.
73
RESULTSThe
DMTIG1285CmutationcausesaGlutoAspsubstitutionatposition399
(E399D)inthe4
thpredictedintracellularloopofDMTI(FigurelA).E399formspartof
ahighlyconservedtransportsignaturethatdefinestheNrampfamily.E399isinvariantin
mouse,zebrafish
(cdy),andyeast(Sm/Ip)relativesofhumanDMTI(FigurelB),
suggestinganimportantstructuralorfunctionalrole
1.AmouseDMTI-HAisoformII
(non-IRE)cDNAtemplatewasusedtocreatemutantsE399D(from
DMTIG1285C
mutation),E399Q(lossofcharge,sidechainsizeretained),andE399A(lossofcharge,
smallersidechain).Wildtype(WT)andmutant
DMTIvariantsweretransfectedinLLC
PKIporcinekidneycells,andcellclonesstablyexpressingindividualproteinswere
selectedforanalysis.LLC-PKIwaschosensinceisderivedfromproximaltubule,asite
ofabundantDMTIproteinexpressioninvivo,andthematuration,targetingandtransport
function
ofDMTIhavebeenextensivelycharacterizedinthesecells51;148.Immunoblot
analysis
ofwholecellextractspreparedfromeitherWTormutant-expressingcells
(Figure2A)usinganantibodydirectedagainstanepitopetagintroducedinallconstructs
identifiedrobustexpression
ofE399DandE399QsimilartoWT,whileE399Awas
expressedatalowerlevel.WTandmutant
DMTIvariantsweredetectedas2
immunoreactivespecies
ofsizes~60and~90kDa.Previousstudieshaveshownthatthe
90kDaspeciescorrespondstothefullyprocessed,”complexglycosylated”form
of
DMT1,whilethe60kDaspeciescorrespondstothefull-Iengthunprocessedor”core
glycosylated”protein
51.Studieswiththetranslationinhibitorcyloheximidefailedto
detectamajoreffect
ofanyofthe3mutationsonproteinstabilityinLLC-PKIcells(data
notshown).TheseresultssuggestthatmutationsatE399(includingE399D)donotaffect
expression,stability
ormaturationofDMT1.
PreviousstudiesfromourlaboratoryhaveshownthatDMTlmutationscanaffect
stability,maturation,targeting,andtransportactivity
28;148.Thus,weinvestigateda
possibleeffect
ofE399mutationsontheseparameters.ThetransportactivityofWTand
mutant
DMTlvariantsforFe2+andC02+wastestedinintactLLC-PKItransfectants,
usingafluorescencequenchingassay
26;28.Transport,measuredasinitialratesfrom
calceinquenchingcurves,wasverysimilarforthe3mutantsandwithintherange
ofthat
74
A
BMouseNramp2
RumanNramp2
Zebrafish
RumanNrampl
MouseNramplWA”‘.1.JJ-ln.n
Drosophila(Mvl)
Yeast(Smflp)
Salmonella(MntR)
CYTOPLASM
KWSRFAR
KWSRFAR
RWSRFAR
RWSRFAR
RWSRFAR
QWPRWCR
KLQPWQR
FHIPLWVR
Figure1.PositionandconservationofE399residueinDMT1.(A)Asche
maticrepresentation
ofDMT1-HAshowingthepositionoftheE399Dmutation
inthefourthpredictedintracellularloopoftheprotein.(8)Multiplesequence
alignments
ofDMT1orthologsforresiduesformingthefourthpredictedintra
cellularloop.Theposition
ofE399ishighlighted(black)alongwithresidues
definingthehighlyconservedconsensustransportmotif(grey).
measuredinindependentcellclonesexpressingWTDMTl(Figure2B).Themutations
didnothaveamajoreffect
ofeitherpreferenceforFe2+andC02+ions(Figure2B),oron
thepH-dependence
oftransport(datanotshown)28.Theseresultsdemonstratethat
residueE399isnotcriticalfortransportactivityofDMT1.Thesub-cellularlocalization
ofWTandE399D,E399QandE399AvariantsexpressedinLLC-PKIcellswasnext
analyzed
bydoubleimmunofluorescencelabelingusingananti-
DMTlantibody,andadditionalmarkersofendomembranecompartments42.Having
previouslyshownthatDMTI-HAisoform
ilisexpressedinsyntaxin13-positive
recyclingendosomes
51,weexaminedtheeffectofthemutationsontargetingtothis
compartment.AIlthreemutationsatE399showedstrongco-IocalizationwithGFP
syntaxin
13(Figure3A;introducedbyco-transfection),andlittleifanyoverlapwiththe
lysosomalmarkerGFP-LamplortheER-markerGFP-Sec
61(notshown).These
findingssuggestthatE399isnotcriticalforpropertargeting
ofDMTltorecycling
endosomes.Finally,weusedanELISA-basedsurfacelabelingmethodtoquantifytheamount
ofWTormutantDMTltargetedtothesurfaceoftransfectedLLC-PK
Icells.
Two
independentclones
ofWTDMTI-HAanalyzedinthismannerwerefoundtoexpress
22.9±1.5%and33.1±4.4%
oftotalDMTI-HAatthecellsurface(Figure3B).Mutants
E399D(26.7±2.5%)andE399A(28.2±6.9%)showedsimilarcellsurfaceexpression
levelscomparedtotheWTprotein(Figure3B).MutantE399Q(49.2±7.0%)showed
slightlyhighercellsurfaceexpressioncomparedtoWT.Theseresultsclearlyshowthat
theE399Dmutationdoesnotaffectcellsurfacetargeting
ofDMT1.
76
A
B
ID+-‘coa:::
..c.0cID:::J
a
107
81
48.7
0.010
0.0080.006
0.004
0.0020
00«0>0>0>0>0>0>
MMMUJUJUJ
~..
“1””””1″””N~
~~a..1()…J
…J
Figure2.StableexpressionandmetaltransportactivityofE399mutants.
(A)EquivalentamountsoftotalcellextractsfromLLC-PK1cellsstablyexpressing
eitherWTDMT-HA
ormutantconstructs(identified)wereseparatedbySDS
PAGEfollowedbyimmunoblottingwithananti-HAmonoclonalantibody.Size
of
molecularweightmarkers(inkDa)areidentifiedtotheleftoftheblot.(8)Cells
loadedwiththemetal-sensitivefluorescentdyecalcein,wereincubatedwithFe2+
orC02+inacidicbuffer(pH5.0),andtherateoffluorescencequenchingwas
measuredovertime.Theresultsareexpressedastheinitialrate
offluorescence
quenching.Errorbarsrepresentstandarderror
ofthemeansofthreeormore
independentexperiments.
A
B
~~..,”,’-.
1-
,’.:~:/:~;;;~,:_
,(,
80%
t:0(n
60%
(f)~a.xWQ)
40%
(.)-ê::lCf)Q)
20%
ü
0%
..-
C’I
~~
Figure
3.
Subcellular
and
ceU
surface
expression
of
E399
mutants
at
steady-state.
(A)LLC-PK1cellsstablyexpressing
WT
or
mutantDMT1-HAweretransientlytransfectedwithGFP-syntaxin13tolabelrecyclingendosomes.Sevenhourslater,
ceUs
were
fixed,permeabilizedandstainedwithapolyclonalanti-DMT1antibody.DMT1moleculeswerevisualizedusingasecondaryanti
rabbitantibodycoupledtofluorescentCy3.Imageswereacquiredbyepifluorescencemicroscopy.Insetsshowmagnifications
of
the
areaboxedinthefigure.
(8)
FixedLLC-PK1cellswereincubatedwithanti-HAprimaryantibodywith
or
withoutpriordetergent
permeabilizationasdescribedinMaterialsandMethods.CellswerethenincubatedwithanHRP-coupledsecondaryanti-mouse
antibody,andtheamount
of
boundprimaryantibodypresentwasdeterminedforbothconditionsbyacolorimetriereactionusing
o-phenylenediaminedihydrochloride(OPD)followedbyspectrornetry.Theamount
of
DMT1-HAexpressedatthecellsurface(in
non-permeabilizedcells)isshownasafraction(%)
of
totalproteinexpression(inpermeabilizedcells).
DISCUSSIONTakentogether,ourresultsstronglysuggestthattheE399Dvariantcreatedbythe
DMT1Gl285Cmutationisfunctional.ThisindicatesthatDMT1G1285Cmutationisnota
complete,butratherconstitutesapartialloss-of-function,andthereforethepatientindeed
expressesasmallamount
offunctionalE399D.Thisproteinisproducedinadditiontothe
loss-of-function
~TM-8variantgeneratedbyexon12skipping.Thissmallieveiof
activityisclearlyinsufficienttomaintainminimumphysiologicalfunctioninthe
erythroidcompartment,andcausesmicrocyticanemia.Thisminimumthresholdactivity
ispresumedtobebetweenthe
~10%leveloffulliengthtranscript(E399D)seeninthe
DMT1Gl285Cpatient,andthe~50%levelsobservedinherheterozygotephenotypically
normalsibling
4.
Thesefindingsalsohelpexplaintheapparentphenotypicdifferencesbetween
rodents(microcyticanemia)andhumans(microcyticanemiaplusironoverload)with
mutations
atDMT1.ThesmallamountoffunctionalE399DproducedintheDMT1Gl285C
patientmaybesufficientforacquisitionofnutritionalironatthebrushborderandstorage
inliver(hemosiderosis),butnotsufficientenoughforefficientuseandrecyclinginthe
erythroidcompartment(microcyticanemia).ThisproposaIwouldsuggestthatthe
minimumDMT1activityrequiredforphysiological”sufficiency”intheerythroid
compartmentforhemoglobinproductionishigherthanforacquisition
ofnutritionaliron
atthebrushborderintheformofinorganicmetal(Fe
2+)orofheme-iron.Thissituation
maybeexacerbatedinrodentswherethelaboratorydie!isfree
ofhemeiron.
Finally,thecombinedfindingsthatthe
DMT1Gl285Cpatientistheonlyhuman
knowntocarryanalterationin
DMT1,butyetthatthismutationbehavesasapartialbut
notcompleteloss-of-function,indirectlysuggeststhattruenullmutationsat
DMTlmay
notbecompatiblewithlife.
ACKNOWLEDGEMENTSWewouldliketothankLaurenHamlin-DouglasandRachelBeckermanforvaluable
technicalassistance.
79
Chapter4:
AnovelR416CmutationinhumanDMTl
(SIc11a2)dispIayspIeiotropiceffectson
functionandcausesmicrocyticanemiaand
hepaticironoverload
80
ABSTRACTApatientsufferingfrommicrocyticanemiaandhepaticironoverloadwas
foundtobecompoundheterozygoteforpolymorphismsintheirontransporter
DMTl(Nramp2,SLCllA2),inc1udinga3basepairdeletion(DMTldeICIT)in
intron4thatpartiallyimpairssplicing,andanaminoacidsubstitution
(DMTlCl246T,R416C)ataconservedresidueintransmembranedomain9ofthe
protein.Thefunctionalpropertiesandpossiblecontributiontodisease
ofthe
DMTlR416Cmutationwerestudiedinindependentmutantsatthatposition
(R416C,R416A,R416K,R416E)expressedinLLC-PK
1kidneycells.Non
conservativesubstitutionsatR416(C,A,E)causemultiplefunctional
deficienciesinc1udingdefectiveproteinprocessing,loss
oftransportactivity,
impairedcellsurfacetargetingandrecyc1ingthroughendosomes,concomitant
withretention
ofthetransporterintheendoplasmicreticulum.Conversely,a
conservativeisoelectricsubstitution(R416K)waslessvulnerable,resultingina
functionaltransporterthatwasproperlyprocessedandtargetedtothecellsurface
andtorecyc1ingendosomes.Weproposethat
DMTlC1246T(R416C)representsa
completeloss-of-functionandthataquantitativereductionin
DMTlexpressionis
thecause
ofthemicrocyticanemiaandironoverloadinthepatient.
81
INTRODUCTIONThemechanisms
bywhichnutritionalironisacquired,re-distributedandrecycled
involveanumber
ofstructurallyandfunctionallydistinctmembranetransporters
includingthe
DivalentMetalTransporter1(DMT1,alsoknownasNramp2orSLCllA2)
251.DMTlisanintegralmembranephosphoglycoproteinexpressedatthebrushborderof
theduodenum,whereitimportsdietaryironinabsorptiveepithe1ialcells161.DMTlis
alsoubiquitouslyexpressedinrecyclingendosomes
ofmanycelltypesandisabundantin
erythroidprecursors,whereitisrequiredfortransport
oftransferrin-assocÏatedironinto
thecytoplasm
169.DMTIrequiresaprotongradienttotransportFe2
+asweIlother
divaientcations(Mn2
+,C02+,Zn2+)in
apH-dependentmanner40.Muchofourknowledge
ofthefunctionofDMTIinvivocornesfromstudiesofrodentmodelsofmicrocytic
anemiaandirondeficiencysuchas
mkinmiceandBelgradeinrats,thatarebothcaused
bythesamenaturaIly-occuringmis-sensemutationinDMTI(GI85R)136;141.Asimilar
butmoreseverephenotypehasrecentlybeenreportedforamousemutantwitha
completeinactivation
ofDMTl(DMTl-I-)258.Furthermore,studiesinvolvingtissue
specificinactivation
ofDMTlhavedemonstratedthecriticalroleofthetransporterin
intestinalironabsorptionandinerythroidironutilization,butsuggestedalesscriticalrole
ofDMTIinliverironuptake258.
hlhumans,Priwitzerovaetalfirstreportedahumanpatienthomozygotefora
mutationin
DMTI(DMT1G1285c).Theyoungfemalepatientistheproductofa
consanguineousunionwhosufferedfromseverecongenitalhypochromicmicrocytic
anemiaandironoverload
4;259.ThephenotypeissimilartothatofDMTlmousemutants
withthenotableexception
ofelevatedhepaticironstoresthatarespecifictothehuman
patient.Themutationhasadualeffect,partiallyimpairingsplicing
ofexon12ofDMTl
(1285G>C)andintroducinganaminoacidsubstitution(E399D)intheremaining
properlysplicedtranscriptfoundinthepatient.WerecentlyshowedthattheE399D
mutationdoesnotinitselfaffectexpression,function,
ortargetingoftheDMTlprotein
260-262,andthusreducedDMTIfunctioninthispatientislikelycausedbyaquantitative
reductionin
DMTlrnRNAlevelsduetoimpropersplicing.
82
Recently,someofusreportedasecondhumanpatientwhowascompound
heterozygotefortwonovelmutationsin
DMTl263.Thepatientisa5-year-oldmalewho
suffersfromseverehypochromicmicrocyticanemiaandwhohasdevelopedhepaticiron
overloadshortlyafterbirth.Hematologicalvaluesandironmetabolismindicesforthis
patientandforthepatientreported
byPriwitzerovaetalweresimilar:bothhadlow
hemoglobin,lowmeancorpuscularvolume,lowmeancorpuscularhemeoglobin,high
serumironandserumferritin,andelevatedtransferrinsaturation.However,thedegree
of
anemiawasmoresevereinthechildpatientwhorequiredbloodtransfusionsuntil
erythropoietintreatmentsufficientlyamelioratedtheanemiatoallowtransfusion
independence.Thepatientpossessedtwo
nove1mutationsinDMT1:a3basepair
deletion(deICTT)inintron4andaCtoTtransitionatnucleotide1246
(DMTlC1246T)in
exon13thatresultsinanArgtoCyssubstitutionatposition416(R416C)ofDMTl.The
CTTdeletiondisruptsnormalsplicing
ofDMTlpre-mRNAbyaffectingtheconsensus
splicingacceptorsite
ofintron4,causingapartial(30-35%)skippingofexon5263.This
truncated
DMTlvariantlacks40aminoacids,inc1udinganentiretransmembranedomain
(TM2),andappearstorepresentaloss-of-functionthatisinherited
inafullyrecessive
manner.
Incontrast,theeffectsoftheR416CmutationonDMTlexpression,targeting,
andfunctionisunknown.Previousstudies
byourgrouphavesuggestedthatR416maybe
criticalfor
DMTlfunction28.AnalaninesubstitutionmutantatR416(R416A)failedto
restoregrowthundermetallimitingconditionstoayeastmutantdeletedfortwo
ofthe
threeendogenousDMTlhomologs(smfllsmj2)despiterobustmembraneexpressionof
thismutant.TheR416AvariantmaynotbestablyexpressedintransfectedCHûcells
prec1udingitsfunctionalanalysis.
Theeffects
oftheR416CmutationonDMTlfunction,inc1udingapossible
contributiontotheironmetabolismdisorderseeninthispatientremainunknownand
werestudied.WehavecreatedindependentmutationsatR416andanalyzedtheeffecton
proteinexpression,glycosylation,targetingandfunctionintransfectedLLC-PK
1cells.
83
MATERIALSANDMETHODS
Construction,expression,andfunctionalcharacterizationofDMTlMutants
AmouseDMTlisoformII(non-IRE)cDNAbackbonemodifiedby
insertionofahemagglutininepitope(HA)taginthepredictedextracytoplasmic
loopdefined
bytheTM7-TM8interval(DMTI-HA)wasusedformutagenesis26.
MutationsatR4l6wereconstructedusingarecombinantpolymerasechain
reaction(PCR)strategy
28,utilizingprimers5’
GTGATCCTGACATGTTCTATCGCCATC-3′(R4l6C),5’
GATCCTGACCAAGTCTATCGCCATC-3′(R416K),and5’
GATCCTGACCGAGTCTATCGCCATC-3′(R416E).Mutantswereintroduced
intotheplasmidvectorpCB6as
SacI/EcoR1fragments.Constructionofthe
R416AmutationinaDMT1constructbearingtwoc-Mycepitopetagsatthe
carboxylterminushasbeendescribedearlier(R416A-myc)
28.R416A-mycwas
introducedintoDMT1-HAasa
SacI/EcoR1fragment.AlImutantswere
transfectedandstablyexpressedinporcineLLC-PK
1kidneycelIsaspreviously
described
28.DMT1transportofC02+wasmeasuredusingacalceinfluorescence
quenchingassay(pH5.0,20
J..I.Mfinalmetalconcentration)262.Initialratesof
metaltransportwerecalculatedfromtheinitialslopesofthefluorescence
quenchingcurves.
Quantificationofcel!surfaceexpressionbyELISA
QuantificationoftheproportionofDMT1-HAmoleculesexpressedatthe
cellsurfacewas
byanELISAwehavepreviouslydescribed184.Briefly,cellswere
growntoconfluencyin48-wellplatesandfixedwith4%paraformaldehyde(30′).
Cellswerewashed(PBScontaining1
mMMgCh,0.1mMCaCh),blocked(5%
nonfatmilkinPBS),andincubatedfirstwithanti-HAAb(1:500,90min)and
thenwithdonkeyanti-mouse-HRPsecondaryantibody
(1:4000,1h).Fortotal
DMT1-HAexpression,cellswerepermeabilized(0.1%Triton
X-lOOinPBS,30′)
priortoblockingandincubationwithanti-HAantibody.Peroxidaseactivitywas
measuredwithanHRPsubstrate(0.4mg/mLo-phenylenediamine
dihydrochloride),accordingtoconditionsfromthecommercialsupplier(Sigma).
84
Backgroundabsorbancereadingsfromnon-specifiebindingofsecondary
antibody,andnon-specifiebinding
ofprimaryantibodytountransfectedcellswere
subtractedforeachsample.CellsurfacereadingswerenormalizedtototalDMT1-
HAvaluesforeachcloneexpressedasapercentage.
Immunofluorescence
GFPplasmidswerekindgiftsofDr.D.Williams(UniversityofToronto;
GFP-Syntaxin13)andDr.
S.High(UniversityofManchester,Manchester,United
Kingdom;GFP-Sec61).LLC-PK
1cellsgrownoncoverslipsweretransfectedwith
GFPplasmids,fixedwith4%paraformaldehyde,permeabilized(5%non-fatmilk,
0.2%saponin
inPBS),andincubated(1h,20°C)withanti-DMT1NTpolyclonal
antibody(1:300)
42.Coverslipswerethenwashed,incubatedwithgoatanti-rabbit
Cy3
(1:2000)forlh,andmountedonglassslides.
85
A
B
MouseDMTlLN
HumanDMTlLN
Zebrafish(Chy)LNLRWS
MouseNramplLKLRW
Drosophila(Mvl)LNLQW
Yeast(Smflp)INWKLQ
CYTOPLASM
Figure1.ThepositionandconservationofR416residueinDMT1.
AschematicrepresentationofDMT1-HAshowingthepositionofthe
R416Cmutation
intheninthpredictedtransmembranedomain(TM)of
theproteinisshownin(A).AmultiplesequencealignmentofDMT1
orthologsisshown
in(8)forresiduesneighboringTM9(grey).The
position
ofR416ishighlighted(black).
RESULTS
TheCtoTtransitionatnucleotide1246ofDMTlcausesanArgtoCys
substitution(R416C)inthe9
thpredictedmembrane-spanningsegmentofDMT1(Figure
lA).AlthoughR416ispositivelychargedandthermodynamicallydisfavoredinthe
membranelipidenvironment,R416isinvariantamongmouse,zebrafish
(cdy),yeast
(smflp),andfly(mvl)relativesofhumanDMTl(FigurelB).Thissuggestsanimportant
structuralorfunctionalroleforR416inmetaltransport
byDMT11.Toexplorethe
importance
ofR4l6inDMT1function,weusedamouseDMTI-HAisoformII(non-IRE)
backbonetocreateindependentmutationsatthatposition:
R4l6C(fromDMTlc1246T
mutation),R4l6A(lossofcharge,smallersidechain),R416K(chargeretained),and
R4l6E(negativecharge).Theeffectofthesemutationsonproteinexpression,transport
activity,subceUulardistributionandrecyclingfromthemembraneintotheendosome
compartmentwereexaminedintransfectedmammalian
ceUs.
DMTl-HAbearsanexofacialhemagglutinin(HA)epitopetagthatenablesusto
label
ceUsurfaceexpressedDMT1-HAmoleculesinintactcells(FigurelA).Wehave
previouslyshownthatinsertion
ofanHAtagatthispositionpreservesexpression,
transportactivityorsubcellularlocalization
ofDMTl51.Wildtype(WT)andR4l6
mutantDMTlvariantsweretransfectedintoLLC-PKIporcinekidneyceUsandceU
clonesstablyexpressingindividualproteinswereselectedandexpandedforanalysis.
LLC-PK
1ceUswerechosensincetheyarederivedfromkidneyproximaltubule,asiteof
abundantDMTlproteinexpressioninvivo,andthematuration,targetingandtransport
function
ofDMTlhavebeenextensivelycharacterizedinthesecells51;184;184.Figure2A
showsatypicalimmunoblot
ofcellextractspreparedfromceUstransfectedwitheither
WTormutantDMTl-HA.WT
DMTlwasdetectedastwoimmunoreactivespecies,a
minorspeciesat
~60kDaandamajorspeciesat~90kDa.Previousstudieshaveshown
thattheminorspeciescorrespondstotheprecursor”coreglycosylated”proteinwhilethe
majorspeciescorrespondstothemature”complexglycosylated”protein
51.Formutants
R416C,R416AandR416E,onlyexpression
oftheprecursorproteinwasdetected(Figure
2A).Theslightlyslowerelectrophoreticmobility
oftheR4l6Aprecursorproteinreflects
thepresence
ofthetwoadditionalc-MycepitopetagsinsertedattheCterminusofthis
87
A
B
..-~()«~w0-C1CDCDCDCD40%()..-..-
..-..-….J….r….r….r….r….JIl::Il::Il::Il::
110«l30%190…..1-::E0
51Q)20%0
-ê:::l(/)
(i)10%
0.0125
()
-*0Q)0..!!!.0.0100::::>«-;0.0075…..CIlIl::.s::0.00500cQ)0.0025:::l0
0..-~0-..-1….r()Il::….J….J
Figure2.StableexpressionandmetaltransportactivityofR416mutants.
EquivalentamountsoftotalcellextractsfromLLC-PK1cellsstablyexpressingeither
WTOMT-HAorR416mutantconstructswereseparatedbySOS-PAGEfollowedby
immunoblottingwithananti-HAmonoclonalantibody.Atypicalimmunoblotisshown
in(A).Thesizeofmolecularweightmarkers(inkOa)areidentifiedtotheleftofthe
blot.Cellsloadedwithametal-sensitivefluorescentdye,wereincubatedwithC02+
inacidicbuffer(pH5.0),andtherateoffluorescencequenchingwasmeasuredover
time.
In(8),theresultsareexpressedâsinitialratesoffluorescencequenching.
Errorbarsrepresentstandarderrorofthemeans
ofthreeormoreindependent
experiments.Asterixesindicatequenchratesthataresignificantlyhigherthanfor
untransfectedLLC-PK1cells(p
<0.001).In(C),fixedLLC-PK1cellswereincubated
withanti-HAprimaryantibodywith
orwithoutpriordetergentpermeabilizationas
described
inMaterialsandMethods.Theamountofboundprimaryantibodypresent
wasdeterminedforbothconditionswithanHRP-coupledsecondaryanti-mouse.
antibodyandacolorimetriereactionfollowedbyspectrometry.Theamount
of
DMT1-HAexpressedatthecellsurface(innon-permeabilizedcells)isshownasa
fraction
(%)oftotalproteinexpression(inpermeabilizedcells).
mutantthatareabsentintheothervariants.TheseresultssuggestthatmutationsatR416
(inc1udingR416C)affectproperprocessingandpost-translationalmodification
ofDMT1,
inc1udingmaturationtothefullyglycosylatedpolypeptides.Interestingly,onlyforthe
mostconservativeR416Ksubstitutionatthatposition,ismaturecomplexglycosylated
proteindetected,suggestingthatalargepositivelychargeresidueatposition416is
requiredforproperfolding/maturation.Maturation
ofR416Kisc1earlyreducedcompared
toWT
DMTl(Figure2A),demonstratingtheessentialroleofR416inthisprocess.
Earlierstudieshaveshownthatconservedchargedresidueswithinthemembrane
spanningsegments
ofDMTlaremutationsensitive,andplayacriticalroleintransport
activity,cellsurfaceexpression,andsubcellulartargeting.
28Therefore,weinvestigated
thepossibleeffects
ofR416mutationsontheseparameters.Wemeasuredthetransport
activity
ofWTandmutantDMTlvariantsinliveLLC-PK1transfectants,usingacalcein
fluorescencequenchingtechniquewheretheinitialrates
ofquenchingcurvesareusedas
anindicator
ofmetaltransportintothecell.MutantsR416C,R416A,andR416E
demonstratednosignificantmetaltransportactivity,displayingtransportratessimilarto
thatofuntransfectedLLC-PK
1cells(Figure2B).TheseresultssuggestthatR416isnot
onlycriticalforprocessingandmaturation
ofDMTlbutisalsoimportantformetal
transport
bythisprotein.Interestingly,mutantR416Kdisplayedmetaltransportactivity
(0.002748
±0.00024AU/sec;meanquenchrate±standarderror)thatwassignificantly
higher(p
<0.001)thanthatofuntransfectedLLC-PKIcells(0.001472±0.00007
AU/sec)(Figure2B).Significantactivitywasalsodetectedforanadditionalindependent
transfectedcellclone
ofR416K(datanotshown).However,transportactivitydetectedin
R416Kexpressingcellswasclearlylowerthan
WTDMT1,whichmaystemfromeither
reducedtransportand/orlowerlevel
ofexpressionofR416KcomparedtoWT.
Nevertheless,theseresultssuggestthata
DMTlproteinwithanisoelectricsubstitutionat
R416retainspartialmetaltransportactivity.
WeusedanELISA-basedsurfacelabelingmethodtodeterminetheamount
of
WTormutantDMTltargetedtothesurfaceoftransfectedLLC-PKIcells.Cells
transfectedwithWT
DMTI-HAexpressed30.9±3.8%oftotalDMTI-HAatthecell
surface(Figure2C).Thisresultisinagreementwithcellsurface
DMTlexpression
89
valuesreportedpreviouslyforisoformIIIwhichwasusedasabackbonetoconstructthe
mutants
51;184;262.MutantsR416C(3.6±1.0%),R416A(6.9±1.5%),andR416E(2.5±
0.6%)allshowedseverelyreducedcellsurfaceexpression(Figure2C),suggestingthat
R416iscriticalforpropersortingandtrafficking
ofDMTltotheplasmamembrane.
Theseresultsareconsistentwiththedefectiveprocessingandlack
ofactivityobserved
forthesemutants(Figure
2Aand2B).Interestingly,ahigherfractionofR416K(16.8±
2.9%)wasdetectedatthecellsurfacecomparedtotheothervariants(Figure2C),
consistentwiththenotedpartialmaturation/glycosylationandlowtransportactivity
of
thismutant.Thisresultsuggeststhatabasic,positivelychargedresidueatposition416is
requiredforcellsurfacetargeting
ofDMTl.
ThesubcellularlocalizationofWTDMTI-HAandtheR416variantswasnext
analyzed
bydoubleimmunofluorescenceusingarabbitanti-DMTlantiserumaswellas
additionalmarkers
ofdifferentendomembranecompartments.Co-Iocalizationstudies
showthatWT
DMTlisoformIIisexpressedinsyntaxin13-positive(GFP-Syntaxin13)
recyc1ingendosomes(Figure3),inagreementwithourpreviousstudies
51;184;184;262.
However,mutantsR416C,R416A,andR416Ewereexpressedinaperi-nuc1earregion
thatshowedlittleoverlapwithGFP-Syntaxin
13(Figure3).Strikingly,R416Kwasthe
onlyvarianttoshowstrongco-localizationwiththerecyclingendosomemarker(Figure
3),suggestingthatanisoelectricsubstitutionatR416doesnotaffectpropersubcellular
targeting
ofDMTl.WhileWTDMTI-HAandvariantR416Kshowedlittleoverlapwith
theendoplasmicreticulummarkerGFP-Sec61,variantsR416C,R416A,andR416Eall
displayedsignificantco-Iocalizationwiththis
ERmarker(Figure4).Theseresultsimply
thatalargefraction
ofthevariantsR416C,R416A,andR416Earemistargetedandare
retainedintheER,consistentwiththeimpairedglycosylation,lack
ofactivity,and
reducedsurfaceexpressionobservedforthesevariants.
90
Figure3.DoubleimmunofluorescencelabelingofDMT1-HAandtherecy
clingendosomemarkerGFP-Syntaxin13.LLC-PK1cellsstablyexpressing
WTormutantDMT1-HAweretransientlytransfectedwithGFP-syntaxin13
to
labelrecyclingendosomes.Thefollowingday,cellswerefixed,permeabilized
andstainedwithapolyclonalanti-DMT1antibody.
DMT1moleculeswerevisual
izedusingasecondaryanti-rabbitantibodycoupledtofluorescentCy3.Images
wereacquired
byepifluorescencemicroscopy.Insetsshowmagnificationsofthe
areaboxed
inthefigure.
Figure4.DoubleimmunofluorescencelabelingofDMT1-HAandtheendo
plasmicreticulummarkerGFP-Sec61.
LLC-PK1cellsstablyexpressingWT
ormutantDMT1-HAweretransientlytransfectedwithGFP-Sec
61tolabelthe
endoplasmicreticulum.DMT1moleculeswerevisualizedasdescribed
inthe
legend
ofFigure3.Insetsshowmagnificationsoftheareaboxedinthefigure.
DISCUSSIONTakentogether,ourdatastronglysuggestthattheR416Cvariantcreated
bythe
DMTlc1246Tmutationrepresentsacompleteloss-of-functioninwhichthemutantprotein
isnotproperlyprocessedandisretainedinthe
ERfordegradation.Therefore,wepropose
thattheabrogation
ofDMTlfunctionobservedinthepatientisduetoacombinationof:
a)productionofafulliengthnon-functionalproteinfromanullmutationatoneallele(
R416C)andb)amutation
(DMTldelcTT)thatpartiallyimpairssplicing(exon5skipping)
intheremainingallele,generatinga
~TM2truncatednon-functionalvariant,withasmall
butunknownamount
offunctionalproteinproducedbyresidualproperlyspliced
transcript.Theresultingoveralliowlevel
ofDMTlactivityinthepatientisclearly
insufficienttomaintainphysiologicalfunctionintheerythroidcompartment,andcauses
microcyticanemia.
263TheseresultssuggestthatalowlevelofDMTlactivityis
pathologicalandisinsufficienttosupportironmetabolismintheerythroidsystem,yetis
sufficienttoallowsornenutritionalironuptakeinthegut,whichinthelongrunleadsto
ironoverloadintheliver.
Coincidently,thefirstreportedpathologicalhumanmutationin
DMTl
(DMT1G1285c)alsoresultsinimpropersplicingofthegene,causingskippingofexon12
andthecreationofatruncated(dTM8)non-functionalvariant4.However,the
DMT1G1285Csubstitutionmutationleadingtoexon12skippingappearstohaveamore
severeeffectonsplicingthanthe3basepairdeletionmutationleadingtoexon5skipping
(DMTldelCTT).4;263Indeed,inthefemalepatientwhoishomozygotefortheDMT1G1285C
mutation,theremainingsmallieveiofpropersplicingofexon12producesaE399D
variantthatshowswildtypeactivity.Inbothpatients,diseaseappearstoresultfroma
quantitativereductioninfulliengthfunctional
DMTltranscriptbelowacertainminimum
threshold,andthesimilaritiesinconditions
ofbothpatientsseemtosupportthisidea.
Thisminimumthreshold
DMTlactivityappearstobebetweenthelowlevelofactivity
observedinboth
DMTlmutantpatientsandthe-50%activityretainedintheir
phenotypicallynormalheterozygoterelatives
4;263.Finally,anewpatientwasrecently
reportedwithaV114-inframedeletionintransmembrane2andaG212Vreplacementin
transmembrane
5.Thispatienthasaphenotypewhichisverysimilartothepreviously
93
describedsubjects265.However,thefactthatpatientswithacompleteloss-of-functionat
both
DMTlalleleshavenotyetbeenidentifiedsuggeststhatevenalowlevelofDMTI
activity,whileinsufficienttomaintainnormalironhomeostasis,isessentialforlife.
Inadditiontoprovidingbetterinsightintotherole
ofDMTlinironhomeostasis,
characterization
oftheR416Cvariantproducedbythepatienthasalsoshedlightintothe
structureandfunction
oftheDMTlprotein.Ourdatashowsthattheconservedarginine
inTM9
ofDMTl(R416)iscriticalfortheproperfoldingandprocessingofthe
transporter.Thefactthatnon-conservativemutationsatR416(R416C,R416A,R416E)
arenotwelltoleratedandthatanisoelectricsubstitution(R416K)retainspartialfunction
stronglysuggeststhatapositivechargeisrequired
byDMTlatthatposition,andthatloss
ofthatchargeratherthanthegainofacysteineatthatpositionisresponsiblefortheloss
offunction.Onepossibilityisthatthispositivechargeinteractswithanothernegatively
chargedaminoacide1sewhereintheproteintoformasaltbridgethataidsintheproper
foldingortertiarystructureassembly
ofDMTl.Indeed,conservedchargedresidues
residingwithinthehydrophobiemembranespanningsegments
ofproteinshavebeen
shownto
beimportantforthestructureofothermembranetransporterssuchasCFTRand
thelactosepermease
322;323.
Hopefully,theidentificationofmorehumanmutationsinDMTlasweIlas
additionaldatafrominvitromutagenesisexperimentswillhelpusbetterunderstandthe
role
ofthistransporterinironhomeostasisandleadtonoveltreatmentsforiron-related
disorders.
94
PrefacetoChapter5
Whereasthepreviousthreechaptersfocusedontheimportanceofconserved
residuesinNramp2function,thenexttwochaptersfocus
onbetterunderstandingits
subcellulartargetingandtrafficking.Priortotheworkdescribed
inChapters5and6,a
number
ofstudiesshowedthatNramp2(isoformil)isexpressedattheplasmamembrane
andintransferrin-positiverecyclingendosomesatsteady-state
42.StudiesbyTouretetal
usinganexofacially-taggedNramp2-HAconstructshowedthatNramp2(isoformil)
moleculespresentatthecellsurfaceandinrecyc1ingendosomeswereindynamic
equilibrium,withsurfacetransportersbeingcontinuouslyintemalizedviaac1athrinand
dynamin-dependentprocess.ThesestudiesgavefurthersupporttotheideathatNramp2
andtransferrinreceptorarefunctionallycoupledtoeffectpH-dependentironuptake
acrosstheendosomalmembrane.Examination
ofthesequenceofproposedcytoplasmic
domains
ofNramp2isoformilrevealedthepresenceofthreeputativeintemalization
motifs
intheaminoandcarboxylterminalregionsofthetransporter.Therefore,we
decidedtouseasite-specificmutagenesisapproachtoidentifycytoplasmicmotif(s)
responsibleforintemalizationandrecyc1ing
ofthetransporterfromtheplasma
membrane.ThisworkisdescribedinChapter
5.
95
Chapter5:
Carboxylterminusdeterminantsoftheiron
transporter
DMTl/SLCllA2isoformII
(-IRE/lB)mediateinternalizationfromthe
plasmamembraneintorecyclingendosomes
96
ABSTRACT
MutationsinDMT](Nramp2,S/cl]a2)impairironmetabolismandcause
microcyticanemia.
DMTlisexpressedattheduodenalbrushborderwhereitcontrols
uptake
ofdietaryiron,andispresentattheplasmamembraneandinrecyclingendosomes
ofmostceIls,whereitisnecessaryforacquisitionoftransferrin-associatediron.Thegoal
ofthisstudywastoidentifysignal(s)inthecytoplasmicsegmentsofDMTlresponsible
foritssubcellulartargetingandintemalizationfromtheplasmamembraneintorecycling
endosomes.Weintroducedmutationsintheaminoterminus(LlNT),carboxylterminus
(LlCT),asweIlasin
NPAy28-31,YSCF62-65andYLLNT555-559motifsofaDMTlconstruct
bearinganexofacialepitopetag,whichallowedlabeling
ofthetransporteratthecell
surfaceforkineticstudies.Mutantswerestablyexpressed
inLLC-PKIkidneyceIlsand
werestudiedfortransportactivity,sub-cellularlocalization,cellsurfaceandrecycling
pooldistribution,andintemalizationfromtheplasmamembrane.Kineticstudiesshowed
thatcarboxylterminusmutants
(LlCTandLlYLLNT)hadanincreasedfractionofthe
"recyclingpool"thatwasexpressedatthecellsurfaceduetoimpairedintemalization
fromtheplasmamembrane.Furthercellsurfacelabelingandimmunofluorescencestudies
inintactcellsshowedthatthe
LlYLLNTandLlCTmutantsweretargetedtothelysosomal
compartmentuponintemalization.Theseresultssuggestthatthemajorsignalfor
intemalizationandrecycling
ofDMTIisoformII(-IRE/lB)residesinitscarboxyl
terminusandthatremoval
ofthissignalleadstoadefaultlysosomaltargeting.
97
INTRODUCTION
TheDivalentMetalTransporter1(DMT1,alsoknownasNramp2,DeT1,
SIcIla2)playsadualroleinintestinalironacquisitionandinironuptakebyperipheral
tissues.
DMTlispartofalargefamilyofhighlyconservedmetaltransporters.Itisan
integralmembranephosphoglycoproteinconsisting
oftwelveputativetrans-membrane
(TM)domains,withthepredictedaminoandcarboxylterminipositioned
ontheintra
cytoplasmicside
ofthemembrane.StudiesinXenopusoocytesandtransfected
mammalian
celllineshavedemonstratedthatDMTltransportsanumberofdivalent
metals(Fe2
+,Mn2+,C02+,Cu2+,Cd2+,Ni
2+,Pb2+,Zn2+)in
apH-dependentfashionbya
protonco-transportmechanism
26;27;40.DMTlisexpressedatthebrushborderofthe
absorptiveepithelium
ofduodenalvilli161,whereitisresponsiblefortheuptakeofnon
hemedietaryironfollowingreductionfromitsferric(Fe3+)to
ferrous(Fe2+)statebya
ferricreductase.
Innon-epithelialcells,ironabsorptionoccursthroughinternalizationof
transferrin-boundironviathetransferrinreceptorpathway.DMTlisalsoexpressedatthe
plasmamembraneandintransferrin-positiverecyclingendosomes
ofmostcellsand
tissues
40;42.Itcatalyzestheeffluxofironfromacidifiedendosomes,acrossthe
endosomalmembraneintothecytosol
324.VacuolarATPase-mediatedendosomal
acidificationsimultaneouslyfacilitatesrelease
ofironfromtransferrinandprovidesthe
pHgradientrequiredforDMT1function51.Naturally-occurringmutationsinDMTl
causesevereirondeficiencyandmicrocyticanemiainmkmiceandBelgraderats136;141.
Bothmutantsshowthesamealteration(G185R)inpredictedTM4ofDMT1,which
abrogatesbothintestinalironabsorptionandendosomaltransportoftransferrin-iron
137;138;146;155;325.Finally,DMT1isalsoexpressedatthebrushborderofepithelialcellsof
proximaltubulesofthekidney172,whereitmayfunctionasare-uptakesystemfor
divalentmetals.
The
DMTlgeneproducesatleasttwodistinctmRNAsbyalternativesplicingof
two3'exonsencodingdifferent3'untranslatedregions(UTR)andproteinproductswith
distinctC-termini
153;154.DMTlisoforml(+IRE)containsaniron-responsiveelement
(IRE)inits3'UTR.DMTlisoformlisexpressedatthebrushborderofduodenal
enterocyteswhereitsexpressionisinducedbydietaryirondepravation161;253.DMTl
98
isoform1isalsoexpressedinthekidney161j172.DMTIisoformII(-IRE)lacksanIREand
encodesaproteinwhichhasadifferentC-terminal25aminoacidsegment.
DMTI
isoformIIisexpressedpreferentiallyinnon-epithelialcells,andisveryabundantin
reticulocytes
169.Recently,additionalisoformsofDMTImRNAshavebeenidentified
basedonaltematepromoterusageat
DMTlexon1(exonlAvs.lB)154.Thisaltemate
promoterusageispredictedtoproducea
DMTIproteinbearinganadditional29amino
acids(exon
lA)upstreamofthepreviouslyidentifiedstartcodonofDMTIisoforms1
andII(exonIB).Therole
oftheseadditionalresiduesintheexpression,function,and
targeting
ofDMTIhasnotyetbeenstudied.Touretandcolleagueshavedemonstrated
that
DMTlisoformII,whenexpressedintransfectedCHOandLLC-PK1cells,ispresent
attheplasmamembraneandinanacidictransferrinreceptor-positiveendomembrane
vesicularcompartment.Kineticstudies
ofanexofacially-taggedDMTlmoleculeshowed
similaramounts
ofproteininendomembranecompartment(s)andatthecellsurface.The
twocomponentsareindynamicequilibrium:surfacetransportersbeingcontinuously
intemalizedviaac1athrinanddynamin-dependentprocess
51.Therefore,proper
physiologicalfunction
ofDMTlinironmetabolisminvolvesnotonlypropertargetingto
theplasmamembrane,butalsorequiresefficientintemalizationandrecyclingvia
recyclingendosomes.
Tyrosine-basedmotifs(NPXYand
YXX,where=bulkyhydrophobie)have
beenshowntoactasintemalizationsignaIsforcIathrin-mediatedendocytosis
183.The
NPXY
motifwasinitiallydiscoveredasamutation(Y807C)inthecytoplasmicdomain
oftheLDLreceptorthatabolishesintemalizationofthereceptor326inapatientwith
familialhypercholsterolemia.Mutagenesisstudies
327showedthatNPXYisan
intemalizationsignalforsurfaceproteinssuchastheLDLreceptor-relatedprotein1
(LRPl)328,theEGFreceptor329andmegalin330.Severalproteins,inc1udingc1athrin,AP-
2,andDab2,appeartofunctionasrecognitionproteinsfor
NPXYsignals.183TheYXX
motifhasalsobeenshowntoactasaplasmamembraneintemalizationmotiffor
endocyticreceptorssuchasthetransferrinreceptorandiontransporterssuchasthecystic
fibrosistransmembraneconductanceregulator(CFTR)
183.YXXmotifshavealsobeen
implicated
intargetingmembraneproteinssuchasLAMP-IandLAMP-2tolysosomes.
99
YXX
recruitclathrinmolecules331-333.Cytoplasmicdileucinebased(LL)motifsofmembrane
proteins(suchasGLUT4)havealsobeenshowntoactassignaIsleadingtoclathrin
mediatedendocytosis
ortargetingtoendosomal-Iysosomalcompartments183.Residues
neighboringtheLLsignaIsappeartodictatewhetherclathrinrecruitmentoccursviaAP
complexesorviaARF-dependentclathrinadaptors.
Examination
ofthesequenceofproposedcytoplasmicdomainsofDMTIreveals
thepresence
ofthreeputativeintemalizationmotifs:twotyrosine-basedmotifsofthe
formsNPXYand
YXX<1>(NPAy28-31andYSCp62-65)in
theaminoterminalregion,anda
di-leucine
motif(LL556-557)inthecarboxylterminalregion.Inthepresentstudy,we
soughttoidentifythespecifiesignales)inthecytoplasmicsegments
ofDMTIisoformII
responsibleforintemalizationandrecycling
ofthetransporterfromtheplasma
membrane.
100
MATERIALSANDMETHODS
Materials
Reagent-gradechemicalswerepurchasedfromSigmaChemical(St.Louis,MO).
Monoclonalmouseantibody(Ab)HA.11directedagainsttheinfluenzahemagglutinin
epitope(HA)waspurchasedfromCovance(Princeton,NJ).Cy2-andCy3-labeledanti
mouseantibodiesandHRP-coupleddonkeyanti-mouseantiserawerepurchasedfrom
JacksonImmunoResearchLaboratories(WestGrove,PA).TheplasmidencodingGFP
fusionproteinGFP-syntaxin
13wasthekindgiftfromDr.D.Williams(Departmentof
Biochemistry,UniversityofToronto).TheGFP-LamplplasmidwasakindgiftfromDr.
PatriceBoquet(Institutnationaldelasantéetdelarecherchémédicale,France).
Plasmidsandconstructs
Full-IengthmurineDMTl(Nramp2,DeTl,SIc11a2,GenBankaccessionno.
L334l5)isoformII(non-IRE,isoformlB)cDNAwasmodifiedbythein-frameaddition
ofanHAepitopeataminoacidposition330,aspreviouslydescribed26.Deletionand
truncationmutationsinDMT1-HAwerecreated
bysite-directedmutagenesisusing
oligonucleotideprimerslistedinTable1
byarecombinantpolymerasechainreaction
protocol
308.For~NT,aprimerwasdesignedtodeIetethefirst67aminoacidsofDMTI,
uptofirstmembrane-spanningsegment,withtheadditionofaninitiatormethionine
(Table
1).For~CT,aprimerwasdesignedtoremove34aminoacidsfromthecarboxyl
terminus
ofDMT1,withtheinsertionofastopcodonafterG534andanEcoR1sitefor
subsequentcloning.Mutantswereconstructedintothemammalianexpressionvector,
pCB6,usingrestrictionenzymesites
Xho1andBstEIIformutants~NPAY,~YSCF,
~NTandsitesSac1andEcoR1formutants~YLLNT,~CT.
Cel!culture,transfection,andimmunoblotting
LLC-PK1cellsweregrownat37°Cina5%C02incubatorinDulbecco’s
modifiedEagle’smedium(Invitrogen)supplementedwith10%fetalbovineserum
(growthmedia).LLC-PK
1cellsweretransfectedwithDMT1-HAlpCB6vectorsusinga
calcium-phosphateco-precipitationmethod.Selection
ofstablytransfectedcloneswas
101
Table1.Oligonucleotidesusedformutagenesis.
PrimerName
ANPAYFor
AYSCFFor
AYLLNTFor
ANTFor
ACTRevNucleotidesequence(5’to3′)
ccttggcgccatcagcaactcatccc
cctgaggaggagagctttcgtaaac
gctcagcctgaactcgtggatgctgactcag
ccgctcgaggccaccatgctgaaactctgggcgttcacggggc
cggaattcttaacccaatgcaatcaaacactgc
Forindicatesforward;Rev,reverse.Boldtypeindicatestheinsertedinitiatormethioninecodon
forANT.TheinsertedstopcodonforACT
isunderlined.
Table2.SummaryofsubcellularlocalizationofWTandmutantDMT1-HA.
WT
ANPAY
AYSCF
AYLLNT
ANT
ACT
PM
+
+
+
+
+
RE
+
+
+
LE/LysER
+
+
+
PM:Plasmamembrane;RE:Recyclingendosomes;LE/Lys:Lateendosomes/lysosomes;ER:
Endoplasmicreticulum.
102
doneusing1.4mg/mLG418(Invitrogen)forlOto14days.Individualcolonieswerethen
pickedandexpanded.Totalcelllysateswerepreparedandseparated
bySDS-PAGE.
ClonesshowingrobustDMT1-HAexpressionwereidentified
byimmunoblottingwith
mouseanti-HAantibody,aspreviouslydescribed
28.
Calceindivalentmetaltransportassay
Ca1ceinacetoxymethylester(calcein-AM,MolecularProbes)waspreparedasa
500
f.lMstocksolutionindimethylsulfoxide.Fe2+andC02+solutionswerefreshly
preparedindeionizedwateras2
mMstocksolutionsofferrousammoniumsulfateand
cobaltchloride,respectively.Measurement
ofFe2+andC02+transportinDMT1-HA
transfectedLLC-PKlcellswasdoneusingafluorescencequenchingassay,exactlyaswe
havepreviouslydescribedwithCHOcells
26.Initialratesofmetaltransportwere
calculatedfromthefluorescencequenchingcurvesandresultsforeachtransfectantwere
normalizedtoactivityofwild-typeDMT1-HAexpressingcells.
ELISAForallELISAassays,2.8×10
5cells/wellwereseededin48-welltissueculture
platesandgrowntoconfluency(16to24h).Cellswerefixedinphosphatebuffered
saline(PBS)containing4%paraformaldehydefor30minat20°C.Blocking,
permeabilization,andincubationswithantibodywerecarriedoutat37°C.Asolution
of
5%nonfatmilkinPBSwasusedforblockinganddilutionofallantibodiesunless
otherwisenoted.WasheswereperformedthreetimeswithPBScontaining1
mMMgCh
and0.1mMCaCh(PBS++).Afterdonkeyanti-mouse-HRPsecondaryAbincubation,
peroxidaseactivitywasdetected
byincubatingcellswithHRPsubstrate(0.4mg/mL0-
phenylenediaminedihydrochloride,SigmaFASTOPD;550f.lL/well)accordingto
manufacturerinstructions.Reactionswerestoppedwith150
f.lLof3MHCIperwelland
absorbancereadings(492nm)weretakenwithaspectrometer.
Forallassays,background
absorbancereadingsfrom(a)non-specificbindingofsecondaryAb,and(b)non-specific
binding
ofprimaryAbtovector-transfectedcells,weresubtractedforeachsample.
103
ForquantificationofcellsurfaceexpressionofDMT1-HAatsteadystate,cells
werewashedwithcoIdPBS++,fixed,blocked,incubatedwithanti-HAAb
(1:500)for90
min,washed,andpermeabilized30minin
0.1%TritonX-100IPBS.Cellswereblocked
for30min,andlabe1edwithsecondaryAb(donkeyanti-mouse-HRPAb,1:4000)for1
h.
ForquantificationoftotalDMT1-HAexpression,cellswerepermeabilizedpriorto
incubationwithanti-HAAb.CellsurfacereadingswerenormalizedtototalDMT1-HA
valuesforeachcellcloneandwereexpressedasapercentage.
ToquantifycellsurfaceDMT1-HAexpressionasapercentage
oftherecycling
pool,cellswereincubatedinanti-HAAb
(1:200)dilutedin2%nonfatmilk/RPMIfor3h
at37°C.TomeasuresurfaceDMT1-HAmolecules,censwerewashedwithPBS,fixed,
blocked,andincubatedwithsecondaryAb.TomeasurerecyclingpoolDMT1-HA,cens
weresequentianypermeabilizedwith
0.1%TritonX-1OOIPBS,blocked,andincubated
withsecondaryAb.Cellsurfacevalueswereexpressedasapercentage
oftotalrecycling
DMT1-HAforeachclone.
Immunostaining
censwerefixedwith4%paraformaldehydedilutedinPBSfor15to20minat
20°C,andwhereindicated,werepermeabilizedwith
0.1%TritonX-100inPBSfor30
minatroomtemperature.
censwereblockedfor30minin5%nonfatmilkinPBS,
followed
byconsecutiveincubationswithprimaryantibodies(mouseanti-HA,1:500;
goatanti-EEA1,1:100;dilutedinblockingsolution)andsecondaryantibodies(goatanti
mouse-Cy3,Donkeyanti-mouse-alexaFluor488,donkeyanti-goat-alexaFluor488;
an
usedat1:2000)eachfor1hat20°C.Forexperimentswithlivecens,theanti-HA
antibodywasdiluted
(1:200)in2%nonfatmilkinRPMIand(whereindicated)censwere
chasedbywashingtwiceandincubatingthecellsingrowthmediafor90minat37°C.
Forco-IocalizationwithGFP-fusionproteins,cellsweretransfected24hpriortofixation
usingLipofectamine2000(Invitrogen)accordingtothemanufacturer’sinstructions.Cells
werevisualizedusingaLeicaIREDR2microscopeusinga100xoilimmersion
objective.DigitalimageswereacquiredwithanOrca
IIERcamera(Hamamatsu)
104
operatedwiththeOpenlab3software(Improvision)installedonanAppleG4computer.
Imageswerecropped,assembledandlabeledusingAdobePhotoshopsoftware.
Cel!surfacebiotinylation
Cellsurfacebiotinylationwasperformedbased
onasimilartechniqueusedto
studytheintemalization
ofCFTR334.LLC-PKIcellsweregrownin100mmculture
dishesonedaypost-confluency,followedby2washesinPBS++andonewithcoIdborate
bufferpH9.0(10
mMborieacid,154mMNaCI,7.2mMKCI,1.8mMCaCh).Cells
werebiotinylatedfor1hat4°Cinboratebuffercontaining0.7to1.0mg/mLsulfo-NHS
SS-biotin(Pierce).Aftertwo5minwasheswithcoldRPMI,cellswereincubatedinpre
warmedRPMIfor0,30,60,or90minat37°Cin5%C02atwhichpointendocytosis
washalted
bythreewasheswithcoldPBS++.Cellsurfaceassociatedbiotinmolecules
wereremoved
(3washes,30mineachat4°C)bytreatingwiththemembrane
impermeablereducingagent2-mercaptoethanesulfonicacid(MESNA,100
mMsolution
with50
mMTris-ClpH8.6,1mMMgCh,0.1mMCaCh).Forquantificationoftotal
surfacelabeling,cellsweresimilarlytreatedbutwithfinalwashesinbufferlacking
MESNA.Biotinylatedcellswerecollectedandsolubilizedfor30minonicein350ilL
of
lysisbuffer(1%TritonX-lOO,0.2%SDS,50mMTris-ClpH7.4,150mMNaCI,20%
glycerol)supplementedwithproteaseinhibitors
(1mMphenylmethylsulfonylfluoride,1
IlMpepstatin,0.3IlMaprotinin,1IlMleupeptin).Lysateswerepre-clearedby
centrifugation(20min,1O,000g,4°C)andproteinsinthesupematantwerequantifiedby
Bradfordassay(BioRad).Biotinylatedproteins(250
Ilgtotalproteinlysate)wereisolated
byovemightincubationat4°C
of100ilLImmunoPureimmobilizedstrepavidinslurry
(Pierce)inafinalvolume
of1mLinlysisbufferwithproteaseinhibitors.Strepavidin
beadswerewashed4timeswithcoldlysisbufferandboundproteinswereelutedwith2x
Laemmlibufferatroomtemperaturefor30min.Proteinswereseparated
bySDS-PAGE
followedbyimmunoblottingwithanti-HAAb.Intensityofimmunoreactivebandswere
quantifiedbydensitometryanalysis
ofexposedfilmsusingaFujiLAS-1000.Background
intensityreadings(after0minendocytosis)weresubtractedfromallreadingsandresults
wereexpressedasapercentage
oftotalsurfacelabelingforeachclone.
105
AB
314-358
230-256
VI124-143277-294::::lr:::’E….
c..<1<1<1<1
114
88,..
-~..~-.(.f~""~
50.7
B1.4...---~---------~-----,
1.2
~
:>1.0
ü
«0.8Q)
;:0.6ct!Q)c:::0.4
0.2
o
-Cobalt
Dlron
Figure2.ExpressionandmetaltransportactivityofDMT1-HAmutantsin
transfected
LLC-PK1cells.(A)TotalcellextractsfromLLC-PK1cellsstably
transfectedwitheithervectoralone(pCB6),wild-typeDMT1-HA(WT),
orvarious
DMT1-HAdeletionmutantswérequantifiedbyBradfordproteinassayand25
~g
ofeachIysatewasseparatedbySDS-polyacrylamidegelelectrophoresis,
followedbyimmunoblottingwithananti-HAmonoclonalantibody.Thesizes
of
themolecularmassmarkersareindicated.(B)MetaltransportactivitybyWT
andmutantDMT1variantswastestedusingafluorescencequenchingassay.
Cellsloadedwithametal-sensitivefluorescentwereincubatedwithFe2+or
Co2+inacidicbuffer(pH6.0),andtherate
offluorescencequenchingwas
measuredovertime.Theslopesfrominitialquenchingcurveswerecalculated
andtransportactivity
ofthemutantswasexpressedasapercentageofWT
activity(relativeactivity).Errorbars
=standarderrorofthemeansofthreeor
moreindependentexperiments.
(Figure2B).DMT1variants~NPAY,~YLLNT,and~CTwerefullyactiveforFe2+and
C0
2+transport,suggestingthattheyareexpressedatthecellsurfaceandareproperly
foldedinatransport-competentconformation.Cellsexpressingvariants
~YSCFand~NT
showednoactivity,displayingtransportratessimilartocellstransfectedonlywiththe
emptyvector(Figure2B).Theseresultssuggestthat
~YSCFand~NTareeithertransport
inactiveand/orarenotefficientlytargetedtothepropertransportsite(plasmamembrane
and/orrecyclingendosomes).
SubcellularLocalization
TodeterminewhetherthemutationsweintroducedaffectedDMT1targeting,the
subcellularlocalization
ofthedifferentvariantswasdeterminedbyimmunoflurescence
usingananti-HAmonoclonalantibody.Infixedandpermeabilizedcells,wildtype
DMT1-HAfluorescencewasdetectedasapunctateandjuxtanuclearpattern(Figure3A)
42;51.HavingpreviouslyshownthatDMT1-HAisoformIIispresentandactivein
syntaxin13-positiverecyclingendosomes
51,weexaminedtheeffectofthedifferent
mutations
ontargetingtothiscompartment.Recyclingendosomeswerelabeledby
transienttransfectionofaGFP-syntaxin13construct,andDMTI-HAvariantswere
visualizedusingasecondaryantibodycoupledtoCy3(Figure3A).The
~NPAYand
~YSCFvariantsshowedextensiveco-Iocalizationwithsyntaxin13andsharedapattern
similartothatseenfor
WTDMTI-HA.~NPAYand~YSCFalsoshowedextensiveco
stainingwithaGFP-transferrinreceptorconstruct(datanotshown),confirmingthatthese
variantsarepresentinrecyclingendosomes.Theseresultsindicatethatthecytoplasmic
motifsNPAYandYSCFdonotplayamajorroleintargeting
DMTltoearlyand
recyclingendosomes.Bycontrast,the~NTvariantshowednosignificantco-Iocalization
withGFP-syntaxin
13anddistributedinamoreperinuclearpatternpossibly
correspondingtotheendoplasmicreticulum(Figure3A).The
~YLLNTand~CTvariants
showedsorneoverlapwithGFP-syntaxin
13(Figure3A)andwithGFP-transferrin
receptor(datanotshown).However,therewasasignificantfraction
of~YLLNTand
~CTthatdidnotco-Iocalizewiththerecyclingendosomemarkers.
Todetermine
ifanyofthemutantsweremistargetedtolateendosomesand/or
lysosomes,
welabeledtheseorganellesbytransienttransfectionwithaGFP-Lamp1
109
Figure3.SubcellulardistributionofWTandmutantDMT1-HAinendomem
branecompartments.LLC-PK1cellsstablyexpressingWTormutantDMT1-HA
variants(identified)weretransientlytransfectedwitheitherGFP-syntaxin13(A)
tolabelrecyclingendosomes,orGFP-Lamp1(8)tolabellysosomes.24hours
later,cellswerefixed,permeabilizedandstainedwithamonoclonalanti-HA
antibody.DMT1-HAmoleculeswerevisualizedusingasecondaryanti-mouseAb
coupiedtofluorescentCy3.Imageswereacquiredbyepifluorescencemicros
copy.Insetsshowmagnifications
oftheareaboxedinthefigure.
construct.WTDMTI-HA,asweIlastheilNPAYandilYSCFvariantsshowedno
significantco-stainingwithGFP-Lampl.Theseresultsareconsistentwiththeextensive
co-Iocalization
ofWT,ilNPAYandilYSCFwithrecyc1ingendosomes(Figure3A).The
ôNTvariantsimilarlydidnotco-IocalizewithGFP-Lamplpositivelysosomes(Figure
3B).TheseresultssuggestthattheN-terminus
ofDMTlisessentialforpropertargeting
anditsdeletionresultsinaproteinthatisnotproperlyglycosylated(Figure2A),non
functional(Figure2B),andismistargetedtoaperinuc1earcompartment(Figures
3Aand
3B).Incontrasttothe
WTandotherDMTI-HAvariants,ôYLLNTandilCTboth
displayedsignificantco-stainingwith
GFP-Lampllysosomes(Figure3B).Theseresults
suggestthattheC-terminus
ofDMTI-HAcontainsinformationthatiscriticalforoptimal
targeting
ofthetransportertorecyc1ingendosomes,andthatdeletionofthisinformation
results
inmis-targetingoftheproteintolateendosomes/lysosomes.
DeterminantsofExpressionattheCel!surfaceandintheRecyclingCompartment
Wenextexaminedtheeffectofthemutationsona)targetingofDMTltothecell
surfaceandb)dynamictraffickingthroughtherecyc1ingendosomecompartment.The
fraction
ofeachDMT1-HAvariantpresentatthecellsurfaceatsteady-statewas
determined
byexposingfixedLLC-PKItransfectantstoanti-HAantibody,withor
withoutpriorpermeabilizationwithmilddetergent.Theamountofboundanti-HA
antibodywasquantifiedusingasecondaryantibodycoupledtohorse-radishperoxidase.
WTDMT1-HAtransfectantsexpressed29.5
±1.4%(mean±S.E.)ofthetotalDMTI
HAattheircellsurface(Figure4),whichiscomparabletothefractionwepreviously
determinedusingradiolabeledantibodies
51.Asignificantlyhigherproportionofvariants
ilNPAYandilCTwasdetectedatthecellsurface(50.1±3.9%and40.2±2.7%,
respectively),whilethe
ilYSCFandilYLLNTvariantsshowedalowerfractionofcell
surfaceexpression(17.5
±0.2%and20.5±1.2%,respectively).FinaIly,onlyasmall
fraction
oftheilNTvariantwasexpressedatthecellsurface(8.8±1.8%).Theseresults
suggestthatdefinedsequencesintheaminoandcarboxylterminaldomains
ofDMT1
playaroleindeterminingthelevelofDMT1proteinexpressionattheplasmamembrane
atsteadystate.
111
60%
«50%
:r:1~~40%::0
Cl>30%0
-ê::J(/)
(j)20%
(,)
10%0%
~~
LL~~~(,)ZZ(,)
a.:CI)….J<1<1
Z~
....J
<1~
Figure4.QuantificationcellsurfaceDMT1-HAexpressionatsteady
state.
LLC-PK1cellsstablyexpressingindividualDMT1-HAvariantswere
fixedandincubatedwithanti-HAprimaryantibodywithorwithoutprior
detergentpermeabilization(seeMethods).CeIlswerethenincubatedwith
anHRP-coupledsecondaryanti-mouseantibody,andtheamountof
boundprimaryantibodypresentwasdeterminedforbothconditionsbya
colorimetriereactionusingo-phenylenediaminedihydrochloride(OPD)
followedbyspectrometry.Theamount
ofDMT1-HAexpressedatthecell
surface(innon-permeabilizedcells)isshownasafraction
(%)oftotal
proteinexpression(inpermeabilizedcells).
Becauseimmunofluorescencestudies(Figure3)indicatedthatcertainvariantshad
alargerfraction
ofproteinassociatedwitheitheraperinuclearcompartment(8NT)or
withlysosomes(8YLLNT,8CT),weattemptedtovisualizetheproportionofthe
recyclingpoolofWTandvariantDMT1-HAthatisexpressedatthecellsurface.Forthis,
liveLLC-PKItransfectantswereincubatedwithanti-HAantibody(2h,37°C),followed
byfixation,andincubationwithasecondaryanti-mouseantibodycoupledtoared
fluorophoretolabelDMT1-HAproteinsatthecellsurface.Thesamecellswerethen
permeabilizedundermilddetergentconditions,andtotalDMT1-HAmoleculeswere
labeledwithasecondaryanti-mouseantibodycoupledtoagreenfluorophore.
Inthese
experiments,DMT1-HAmoleculesatthecellsurfacearelabeled
inredwhiletotal
moleculeswerelabeledingreen,withtheyellowcolorinmergedimagescorresponding
tothefraction
oftotallabeledDMT1-HAmoleculesexpressedatthecellsurface(Figure
SA).Thisprotocolselectivelylabels
onlythoseDMT1-HAmoleculesthatwere
intemalizedfromtheplasmamembranewithin2h,allowingabetterdetermination
ofthe
proportion
ofDMT1-HAatthecellsurfacevs.theintracellularrecyclingpool.These
studiesshowedthatonlyasmallproportion
oflabeledDMT1-HAisexpressedatthecell
surfacefor
WTand8YSCF,withthemajorityofproteinappearingtobeintracellular
(FigureSA).
Onthecontrary,incellsexpressingvariants8NPAY,8YLLNT,and8CT,a
significantlylargerproportion
oftheproteinappearstobeatthecellsurface(yellow
staininginFigureSA).Resultsforvariants
8NPAYand8CTareconsistentwiththe
higherexpressionatthesurfacedetectedforthesemutants
inFigure4.Ontheotherhand,
thehighassociation
of8YLLNTwiththelysosomesnotedinFigure3Bmaybe
responsiblefortheapparentlowersurfaceexpressionnotedinFigure4.Cellsexpressing
the
8NTvariantshowedlittleifanysurfacelabelingbythismethod,inagreementwith
thealmostcompleteassociation
ofthismutantwiththeaperinuclearcompartmentnoted
inFigure3.
Thehigherproportion
ofcellsurfaceexpressionfromthetotalrecyclingpool
detectedforvariants
8NPAY,8YLLNT,and8CTbyimmunofluorescence(FigureSB)
wasfurtherquantified
byanELISA-basedassayasdescribedinMaterialsandMethods.
InthecaseofWTDMT1-HA,19.3±4.8%ofthelabeledrecyclingcompartmentwas
113
A
B
ë(1)E1::
80%
~
60%
Eo()Clc:'5~Cl::~~::l(1)Qi()
1-Z..J
..J
internalization(Figure7B).TheseresultssuggestthattheYLLNTmotif,andpossibly
otherresiduesintheC-terminus
ofDMTlisoformII,arecriticalfortherecyclingofthe
transportertothecellsurface.Furthermore,thisstudyshowsthatcriticalmutationsinthe
C-terminus
ofDMTlisoformIIresultinatransporterthatis:(a)internalizedwithslower
kineticsfromthecellsurface,(b)notefficientlyrecycledbacktothecellsurface,and(c)
eventuallytargetedtothelysosomalcompartment.
118
B
Internalized
DMT1-HA
Figure7.SubcellularlocalizationofinternalizedDMT1-HAmolecules.(A)LLC-PK1
transfectantsexpressingeither
WTormutantDMT1-HAwereincubatedwithanti-HAAb
for2htolabelce”surfaceandrecyclingDMT1-HAmolecules.
CeUswerefixed,permea
bilizedandincubatedwithanantibodyagainsttheearlyendosomalmarkerEEA
1.(8)
LLC-PK1ce”sweretransientlytransfectedwiththeIysosomalmarkerGFP-Lamp1.24h
later,surfaceandrecyclingDMT1-HAmoleculeswerelabeledwithanti-HAAbfor3h,
washedandchasedbyincubationingrowthmediafor90minat
3rC.CeUswerefixed,
permeabilized,andstainedwithananti-mousesecondaryAbtovisualizeDMT1-HA
molecules.Imageswereacquiredbyepifluorescencemicroscopy.Insetsshowmagnifi
cations
oftheareaboxedinthefigure.
DISCUSSION
Inthisstudy,wesoughttoidentifyspecifiesignales)incytoplasmicdomainsof
DMTlresponsibleforintemalizationofthetransporterfromtheplasmamembraneinto
recyc1ingendosomes.Truncation
oftheaminoterminaldomainofDMTl(~NT)had
drasticeffects
onproteinexpressionandtargeting.~NTwasnotproperlyglycosylated,
wastransportinactive,andwasexpressedneitherattheplasmamembrane,
norin
syntaxin13-positiverecyc1ingendosomes
orLampI-positivelysosomes.~NTwasfound
almostexclusivelyinaperinuc1earcompartmentwhichwaspositivefortheendoplasmic
reticulummarkercalnexin(datanotshown).Theseresultssuggestthatanintactamino
terminus
ofDMTlisessentialforprocessingandstabilityoftheprotein.
Deletion
oftheNPAy28
-31motifintheaminoterminusoftheproteindidnothave
amajoreffect
oneitherproteinfunctionorsubcellulartargeting.The~NPAYvariantwas
transport-competent(Figure2B)andshowedasub-cellulardistributionindistinguishable
fromthat
ofWTDMTl(Figure3).Ontheotherhand,the~NPAYmutantshowed
increasedsurfaceexpression(Figure4)andanincreasedproportion
oftherecyc1ingpool
waspresentatthecellsurface(Figures4,5)comparedtoWT.
~NPAYshoweda
somewhatslowerrate
ofintemalization(Figure6B).Theseresultsraisethepossibility
thattheNPAY
motifmayfurthercontributetointemalizationofDMTlinLLC-PK1
cells.However,thismotifappearstobeoflimitedimportancewhencomparedtosignaIs
inthecarboxylterminusofDMTI(seebelow).Finally,wecannotexc1udethepossibility
thattheNPAY
motifmayplayadditionalcell-specificrolesintargetingandtraffickingof
DMTl,whichmayhavegoneundetectedinLLC-PK1kidneycells.
Deletion
oftheothertyrosine-basedmotif,YSCF62
-65
,didnothaveamajoreffect
onthesub-cellularlocalization
oftheproteininLLC-PKIcells.LikeWTDMTl,this
mutantwaslimitedtoplasmamembraneandrecyc1ingendosomes(Figure3).
Distribution
ofthe~YCSFmutantbetweenthecellsurfaceandendomembranesofthe
recyc1ingpoolwasalsosimilarto
WTprotein.Thus,theYSCFmotifappearstobe
neithercriticalforDMTltargetingnorrecyc1inginLLC-PKIcells.However,~YSCF
wasc1earlytransportincompetent(Figure2B),suggestingthatsorne
orailoftheresidues
inYSCF
62-65arerequiredfortransport.Thisrequirementmayinvolveadirectrolefor
120
theseresiduesinthetransportprocess.However,thelowerleve1ofexpressionofthis
mutant(Figure2A)mayalsocontributetothemutant’sinactivity.
Ontheotherhand,alterationsinthepredictedcarboxylterminalintracellular
domain
ofDMTIhadthemostdramaticeffectsonDMTItargetingandrecyc1ing.
Truncation
oftheentirecarboxylterminaldomain(iCT)ordeletionoftheYLLNT
(iYLLNT)
motifdidnotsignificantlyaffecteitherthelevelofproteinexpressionin
LLC-PK1transfectants,ortheirtransportproperties(Figure2),suggestingthatthese
regionsarenotcriticalforactivityorstability.Asmallfraction
ofiCTandiYLLNTwas
detectedinthesyntaxin13-positiverecyc1ingendosomes(Figure3A),suggestingthata
part
ofthesevariantswasproperlytargeted.However,theC-terminaldomainvariants
showedrobustco-IocalizationwithLampl-positivelysosomes,bothatsteadystate
(Figure3B)andafteratransientlabelingofintemalized
DMTI-HAmolecules,90min
afterendocytosis(Figure7B).Interestingly,bothiCTand
iYLLNTvariantsdisplayed
anincreasedfraction
ofthe”recyc1ingpool”expressedatthecellsurfacewhencompared
toWT(Figure5).Thisincreasedaccumulationattheplasmamembranemaybe
explained
byadefectinintemalizationofthesevariantsthatwemeasuredbysurface
biotinylation(Figure6).TheseresultsimplicatetheYLLNTsequence
ofDMTlasa
critical
motifforintemalizationfromtheplasmamembrane.Othercarboxylterminal
residues
ofDMTlmayadditionallycontributetointemalizationsincetruncationofthe
entirecarboxylterminuscausesamoreseveredefectthanthatseen
intheYLLNT
mutant.
PreviousstudieshaveshownthatWT
DMTlisoformIIisrapidlyintemalizedvia
ac1athrinanddynamindependentmechanismandisefficientlytargetedbackthecell
surface
bymeansofrecyc1ingendosomes51.Theimpairedintemalizationand
accumulationinthelysosomes
oftheC-terminaldomainmutantssuggestsamodel
whereby,
intheabsenceoftheYLLNTmotif,DMTlissubjecttoadefaultlysosomal
targeting.Inthismodel(Figure8),
DMTlrecruitsspecifieadaptorproteinsrequiredfor
rapidc1athrin-mediatedendoeytosisthroughbindingtoitsYLLNTmotif.
DMTlis
intemalizedintoearlyendosomesandthereeruitedadaptorcomplexesareinvolvedin
signalingthereeycling
ofthetransportersbaektotheeellsurfaceviarecyc1ing
121
Non-clathrinendocytosis
non-CCV
f
Q’e-
Jf14
‘e-
~..siW
Clathrin-mediatedendocytosis
o
WTDMT1
o
LlYLLNTILlCT
,Transferrinreceptor
+
Lamp1
{Clathrin/adaptin
Figure
8.
Schematic
model
for
endocytosis
of
cell
surface-expressed
wr
and
C
terminus
mutant
DMT1
molecules.
Clathrinmoleculesinteractwithcellsurface
DMT1
moleculesviaadaptins(Ap-1,Ap-2)thatspecifi
callyrecognizethetyrosine-basedmotifYLLNT.Thiscausesformation
of
DMT1
enrichedclathrin-coatedpitson
thecellsurface,whicharerapidlyintemalized(step
1)
bya
dynamin-dependentprocessintoclathrin-coated
vesicles(CCV).DMT1-containingCCVsarethensortedtotheearlyendosomecompartment(EE,step2)and
eventuallyrecycledbacktothecellsurfaceviasortingtotherecyclingendosomecompartment(RE,steps3and
4)alongwiththetransferrinreceptor.Incontrast,
DMT1
C-terminusmutantswhichlackthetyrosine-basedsorting
motif
(Ll
YLLNT,LlCT),areintemalizedlessrapidlybyaclathrin-independentmechanism(suchasbulkpinocytosis)
intonon-CCVs(step5)andaresortedtoEE(step6).Themutant
DMT1
proteinsarerecycledmuchlessefficiently
comparedto
WT
andareeventuallysortedtotheLamp1-positivelateendosomeandlysosomecompartments
(steps7and8).
endosomes(Figure8).Indeed,properinteractionwithclathrinhasbeenpreviouslyshown
tobecriticalforintemalizationandtrafficking
ofcertainmembraneproteinsthrough
recyclingendosomes,mostnotablythetransferrinreceptor
336;337.Therecruitmentof
adaptorcomplexes(Ap1I1l1B)hasbeenshowntobecriticalforefficientrecyc1ingofboth
thetransferrinandLDL-receptorstothebasolateralmembraneinepithelialcells338.In
contrast,theC-terminaldomainmutants,whichlacktheYLLNTmotif,areunableto
recruitc1athrin/adaptorcomplexesandareintemalizedintoearlyendosomes
bya
kineticallyslowermechanismsuchasbulkpinocytosis(Figure8).However,thefailure
of
theC-terminusmutantstorecruitspecifieadaptorsdisruptstheirpropersortingto
recyc1ingendosomesandleadstoanaccumulation
inlysosomes.Ultimately,ourdata
doesnotfavourtheexistence
ofatruelysosomaltargetingmotifinDMTlisoformII,but
ratherthatthelysosomemaybethedefaultpathwayforthetransporter.
Recently,TabuchiandcolleaguesobservedthatintransfectedHEp-2larynx
carcinomacells,the
DMTIisoform1istargetedtothelateendosome/lysosome
compartment,whileisoformIIistargetedtotheearlyendosomes
ofthesecells155.They
demonstratedthatthe36carboxyterminalresidues
ofDMTlisoformIIareimportantfor
targetingtoearlyendosomesandalterationsintheYXLXX
motif(YLLNT)inisoformII
impairtargetingtoearlyendosomesandcauseaccumulation
ofthemutantsinLamp2-
positivelateendosomes/lysosomes.Theauthorsconc1udedthatthetargetingsignalfor
earlyendosomesdominateoversignaIsforlateendosomes/lysosomesin
DMTlisoform
II.OurresultsagreethatcriticalC-terminusmutationsin
DMTlisoformII(ôYLLNT,
ôCT)causesignificantcolocalizationofthetransporterwithLampll2-positive
lysosomes.However,ourdatasuggeststhattheYLLNT
motifismorecriticalfor
transporterrecyc1ingsince,uponintemalization
ofÔYLLNT/ôCTfromthePM,there
waspartialco-IocalizationwithEEAI-positiveearlyendosomesbutnotSyntaxin13-
positiverecyc1ingendosomes(Figure7A).Tabuchiandcolleaguesalsoshowedthatthe
C-terminus
ofDMTlisoformII,whenfusedtoaplasmamembranemarker(TAC
antigen),results
inearly/recyc1ingendosometargeting155.Thisresultisconsistentour
cellsurfacebiotinylationdata(Figure6)thatshowstheYLLNT
motifisalsocriticalfor
endocytosisfromtheplasmamembrane.
123
Cytoplasmicdileucine-based(LL)motifsofmembraneproteinshavebeenshown
toactassignaIsIeadingtoclathrin-mediatedendocytosis
inothertransporterssuchasthe
glucosetransporterGLUT
4339andthecoppertransporterMenkes(MNK)340-342.Inmost
proteins,residuesprecedingtheLLsignaIsappeartodictatethemethod
ofc1athrin
recruitment.Dileucine-basedsignaIsusuallyfitthe[DE]XXXL[LI]
orDXXLLconsensus
motifs.[DE]XXXL[LI]signaIsarespecificallyrecognized
byAPcomplexes.ConverseIy,
DXXLLsignaIsarerecognized
byanotherfamilyofadaptorsknownasGGAs,arecently
describedfamily
ofARF-dependentc1athrinadaptors183.Theresiduesprecedingthe
dileucine
motifinDMTlisoformII(ELYLL553-557)donotcorrespondtothetraditional
[DE]XXXL[LI]
motifbutresembletheDXXLLconsensussignature.Ithasbeen
demonstratedthatmutationsateithertheDorLLresidues
oftheDXXLLcauseretention
ofthemannose-6-phosphatereceptoratthecellsurface343,abehaviorresemblingthatof
DMTlmutants~CTand~YLLNTmutantsstudiedhereinLLC-PKIcells.Thissuggests
thatthe
ELYLL553-557sequenceofDMTlmayfunctionasaDXXLLsignal.Although
thedileucine
motifinDMTlresemblestheDXXLLconsensuswithaglutamate(E)
substitutingforaspartate(D),itremainstobedeterminedwhether
ornottheyactthe
samewayandwhether
DMTlrecruitsGGAsviathismotif.Indeed,previousstudiesof
M6PRmutantshaveshownthattheisoelectricsubstitutionofaspartateintheDXXLL
consensussequenceforglutamateisnotweIltolerated
344.Furthermore,theaspartatein
classicalDXXLLmotifsisgenerallyfoundinthecontext
ofac1usterofacidicresidues,
whichisnotthecaseforthe
DMTldileucinesignal.Thus,theDMTldileucinemotif
ELYLL553-557maybeanovelfunctionalvariantofDXXLL,ormayrepresentanovel
signalforendocytosis
ofmembraneproteinsfromthecellsurface.
Takentogether,ourresultsindicatethatdeterminants
ofthecarboxylterminus
cytoplasmicdomain
ofDMTlplayacriticalroleinintemalizationoftheproteinfromthe
plasmamembraneandforrecirculation
inrecyclingendosomes.Eliminationofthese
signaIsimpairsintemalization,andre-directstheintemalizedDMTlproteinstothe
lysosomalcompartment,whichappearstoactasadefaultpathwayforsuch
DMTl
variants.
124
PrefacetoChapter6
AlternatesplicingofNramp2pre-mRNAat3’exonsgeneratestwoprotein
isoforms
(1andII)differingonlyintheircarboxylterminalsegments153.Although
simultaneousexpression
ofbothisoformsofNramp2mRNAwasdetectedinsorne
tissues,isoform1ispredominantlyexpressed
inepithelialcellswhileisoformIIis
predominantlyexpressedinerythroidcells
40;155;160.Thesignificanceofthistissue
specificexpressionpattern,inc1udingapossiblephysiologicaladvantage,wasunc1earand
neededtobeinvestigated.
Atthesubcellularlevel,itwasknownthatbothisoformsareexpressedatthe
plasmamembranebutexhibitdifferentsteady-statetargeting.WhileisoformIIis
expressed
inearlyandrecyc1ingendosomes,otherstudiessuggestedthatisoform1is
presentinlateendosomesandlysosomes
42;51;155.WorkbyTabuchiandcolleagues
showedthataYLLNT
motifinthecarboxylterminusofNramp2isoformIIisresponsible
fortheearlyendosometargeting
oftheprotein155.Theworkdescribedintheprevious
chaptershowedthattheSameYLLNT
motifinisoformIIisresponsiblefor
internalization
ofNramp2fromtheplasmamembraneandrecyc1ingofthetransporter
backtothecellsurface
184.Incontrast,muchlesswasknownaboutthesubcellular
distribution,targeting,anddynamictrafficking
ofNramp2isoform1.Therefore,we
decidedtoinvestigatepossibletraffickingdifferencesbetweenisoforms1andII
by
expressingandcharacterizingexofaciallytaggedNramp2-HAproteinsinanepithelial
cellline.Thisworkisdescribedinthenextchapter(Chapter6).
125
Chapter6:
Distincttargetingandrecyclingpropertiesoftwo
isoforms
oftheirontransporterDMTl
(NRAMP2,Slclla2)
126
ABSTRACT
ThemetaltransporterDMTl(S1clla2)playsavitalroleinironmetabolism.
Alternativesplicing
ofthe3’exongeneratestwoDMTlisoformswithdifferentC
terminalproteinsequences,and
3’untranslatedregionharboring(isoforml,+IRE)ornot
(isoformII,-IRE)anironresponsiveelement.Isoform1isexpressedattheplasma
membrane
ofcertainepithelialcens,inc1udingtheduodenumbrushborderwhereitis
essentialforabsorption
ofnutritionaliron.IsoformIIisexpressedinmanycensandis
essentialforacquisitonoftransferrinironfromacidifiedendosomes.Thetargetingand
traffickingproperties
ofDMTlisoforms1andIIwerestudiedintransfectedLLC-PKI
kidneycens,withrespecttoisoform-specificdifferencesinfunction,subcenular
localization,endocytosiskinetics,andfateuponinternalization.Isoform1showedhigher
surfaceexpressionandwasinternalizedfromtheplasmamembranewithslowerkinetics
thanisoformII.AsopposedtoisoformII,whichisefficientlysortedtorecyc1ing
endosomesuponinternalization,isoform1wasnotefficientlyrecyc1edandwastargeted
tolysosomes.Thus,alternativesplicing
ofDMTlcriticallyregulatesthesubcellular
localizationandsite
ofFe2+transport.
127
INTRODUCTION
Ourknowledgeandunderstandingofironmetabolismhasincreasedremarkablyin
recentyears.Muchofthisknowledgestemsfromthediscoveryofproteinsthatplaykey
rolesinironabsorptionandregulation,includingtheidentificationofmembraneiron
transporters
251.Onesuchtransporter,thedivalentmetaltransporter1(DMT1,alsocalled
Nramp2
orSlc11a2),isessentialforintestinalironacquisitionandforironuptakeby
peripheraltissues.DMTlisanintegralmembranephosphoglycoproteinconsistingof
twelvepredictedtrans-membranesegments(TM).DMTlispartofalarge,highly
conservedfamily
ofmetaltransportersandhasbeenshowntotransportanumberof
divalentmetals(Fe2+,Mn2+,C02+,Cu2+,Cd2+,Ni2+,Pb2+,Zn2+)ina
pH-dependent
fashion
byaprotonco-transportmechanism26;27;40.
GeneticstudieshaveshownthatDMTlplaysakeyroleinironmetabolism.A
G185Rmutation
inDMTlcausesmicrocyticanemiaandirondeficiencyinthemkmouse
and
intheBelgraderat,tworodentmodelsofirondeficiency136-138;141;146;148.These
animaIsshowimpairedironuptakeattheduodenalbrushborder,
butarealsodefectivein
ironacquisitioninperipheraltissues,includingerythroidprecursors138;140;143;144.The
G185Rmutationlikelycausesmis-folding
oftheprotein,resultinginalteredsubcellular
localization
147;148;169;172,impropermaturation,increasedrateofdegradation148,and
impairstransportactivity.Recently,a
humanpatientsufferingfromseverecongenital
hypochromicmicrocyticanemiaandironoverload,
wasshowntobehomozygotefora
mutationin
DMTl(DMTIG1285C)4;259.Thehumanmutationhadtwoeffects:itseverely
impairedpropersplicing
ofDMTlmRNAandintroducedanaminoacidpolymorphism
(E399D)
intheremainingproperlysplicedtranscriptfoundinthepatient.TheE399D
mutationdoesnot
initselfaffectexpression,function,ortargetingoftheDMT1protein
262;345,andthusreducedDMTlfunctioninthispatientiscausedbyreducedlevelsof
DMTlexpression(impropersplicing).
Two
majorDMTlproteinisoformsgeneratedbyalternativesplicingat3’exons
havebeenidentified
153.Isoform1(+IRE)hasanironresponsiveelement(IRE)inthe3’
untranslatedregion,whereasisoform
il(-IRE)lackstheIRE.Inaddition,theC-terminal
18aminoacids
ofisoform1arereplacedbyanalternate25-aminoacidsegmentin
128
isofonnII.DMT!isofonn1ispredominantlyexpressedinepithelialcellswhileisoform
IIispredominantlyexpressedinerythroidcells.Indeed,isoform
1proteinisexpressedin
enterocytesattheduodenalbrushborder
161andinepithelialcellsliningthekidney
proximaltubule
172,whileisoformIIisexpressedabundantlyinreticulocytes169.
However,preferentialexpressionofeachisofonnisnotnecessarilymutuallyexclusive:
simultaneousexpression
ofbothDMTlisoforms1andIImRNAslevelhasbeenobserved
inseveraltissuesincludingkidney,thymus,andliver
40;155;160.Recently,additional
isoforms
ofDMTlmRNAshavebeenidentifiedbasedonalternatepromoterusageat
DMTlexon1(exonlAvs.lB)154.Thisaltematepromoterwouldproduceapredicted
DMTIproteinwithanadditional29aminoacids(exonlA)upstreamofthepreviously
identifiedstartcodon
ofDMTIisoforms1andII(exonlB).However,theroleofthese
additionalresiduesintheexpression,function,andtargetingofDMTIhasnotyetbeen
explored.Althoughboth
DMTIisoformsareexpressedattheplasmamembrane,isoforms1
andIIappeartoshowdifferentsubcellulartargetingatsteady-state.Whileintransfected
LLC-PKl,CHOand
RAWcells,DMTIisoformIIisexpressedinearlyandrecycling
endosomes
42;51studiesintransfectedHEp-2cellsindicatethatisoform1ispresentinlate
endosomesandlysosomes
155.Recentstudiesusinganexofacially-taggedDMTI
moleculehavefurtheredourunderstandingofisoformIItrafficking:isoformIImolecules
presentatthecellsurfaceandinrecyclingendosomesare
indynamicequilibrium,with
surfacetransportersbeingcontinuouslyintemalizedviaaclathrinanddynamin-dependent
process
51.TabuchiandcolleagueshaveshownthataYXLXX555-559motifintheC
terminus
ofDMTIisoformIIisresponsiblefortheearlyendosometargetingofthe
protein,withmutationsinthismotifresultinginlysosomallocalization155.Recently,we
haveshownthatcriticalresiduesintheCterminus
ofDMTIisoformII,includingthe
YXLXX555-559signal,arerequiredforthetransporter’sintemalizationfromthecell
surfaceanditsrecyclingbacktotheplasmamembrane.
184Removalofanintact
YXLXX555-559motifappearstocauselysosomaltargetingbydefault.
While
DMTIisoformIItraffickinghasbeenwellstudied,muchlessisknown
aboutthesubcellulardistribution,targetinganddynamictrafficking
oftheDMTI
129
intestinalisoform1.Whereasisoform1lackstheYXLXXsignalpresentinisoformII,
closeexamination
oftheCterminusofisoform1revealsthepresenceofadileucinemotif
(LL550-551).Dileucine-based(LL)motifspresentinanumberofmembraneproteinshave
beenaltematelyshowntoactassignaIsforclathrin-mediatedendocytosis
ortargetingto
variousendosomes/lysosomes
183.Inthisstudy,wewantedtoinvestigatepossible
traffickingdifferencesbetweenisoform1andII
ofDMTl.Weexpressedexofacially
tagged
DMTI-HAproteinsinaporcinekidneyepithelialcelllineandstudieddifferences
betweenexpression,function,subcellularlocalization,intemalizationkinetics,andfate
uponintemalization
ofDMTlisoforms1andII.WefoundthatDMTlisoform1is
intemalizedwithslowerkineticsfromthecellsurfacecomparedtoisoformII.This
resultsinanincreasedproportion
ofisoform1expressedattheplasmamembrane,
perhapsfavoringirontransportatthissite
inepithelialcells.
130
MATERIALSANDMETHODS
Materialsandplasmids
AlIreagent-gradechemicalswerepurchasedfromSigmaChemical(St.Louis,
MO).Monoclonalmouseantibody(Ab)HA.11directedagainsttheinfluenza
hemagglutininepitope(HA)waspurchasedfromCovance(Princeton,NJ).Cy3-labeled
anti-rabbitandanti-mouseAbsandHRP-coupleddonkeyanti-mouseAbwerepurchased
fromJacksonImmunoResearchLaboratories(WestGrove,PA).PlasmidsencodingGFP
fusionproteinswerekindgiftsfromDr.D.Williams(DepartmentofBiochemistry,
University
ofToronto;GFP-syntaxin13)andDr.PatriceBoquet(Institutnationaldela
santéetdelarecherchémédicale,France;GFP-Iampl).Full-iengthmurine
DMTl
isoform1(+IRE,isoformlA)andisoformII(-IRE,isoformlB)cDNAsweremodified
bythein-frameadditionofanHAepitopeinthefourthextracellularloop,aspreviously
described
26.
Cel!culture,transfection,andimmunoblotting
LLC-PK
1cellswereculturedina37°C/5%CO2incubatorinDulbecco’smodified
Eagle’smedium(Invitrogen)supplementedwith10%fetalbovineserum(growthmedia).
CellsweretransfectedwithDMTl-HAlpCB6vectorsusingLipofectamine2000
(Invitrogen)accordingtothemanufacturer’sinstructions.Selection
ofstablytransfected
cloneswasdoneusing1.4mg/mL
G4l8(Invitrogen)for14days.Individualcolonies
werethenisolatedandexpanded.Totalcelllysateswerepreparedandseparated
bySDS
PAGE.Clonesshowingrobust
DMTl-HAexpressionwereidentifiedbyimmunoblotting
withmouseanti-HAantibody,aspreviouslydescribed
28.
Calceindivalentmetaltransportassay
Calceinacetoxymethylester(ca1cein-AM,MolecularProbes)waspreparedin
dimethylsulfoxide.Fe
2+andC02+solutionswerefreshlypreparedindeionizedwateras2
mMstocksolutionsofferrousammoniumsulfateandcobaltchloride,respectively.
Measurement
ofmetaltransportinDMTl-HA-transfectedLLC-PKIcellswasdoneusing
131
afluorescencequenchingassayaswehavepreviouslydescribed262.Initialratesofmetal
transport(quenchrates)werecalculatedfromthefluorescencequenchingcurves.
ImmunostainingCellswerefixedwith4%paraformaldehydelPBSfor20minandwhereindicated,
wereblockedandpermeabilizedwith0.2%saponin/5%non-fatmilkIPBSfor30min.For
co-IocalizationwithEEA1(Fig.5A),cellswereblockedin5%nonfatmilk(30min)and
permeabilizedwith0.1%Triton
X-1OOIPBS(30min)followingfixation.AlIantibody
incubationswereperformedfor1hatroomtemperatureanddilutedinblockingsolution
unlessotherwiseindicated.PrimaryAbswereusedatthefollowingdilutions:rabbitanti
DMT1,1:200;mouseanti-HA1:100;goatanti-EEA1,1:200;whilecorresponding
secondaryAbs(goatanti-rabbitCy3,goatanti-mouseCy3,donkeyanti-goatAlexa488,
respectively)wereeachusedat
1:1000.Forco-IocalizationwithGFP-fusionproteins
(Fig.2andFig.5B),cellsweretransfectedwithGFP-syntaxin
13orGFP-Iamp1plasmids
24hpriortofixationusingLipofectamine2000.Forlabeling
ofcellsurfaceDMT1-HA
molecules(Fig.3A),cellswerefixed,blockedin5%non-fatmilkfor30min,labeled
withanti-HAprimaryAbandthesecondaryAbwithoutpermeabilizingthecellswith
detergent.Forexperimentswithlivecells(Fig.5),anti-HAantibodywasdiluted
(1:200)
inRPMImediumand(whereindicated)cellswerechased
bywashingtwiceand
incubatingthecellsingrowthmediafor90minat37°C.Cellswerevisualizedusingan
Axiovert200Mepi-fluorescencemicroscopewitha100xoilimmersionobjective.Digital
imageswereacquiredwithaZeissAxioCamHRmcameraoperatedwithAxioVision4.3.
Imageswerecropped,assembledandlabeledusingAdobePhotoshopandIllustrator
softwares.
Measurement
ofsurfaceDMT1-HAatsteady-state
Quantification
oftheproportionofDMT1-HAmoleculesexpressedatthecell
surfacehasbeenpreviouslydescribed
262.Briefly,LLC-PKIcellsweregrownto
confluencyin48-wellcultureplatesandfixedwith4%paraformaldehydefor30min.
Cellswereblockedin5%non-fatmilkinPBSfor30min,incubatedwithanti-HAAb
132
(1:500)for90min,washedandincubatedwithsecondaryAb(donkeyanti-mouse-HRP
Ab,1:4000)for1
h.ForquantificationoftotalDMT1-HAexpression,cellswere
permeabilized
byincubationwith0.1%TritonX-1OOIPBSfor30minpriortoincubation
withanti-HAAb.Peroxidaseactivitywasdetected
byincubatingcellswithHRP
substrate
(004mg/mLo-phenylenediaminedihydrochloride,SigmaFASTOPD)
according
tamanufacturerinstructions.Reactionswerestoppedafter30minwith3M
HCIandabsorbancereadings(492nm)weretakenwithaspectrometer.ForaIlassays,
backgroundabsorbancereadingsfrom(a)non-specificbindingofsecondaryAb,and(b)
non-specifiebinding
ofprimaryAbtovector-transfectedcells,weresubtractedforeach
sample.Cellsurfacereadingswerenormalized
tatotalDMT1-HAvaluesforeachcell
cloneandwereexpressedasapercentage.
Cel!surfacebiotinylation
Measurement
ofDMT1intemalizationbycellsurfacebiotinylationhasbeen
previouslydescribed
184.Briefly,confluentLLC-PK1monolayerswerebiotinylatedat
4°C
inboratebufferpH9.0containing1mg/mLsulfo-NHS-SS-biotin(Pierce).Cells
werewashedandincubatedinpre-warmedRPMIfor0,30,60,
or90minat37°Cat
whichpointendocytosiswashalted
bywasheswithcoIdPBS.Cellsurfaceassociated
biotinmoleculeswereremoved
by3coIdwasheswiththemembraneimpermeable
reducingagent2-mercaptoethanesulfonicacid(MESNA,100
mMsolution).For
quantification
oftotalsurfacelabeling,cellsweresimilarlytreatedbutwithfinalwashes
inbufferlackingMESNA.Biotinylatedcellswerecollectedandsolubilizedinlysis
bufferwithproteaseinhibitors.Lysateswerepre-c1eared
bycentrifugationandprotein
yieldwasquantified.Biotinylatedproteins(200
~gtotalproteinlysate)wereisolatedby
ovemightincubationat4°Cwith100~LofImmunoPureimmobilizedstrepavidinslurry
(Pierce)inafinalvolume
of1mLinlysisbuffer.Strepavidinbeadswerewashed4times
withcoIdlysisbufferandboundproteinswereelutedwith2xLaemmlibufferatroom
temperaturefor30min.Proteinswereseparated
bySDS-PAGEfollowedby
immunoblottingwithanti-HAAb.Intensityofimmunoreactivebandswerequantifiedby
densitometryanalysisofexposedfilmsusingaFujiLAS-1000.Backgroundintensity
133
readings(after0minendocytosis)weresubtractedfromaIlreadingsandresultswere
expressedasapercentage
oftotalsurfacelabelingforeachclone.
134
RESULTS
Expression
andfunctionofDMTlisoforms1andII
Toinvestigatethesubcellularlocalizationandtraffickingpropertiesofthetwo
DMTIproteinisoforms,wemodifiedmouse
DMTlisoforms1andIIcDNAsbyinsertion
ofanexofacialhemagglutinin(HA)tagintotheextracellularloopoftheproteindefined
bypredictedTM7and8(FigurelA).Thisexofacialtagenabledustolabelandtrackcell
surfaceexpressed
DMTI-HAmoleculesinintactcells.Wehavepreviouslyshownthat
insertion
ofanHAtagatthispositiondoesnotaffectexpression,transportactivityor
subcellularlocalization
ofDMTI51.Wegeneratedstablytransfectedcellsexpressing
DMTIisoforms
1andIIintheporcinekidneycelllineLLC-PK
I.
LLC-PK
Icells
were
chosensincetheyarederivedfromthekidneyproximaltubule,anabundantsite
ofDMTl
proteinexpressioninnormaltissues172.Therefore,thesecellsarelikelytoexpressthe
necessarycellularmachineryforproperDMTItrafficking,includingtherecognition
of
sortingandtargetingsignaIs.FigurelCillustratesarepresentativeimmunoblotofcell
extractspreparedfromtwoindependentLLC-PKIcellclonesexpressingdifferentlevels
ofDMTIisoforms1orII.Ineachcase,aminorspeciesat~60kDaandamajorspeciesat
~90kDawereobserved.Previousstudiesfromourgrouphaveshownthatthesetwo
populationscorrespondtocoreandcomplexglycosylatedspecies
ofDMTl,
respectively.51TheseobservationsshowthatbothisoformsofDMTlarestablyexpressed
andglycosylatedtoasimilarextentinLLC-PKIcells.Experimentswiththeprotein
translationinhibitorcycloheximiderevealedthatDMTIisoforms
1andIIshowsimilar
stabilityinLLC-PKIcells(supplementarydata).Finally,wetestedtheability
ofDMTl
isoforms1andIItotransportdivalentmetals(Fe2+andC02+)atacidicpHbycalcein
fluorescencequenching.IndependentclonesfrombothDMTIisoforms
1andIIshowed
robusttransportactivitycomparedtovector-transfectedcells(pCB6).Nodifferencein
transportratesorionselectivitybetweenthetwoisoformswasdetected,indicatingthat
bothisoformsareexpressedatthecellsurfaceandproperlyfoldedinatransport
competentmanner(FigureID).
135
B
IsoformIl(-IRE)
544-568
lTGTlTTT,iIcf[p-XET(YyTlTCTiiflfIvItiIA
*****
533-543tTSTFTlTtiTtTGTFîIs
Isoform1(+IRE)
544-561
lTsTITsTKYVTlT[‘J:S’I’EToYtTsTGT
-ê
Aa>::J<0.....CIlEndocytosis(min)OJCI:!xUCIlCI:!000a.~~0("1)<00')
••
Clone1.'1,~"';5~
IsoformIII
""',.,-'
(-IRE)
'.
Clone2~','<
lsofonn1l
Clone10
(+IRE),
-ê::JCIlXCI:!40%
E
-0
~0…….,
“‘Ca>.!:::!m20%r::L-a>…..r::
~1-~00%306090
Time(minutes)
Figure4.QuantificationoftherateofendocytosisofDMT1isoforms1andIlin
LLC-PK1cells.Acellsurfacebiotinylationassaywasusedtocompareinternalization
rates
ofOMT1isoforms1andIlfromtheplasmamembrane.Weusedamembrane
impermeablecleavablebiotinreagenttolabelsurfaceproteins,whichwasperformedat
4
oCtohaitendocytosis.Internalizationofsurfaceproteinswasallowedtooccurat3rC
for0to90min.Afterthattime,biotinmoleculesremainingatthesurfacewereremoved
andbiotinylatedproteinswereisolatedframcellIysateswithimmobilizedstrepavidin.
ProteinswereresolvedbySOS-PAGEandOMT1moleculesweredetectedbyimmunob-
10Uingwithanti-HAantibody.Typicalimmunoblotsfromindividualexperimentsdoneon
independentclones
(1and2)areshownin(A).Immunoblotswerescannedbydensi
tometry,andtheamount
ofOMT1internalizedovertimeisexpressedasafraction(%)of
thetotalcellsurfaceexpression(Max)in(8).Lysate=20~gofunbiotinylatedcrudecell
Iysateforeachmutant.Max
=totalsurfaceOMT1-HAexpressionforeachclone
(biotinylatedOMT1-HAmoleculesisolatedwithoutpriorstripping).Errarbarscorrespond
tostandarderrars
ofthemeansframthreeormoreindependentexperiments.
inFigure5A).TodetenninethefateofintemalizedDMTlmoleculesafteralonger
incubationperiod,thecellswerelabeledfor3h,washedandchasedinculturemediafor
anadditional90min.Lysosomeswerelabeled
bytransienttransfectionwithGFP-Lampl
24hpriortolabeling.Asexpected,isofonnIIdidnotshowsignificantco-Iocalization
withLampl(Figure5B),indicatingthattheproteinwasefficientlyrecycledbacktothe
cellsurface.Strikingly,incellslabeledfor3hours,isofonn1showedstrongco
localizationwiththelysosomalmarker(Figure5B).Theseresultsshowthat
DMTl
isofonnl,uponintemalizationandpassagethroughearlyendosomes,failstorecycleback
toplasmamembraneandistargetedtolateendosomesandlysosomes.Theseresults
suggestthatthecarboxyltenninus
ofisofonn1notonlylacksanendocytosissignalbut
alsoarecyclingsignalpresentinthecarboxyltenninus
ofisofonnII.Slowerendocytosis
ofisofonn1fromtheplasmamembraneresultsinelevatedcellsurfaceexpressionat
steady-state(Figure3).
142
A
E-‘-w00:::….-o+~………
8
E’-w00:::….-o…!….en
E-‘-w00:::….-o+~-
Figure5.ThefateofinternalizedDMTisoform1andIlmolecules.In(A),
LLC-PK1cellsexpressingeitherDMT1isoforms
1orIlwereincubatedwith
anti-HAantibodyfor2htolabelcellsurfaceandrecyclingDMT1-HAmolecules.
Toinvestigateco-IocalizationofinternalizedDMT1-HAmoleculeswithearly
endosomes,cellswerefixed,permeabilizedandincubatedwith
anantibody
against
the.earlyendosomalmarkerEEA1.DMT1moleculeswerevisualized
usingafluorescentsecondaryantibody.Areasshowingoverlappingstainingare
highlighted(bluearrowheads).
Toinvestigateco-IocalizationofDMT1isoforms
withlysosomes,cellsweretransientlytransfectedwiththeIysosomalmarker
GFP-lamp1in(B).24hourslater,surfaceandrecyclingDMT1-HAmolecules
werelabeledwithanti-HAAbfor
3h,washedandchasedbyincubationingrowth
mediafor90min.Cellswerefixed,permeabilized,andstainedwith
ananti
mousefluorescentsecondaryAbtovisualizeDMT1-HAmolecules.Insetsshow
magnifications
oftheareaboxedinthefigure.Imageswereacquiredbyepifluo
rescencemicroscopy.
DISCUSSION
DMTlisoformIIisexpressedinmanycelltypesbutisparticularlyabundantin
erythroidprecursors
169.StudiesintransfectedcellsinvitrohaveshownthatisoformIIis
rapidlyintemalizedalongwiththetransferrinreceptorfromtheplasmamembrane
bya
clathrinanddynamindependentprocess
51.Endosomalacidificationfacilitatesreleaseof
ironfromtransferrinandprovidestheprotongradientforDMTl-mediatedirontransport
acrosstheendosomalmembrane.Ironisthenstoredboundtoferritin
ortransportedinto
mitochondriaforhemesynthesis.
DMTlisoformIIandtransferrinreceptorare
subsequentlyrecycledbacktothecellsurface.Thecriticalrole
ofDMTlinthisiron
acquisitionprocessishighlighted
bytherecentreportthatapatientwithamutationin
DMTlsuffersfromerythroidhyperplasiawithdefectivehemoglobinization4.In
circulation,themajorityofironisboundtotransferrinandverylittleironexistsinitsfree
cationicform.Thiscorrelateswellourdataaswellasearlierreportsthatlessthan35%
of
totalDMTlisoformIIisexpressedatthecellsurface,withthemajorityofisoformII
residingintransferrinreceptor-positiverecyclingendosomes.
Directional
ortrans-cellulartransportisessentialinepithelialcellslocatedatsites
ofabsorptionorre-absorptionofkeyelementssuchasiron.Dietarynon-hemeironis
mainlyabsorbedattheduodenalbrushborder.
DMTl,expressedattheapicalpoleof
enterocytesliningtheintestinallumen,transportsironacrosstheapicalmembranewhile
ferroportin,expressedatthebasalpole,transportsironacrossthebasolateralmembrane.
Itispredominantlytheisoform1variantofDMTlthatisexpressedattheapical
membrane
ofenterocytes161.Ourfindingthatcellsurfaceexpressionofisoform1in
LLC-PK
1cellsishigherthanisoformIIisinagreementwiththenotedpreferential
expression
ofisoform1atthecellsurfaceofdifferentcelltypesinvivo(comparedto
isoformII),namelythebrushborder
ofepithelialcellsoftheduodenumandofthekidney
proximaltubule.Wealsofoundthatisoform1isnotefficientlyrecycledupon
intemalizationandisultimatelytargetedtolysosomes.Thesefindingsindicatethatiron
transportedbyisoform1(attheduodenalbrushborder)involvesdirecttransfer
ofFe2+
fromtheintestinallumenintothecytoplasmofenterocytes,intheabsenceofanactive
Tf-TfRcycleattheapicalpole
ofthesecells.Ontheotherhand,TfRisexpressedatthe
144
basolateralmembraneandinbasalrecyclingendosomesofintestinalepithelialcells346;347
anditispossiblethatDMTIisoformIImayplayanadditionalroleinironacquisitionat
thatsiteas
we1l155.Inaddition,mutagenesisstudiessuggestedthatN-linkedglyosylation
ofDMTIcontrolsapicalvs.basolateraltargetinginpolarizedcells155.Thus,itispossible
thattraffickingdifferencesbetween
DMTlisoforms1andIIinepithelialcellsmaynot
onlyinvolvedifferentialtargetingtotheplasmamembranebutmayalsobeinfluencedby
differentrates
ofintemalizationofthetransportersfromthecellsurface,asdemonstrated
hereintransfectedLLC-PK
1cells.
Inaddition,recentwork
byJohnsonandcolleaguesin
Caco-2cellssuggeststhatisoform
1traffickingmayberegulatedbycellularironlevels.
Thisresultemphasizestheimportance
ofisoform-specifictraffickinginoverallDMTl
function,includingcouplingtotheTf-TiRcycle160.
WhiletargetinganddynamicsortinglrecyclingofDMTlisoformIIhasbeen
thoroughlystudied
byusandothers,muchlessisknownaboutisoform1.Theprotein
sequencesofisoforms
1andIIdifferonlyintheirC-terminalsegments(FigurelB).In
isoform
II,TabuchiandcolleagueshaveshownthataYLLNT555-559signalintheC
terminus
oftheproteinisrequiredforitsearlyendosometargetinginHEp-2larynx
carcinomacells
155.WehavepreviouslyshownthattheCterminusofisoformII,along
withtheYLLNTsignal,isvitalfortheprotein’sintemalizationfromthecellsurfaceand
recyclingbacktotheplasmamembraneinLLC-PK
1epithelial
cells
184.Criticalmutations
ordeletionsatYLLNTresultinsevere1yimpairedintemalization,accumulation
ofthe
transporteratthecellsurface,andfailureoftheproteintorecycleproperlyresultingin
lysosomaltargeting.
DMTlisoform1lacksanendogenousYLLNTmotifinitscarboxyl
terminus.Interestingly,thetraffickingproperties
oftheisoformIIYLLNTmutants
(includingaC-terminaltruncation)virtuallymirrorthetraffickingproperties
ofDMTl
isoform1reportedhere184.Thesefindingstogetherwiththeresultsofthepresentstudy
togetherdemonstratethecriticalrole
oftheYLLNTsequencemotifasarecyclingmotif
in
DMTl.Furthermore,theseresultsargueagainstthepresenceofafunctionaltargeting
motifintheC-terminalregionofDMTlisoform1.
ExaminationoftheC-terminalsequenceofisoform1revealsthepresenceofa
putativedileucinemotif(LL550-551,Figure·lB).Indeed,cytoplasmicdileucine-based(LL)
145
motifsofmembraneproteinsoftenactassignaIsleadingtoc1athrin-mediatedendocytosis
ortargetingtoendosomaI-Iysosomaicompartments
339-342.ResiduesneighboringtheLL
signaIsappeartodictatewhetherclathrinrecruitmentoccursviaAPcomplexesorvia
ARF-dependentclathrinadaptors
183.Dileucine-basedsignaIsusuallyfiteitherthe
[DE]XXXL[LI]
orDXXLLconsensusmotifs.[DE]XXXL[LI]signaIsarespecifically
recognized
byAPcomplexeswhileDXXLLsignaIsarerecognizedbyGGAs,arecently
describedfamily
ofARF-dependentc1athrinadaptors.However,theaminoacids
precedingthedileucine
motifinDMTlisoform1(SISKV545-549)donotfiteitherofthese
consensusmotifs.Therefore,therelevanceofthisleucinepair(LL550,551)inthetargeting
andsorting
ofisoformIIremainsunc1ear,andawaitsfurthercharacterization.
Based
onpreviousworkanddatareportedhere,weproposeamodelforthe
subcellulartrafficking
ofthetwoisoformsofDMTl.Inthismodel,DMTlproteins
wouldbesynthesizedintheendoplasmicreticulum,post-translationallymodifiedinthe
Golgiapparatus,andtargetedtotheplasmamembrane.IsoformIImoleculesarethen
rapidlyintemalizedfromthecellsurface
byrecruitingspecificadaptorproteinsrequired
forclathrin-mediatedendocytosis
51,whichweproposeinteractwiththeYLLNTmotif
andpossiblyotherdeterminantsintheC-terminalregion
ofthetransporter.IsoformIIis
intemalizedintoearlyendosomesandtherecruitedadaptorcomplexesareinvolvedin
recyclingthetransporterbacktothecellsurfaceviatargetingtorecyclingendosomes
(Figure6).ThispathwayensuresthattheisoformIImoleculesexpressedinerythroid
cellscanworkinconjunctionwithtransferrinreceptorintheuptakeoftransferrin-iron.
DMTlisoform1moleculeslacktheYLLNTmotifwouldbeunabletorecruit
clathrinladaptorcomplexesattheplasmamembrane.Consequently,isoform
1transporters
areintemalizedintoearlyendosomesbyakineticallyslowermechanismsuchasbulk
pinocytosis(Figure6).Theslowerrate
ofendocytosisofisoform1resultsinagreater
fraction
oftheDMTlvariantexpressedatthecellsurfaceatsteady-state.However,
failure
ofisoform1moleculestorecruitspecificrecyc1ingadaptorcomplexesprohibits
theirsortingtorecyclingendosomesandIeadstoanaccumulationinthelateendosomes
andlysosomes.Thistraffickingpathwayensuresahighlevel
ofDMTlisoform1
146
expressionattheplasmamembraneofepithelialcells,favoringtheabsorptionlre
absorption
ofiran.
Overall,ourfindingshighlightthecriticalraIe
ofaltematesplicingatthe3’endof
theDMT1geneforthegenerationofproteinisoformsthattransportironatdifferent
subcellularsitesinatransferrin-dependent(isoformII),andtransferrin-independent
(isoform
1)manner.
147
Non-clathrinendocytosisClathrin-mediated
endocytosis
..•
oDMT1isoformIl(-IRE)TTf-Transferrinreceptor{Clathrin/adaptin
oDMT1isoform1(+IRE)1Lamp1.:Iron
Figure6.AschematicmodelfordistincttraffickingofDMT1isoforms1andIl.
DMT1moleculesaresynthesizedintheendoplasmicreticulum,post-translationally
modifiedintheGolgiapparatus,andtargetedtotheplasmamembranebydefault.
ClathrinmoleculesinteractwithsurfaceDMT1isoformIl(-IRE)moleculesviaadaptins
thatspecificallyrecognizethetyrosine-basedmotifYLLNTandpossiblyotherresidues
presentintheCterminus
ofthevariant.IsoformIlmoleculesarerapidlyinternalized
(step
1)byadynamin-dependentprocessintoclathrin-coatedvesicles(CCV).DMT1-
containingCCVsaresortedfirsttoearlyendosomes(EE,step2)followedbyrecycling
endosomes(RE,step3).Theacidicenvironment
ofREfavorthereleaseofironfrom
transferrinandcreatetheprotongradientrequiredforirontransportby
DMT1isoform
II.Incontrast,surfaceDMT1isoform1molecules,whichlacktheC-terminaltyrosine
basedsortingmotif,areinternalizedlessrapidlybyaclathrin-independentmechanism
intonon-CCVs(step5).Isoform
1moleculesaresortedtoEE(step6)butarenot
efficientlyrecycledbackthecellsurfaceandareeventuallytargetedtolamp1-positive
lateendosomesandlysosomes(steps7and8).
111
93
53.5
111
93
53.5
~IsoformIl(-IRE)
~101357
1
~IsoformIl(-IRE)
~101357
1
Isoform1(+IRE)
11
o1357
Clone1
Isoform1(+IRE)
11
o1357
Clone2
Supplementaryfigure.ThestabilityofDMT1isoforms1andIlproteins.Independent
clones
(1and2)ofLLC-PK1cellsstablyexpressingOMT1isoforms1andIlweretreated
withthetranslationinhibitorcycloheximide(20
J..lg/mL)for0,1,3,5,or7hours.Following
treatment,cellextractswerepreparedandequalamounts
oftotalproteinwereresolved
bySOS-PAGEfollowedbyimmunoblottingwithananti-HAantibody.
PrefacetoChapter7
TheworkdescribedintheprevioustwoChaptersofthisthesisfocusedonthe
subcellulardistributionofNramp2.
Inthesestudies,weadaptedordevelopedanumberof
toolsandtechniquestostudyproteinstability,steady-statetargeting,cellsurface
expression,endocytosiskinetics,andfateuponintemalizationofNramp2.
Inthenext
Chapter,wedecidedtousesorne
ofthesetechniquestocharacterizethesubcellular
targetingproperties
ofNrampl.ItwasknownforsornetimethatNramplisexpressedin
thelysosomes
ofphagocyticcellsandmorerecentdatasupportamodelwherebyNramp1
restrictsthereplication
ofintracellularpathogensbyremovingdivalentmetals(Mn2+,
Fe2+)fromthephagolysosome.However,targetingmotifsresponsibleforthelysosomal
localization
ofNramplwerenotyetknownandneededtobestudied.InChapter7,the
subcellulartraffickingproperties
ofNrampl,inc1udingcytoplasmicsequences
responsiblefortargetingtolysosomes,wereinvestigatedusingNrampl/Nramp2chimeric
proteins.
150
Chapter7:
Identificationofatyrosine-basedmotif(YGSI)in
theaminoterminus
ofNrampl(Slellal)
responsibleforlysosomaltargeting
151
ABSTRACT
Inmacrophages,Nrampl(SIc1lal)isexpressedinlysosomesandrestricts
replication
ofintracellularpathogensbyremovingdivalentmetals(Mn2+,Fe2+)fromthe
phagolysosome.
Nramp2(DMTl,Slclla2)isexpressedbothattheduodenalbrush
borderwhereitmediatesuptake
ofdietaryiron,andubiquitouslyattheplasma
membrane/recyc1ingendosomes
ofmanycelltypes,whereittransportstransferrin
associatedironacross
theendosomalmembrane.InNramp2,aC-terminalcytoplasmic
motif(YLLNT555-559)iscriticalforintemalizationandrecyc1ingofthetransporterfrom
theplasmamembrane.Inthisreport,wehavestudiedthesubcellulartrafficking
properties
ofNramp1andhaveinvestigatedthecis-actingsequencesresponsiblefor
targetingtolysosomes.Forthis,wehaveconstructedandstudiedNrampl/Nramp2
chimericproteinswherehomologousdomains
ofeachproteinwereexchanged.Chimeras
exchangingtheamino(upstream
TMl)andcarboxylterminal(downstreamTMl2)
cytoplasmicsegmentsofbothtransporterswerestablyexpressedinporcineLLC-PK
1
kidneycells,andwerestudiedwithrespecttoexpression,maturation,stability,cell
surfacetargeting,transportactivity,andsubcellularlocalization.
AnNramp2isoformII
chimerabearingtheNterminus
ofNramp1wasnotexpressedatthecellsurfacebutwas
targetedtolysosomes.Thislysosomaltargetingwasabolished
bysinglealanine
substitutionsat
y15and118ofaYGS115-18motifpresentintheNterminusofNrampl.
TheseresultsidentifyYGSIasatyrosine-basedsortingsignalresponsibleforlysosomal
targeting
ofNramp1.
152
INTRODUCTION
Nrampdefinesalarge,highlyconservedfamilyofintegralmembraneproteins
thattransportdivalentmetalsinapH-dependentfashion
41.Thefirstmemberofthis
family,
Nrampl(Slellal),wasidentifiedbypositionalcloningofalocus(Bcg/lty/Lsh)
thatregulatessusceptibilityofmicetoinfectionwithanumberofumelatedintracellular
pathogens
7.Naturallyoccurring(NramplG169D)orexperimentallyinduced(Nrampr/-)
loss-of-functionmutationsatNramplcausesusceptibilitytoinfectionwithseveral
species
ofMyeobaeteria,Salmonella,andLeishmania348;349.Polymorphievariationsat
human
NRAMP1havealsobeenlinkedtoincreasedsusceptibilitytotuberculosisand
leprosyinare
aswherethosediseasesarethemostprevalent112;117;350.Hydropathy
profilingandtopologicalstudiessuggestthatNrampliscomposed
of12putative
transmembranedomainsandisexpressedprimarilyinlysosomes
ofmononuclear
phagocytesandintertiarygranules
ofpolymorphonuclearleukocytes41.Upon
phagocytosis
ofinertparticlesoroflivepathogens,Nramp1israpidlyrecruitedtothe
membrane
ofmaturingphagosomes35;37-39.RecentworkhasshownthatNrampl
functions
asaphagosomalmetaleffluxpumpthattransportsdivalentcationssuchas
Mn2+,Fe2+inapH-dependentmannerdownaprotongradientcreatedbythevacuolarH+
ATPase27;52;94.NrampI-mediatedexclusionofessentialmetalsmayimpairmicrobial
metabolicactivityincludingexpression
ofintracellularsurvivalmechanisms,ormay
directlyenhancetheefficacyofbactericidaleffectormechanisms,orboth.
Nramp2(alsoknownasDMTl,Slella2)isaclosehomologofNrampl40;131,
with66%aminoacidsequenceidentityand77%similaritybetweenthe2proteins1.
Nramp2isexpressedabundantlyandinaniron-regulatedfashionatthebrushborderof
theduodenum161,whereitimportsdietaryironacrosstheabsorptiveepithelium.Nramp2
isalsoexpressedinmanycelltypesandisabundantinerythroidprecursors
169,whereitis
requiredforrecruitment
oftransferrin-associatedironfromrecyclingendosomesintothe
cytosol
251.ExpressionofNramp2hasalsobeendetectedatthebrushborderofepithelial
cellsliningtheproximaltubules
ofthekidney,whereitmayfunctionasare-uptake
systemfordivalentmetals
172.TwomajorNramp2proteinisoformsgeneratedby
alternativesplicingat
3’exonshavebeenidentified.Isoform1(+IRE)hasaniron
153
responsiveelement(IRE)inthe3’untranslatedregion,whereasisoformII(-IRE)lacks
theIRE.Inaddition,theC-terminal
18aminoacidsofisoform1arerep1acedbyan
altemate25-aminoacidsegmentinisoform
II.Nramp2isoform1ispredominantly
expressedinepithelialcenswhileisoformIIispredominantlyexpressedinerythroid
cens.Much
ofourknowledgeofthefunctionofNramp2invivocornesfromstudiesof
rodentmodelsofmicrocyticanemiaandirondeficiencyinc1udingthemkmouseandthe
Belgraderat,thatarebothcaused
bythesamemis-sensemutationinpredictedTM4of
Nramp2(G185R)136;141.Thisphenotypeisrecapitulatedinamousemutantwithtargeted
inactivation
ofNramp2(Nramp2-1-)258.Recently,twohumanpatientswithsevere
hypochromicmicrocyticanemiaandhepaticironoverloadwereshowntoharbor
mutationsinNRAMP2
4;263.Inbothpatients,aquantitativereductionintheexpressionof
functionalNramp2hasbeenidentifiedasthecauseofdisease260-262.
AlthoughNramplandNramp2codeforfunctionallyundistinguishablepH
dependentdivalentmetaltransporterswithsimilarsubstratespecificities
27,theydiffer
sharplyintheirsubcellularlocalizations.Nramp1isfoundstrictlyatthelysosomal
compartment
ofcenswithnoexpressionatthecellsurface35;39.Incontrast,Nramp2is
ubiquitouslyexpressedattheplasmamembraneaswellasinrecyclingendosomes
(isoformII)atsteady-state
42;161.Sequencemotifsfoundinthecytoplasmicterminal
regions
ofmembraneproteinsoftencontroltheirsubcellulartargetingandtrafficking
155;184.Themostcommonmotifsareeithertyrosine-basedsignaIsoftheformsNPXYor
YXX
motifs(LL)
183.ThesubcellulartraffickingofNramplisnotweIlunderstoodandspecifie
cytoplasmicmotifsinvolvedinitslysosomaltargetinghavenotyetbeenidentified.
However,wehavepreviouslyshownthatinsertion
ofahemagglutininepitope(HA)tag
inthefourthextra-cytoplasmicloop
ofNramplcausesthetransportertobemistargetedto
theplasmamembranewhereitdisplaysmetaluptakeactivity
27.Ontheotherhand,
traffickingsignaIsintheaminoandcarboxylcytoplasmicregions
ofNramp2havebeen
identifiedandcharacterized.Weandothershaveshownthata
YLLNT555-559motifinthe
Cterminus
ofNramp2isoformIIcontrolstargetingtotransferrin-positiverecyc1ing
endosomes
155;184.DeletionofthismotifortruncationoftheentireCterminal
154
cytoplasmicsegmentofNramp2isoformIIresultsinamutantproteinthatisintemalized
lessrapidlyfromtheplasmamembrane,isnotproperlyrecyc1edback
tothecellsurface,
andistargetedtolateendosomesandlysosomes(32).Mutagenesisexperimentshavealso
shownthatmotifsintheNterminus
ofNramp2(NPAY,YSCF)playanadditionalbut
lessdeterminantroleintherecycIingendosometargetingofNramp2
184.
Inthisstudy,wehaveinvestigatedthesubcellulartargetingandtrafficking
properties
ofNrampl.Forthis,wehaveconstructedchimericproteinsbyexchanging
homologoussegments
ofNramp1andNramp2(isoformII).Weexpressedthechimeras
instablytransfected
LLC-PK1cellsandstudiedtheirexpression,stability,cellsurface
expression,metaltransportactivity,andsubcellularlocalization.Wedemonstratethata
tyrosine-basedsortingsignal
oftheformYXXfunctionsasalysosomaltargetingmotif
intheaminoterminus
ofNrampl.
155
MATERIALSANDMETHODS
Materialsandantibodies
Reagent-gradechemicalswerepurchasedfromSigmaChemical(St.Louis,MO)
unlessotherwiseindicated.Monoclonalmouseantibody(Ab)HA.11directedagainstthe
influenzahemagglutininepitope(HA)waspurchasedfromCovance(Princeton,NJ).
Cy3-labeledgoatanti-rabbitandHRP-coupleddonkeyanti-mouseAbswerepurchased
fromJacksonImmunoResearchLaboratories(WestGrove,PA).PlasmidsencodingGFP
fusionproteinswerekindgiftsfromDr.
D.Williams(DepartmentofBiochemistry,
University
ofToronto;GFP-syntaxin13)andDr.PatriceBoquet(Institutnationaldela
santéet
delarecherchemédicale,France;GFP-Lampl).Thegenerationofpolyclonal
antibodiesrecognizingtheaminoterminalsegments
ofmurineNramp1andNramp2
proteinshasbeendescribed
35;42.
Plasmidsandconstructs
TheconstructionofmammalianexpreSSIOnplasmidscontainingfull-Iength
murinecDNAsforeither
Nrampi(Nl/pCB6)orNramp2isoformII(N2/pCB6)were
describedearlier
26.Full-IengthmurineNramp2cDNA(N2HAlpCB6)wasmodifiedby
thein-frameaddition
ofanexofacialhemagglutinin(HA)epitope(YPYDVPDYAS)in
thefourthpredictedextracellularloop,
aspreviouslydescribed26.Arecombinant
polymerasechainreaction
(peR)protocolwasusedtogenerateNramp2-HAchimeras
bearingtheamino(NIN-HA)orcarboxylterminal(NIC-HA)segments
ofNrampi.
Similarly,Nrampichimerasbearingtheamino(N2N)orcarboxylterminal(N2C)
segments
ofNramp2werecreated.ForNiN-HAandN2Nconstructs,asilentBstBI
restrictionenzymesitewasintroducedintoN2HAlpCB6andNl/pCB6plasmids(at
nucleotidepositions200and155,respectively)immediatelyprecedingthefirstpredicted
transmembranedomain
ofeachproteinusingprimersN2-BstBIandNl-BstBI(Table1).
NIN-HAlpCB6andN2N/pCB6constructswerethencreatedbyexchangingXhoI-BstBI
fragmentsofN2HAlpCB6andNl/pCB6plasmids.NIC-HAandN2Cconstructswere
createdbyarecombinantPCRamplificationprotocolusingchimericoligonucleotides
N2HA-NICandNl-N2C(Table
1),showingcomplementaritywithNIandN2atthe
156
Table1:Oligonucleotidesusedformutagenesis.
PrimerName
N1-BstBIF
N2-BstBI
F
N1-N2CTF
N2-N1CTF
N1Y15AF
N1G16AF
N1S17AF
N1118AF
Nucleotidesequence(5’to3′)
cattcagcctTCgAaagctgtgg
ttagctttcgAaaactctggg
gttgcatcgccTTGGGTCTGTC
gtttgattgcaCACGGAGCCAC
gcaggcccagtGCtggctccatttc
gcccagttatgCctccatttccag
ccagttatggcGccatttccagc
gttatggctccGCttccagcctgcc
Findicatesforward.CAPITALLETTERSindicatenucleotides
differingfromthetemplatemolecule.The
BstBIrestrictionsiteis
underlined.
157
boundariesofregionstobeexchanged.TheN2HA-N1Camplificationproductwas
insertedintoN2HNpCB6usingrestrictionenzymesites
Sac!andEcoRitogenerate
N1C/pCB6.The
Nl-N2CamplificationproductwasinsertedintoNlIpCB6using
restrictionsites
AvrIlandEcoRitogenerateN2C/pCB6.Alaninesubstitutionmutations
attheYGSI
I5
-18motifofNramp1werecreatedusingmutagenicprimerslistedinTablel.
MutantswereintroducedintoN1N-HNpCB6usingrestrictionenzymesitesXho!and
BstB!.TheintegrityofallmutantandchimericcDNAswasverifiedbyDNAsequencing.
Cel!culture,transfection,andimmunoblotting
LLC-PK1cellswereculturedaspreviouslydescribed184andtransfectedwith
eitherNlIpCB6,N2HNpCB6,orchimericplasmidsusingLipofectamine2000(Gibco)
accordingtoinstructionsfromthemanufacturer.Stablytransfectedcellcloneswere
obtainedafter
14daysofselectioninmediumcontainingG418(l.4mg/mL;Invitrogen),
andindividualcoloniesweresubsequentlypickedandexpanded.Totalcelllysateswere
preparedandresolvedbySDS-PAGE.Clonesshowingrobustexpression
ofthe
transfectedconstructswereidentifiedbyimmunoblottingwitheitheramouseanti-HA
Ab
(forN2HNpCB6,N1N-HNpCB6,andN1C-HNpCB6),arabbitanti-Nramp2NTAb
(N2N/pCB6),orarabbitanti-Nramp1NTAb(N1/pCB6,N2C/pCB6),aspreviously
described
184.Forexperimentswithcyc10heximide(CHX),cellswereincubatedin
growthmediasupplementedwith20
/-lg/mLCHXfortheindicatedtimeintervalspriorto
lysisandSDS-PAGE.
Metaltransportassay
MeasurementofmetaltransportintransfectedLLC-PK1cellswascarriedout
usinga
ca1ceinfluorescencequenchingassay,aswehavepreviouslydescribed26;28.
Calceinacetoxymethylester(calcein-AM,MolecularProbes)stocksolutionswere
preparedindimethylsulfoxideandcellswereloadedatafinalconcentration
of0.25/-lM
calcein-AM.C02+solution(cobaltchloride,2mM)waspreparedfreshindeionized
water.Initialrates
ofmetaltransportwerecalculatedfromtheinitialfluorescence
quenchingcurves(34;35).
Cel!surfacebiotinylation
158
LLC-PKIcellmonolayerswererinsedtwicewithice-coldphosphate-buffered
saline(PBS)andoneewithiee-eoldboratebuffer(10.0mMborieaeid,154mMNaCl,
7.2
mMKCl,1.8mMCaC12,pH9.0),andthenincubated(60minutes,4°C)inthesame
buffercontainingSulfo-NHS-SS-biotin
(1mg/mL;Pierce).Unreactedbiotinwas
removedby3washeswithRPMImedium.Biotinylatedcellswerecollectedand
solubilizedinlysisbuffer
(1%TritonX-100,0.2%SDS,50mMTris-HClpH7.4,150
mMNaCl,20%glycerol)withproteaseinhibitors.Lysateswerepre-clearedby
centrifugation(10
OOOg,30minat4°C)andproteinyieldwasquantifiedbyBradford
assay(Bio-Rad).Biotinylatedproteins(100
/-lgtotalproteinlysate)wereisolatedby
ovemightincubationat4°Cwith50
/-lLofImmunoPureimmobi1izedstrepavidinslurry
(Pierce)inafinalvolume
of1mLinlysisbuffer.Strepavidinbeadswerewashedsix
timeswithcoldlysisbufferandboundproteinswereelutedwith2xLaemmlibufferat
roomtemperaturefor30min.ProteinswereseparatedbySDS-PAGEfollowedby
immunoblotting.
QuantificationofcellsurfaceexpressionbyELISA
QuantificationofNramp2-HAmoleculesexpressedatthecellsurfacebyELISA
wasaspreviouslydescribed
184.Briefly,LLC-PK1cellsweregrowntoconfluencyin48-
wellcultureplatesandfixedwith4%paraformaldehydefor30min.Cellswereblocked
in5%non-fatmilkinPBSfor30min,incubatedwithanti-HAAb
(1:500)for90min,
washedandincubatedwithsecondaryAb(donkeyanti-mouse-HRPAb,1:4000)for1h.
Forquantification
oftotalNramp2-HAexpression,cellswerepermeabilizedby
incubationwith
0.1%TritonX-1001PBSfor30minpriortoincubationwithanti-HAAb.
PeroxidaseactivitywasdetectedbyincubatingcellswithHRPsubstrate(0.4mg/mL
0-
phenylenediaminedihydrochloride,SigmaFASTOPD)accordingtotheinstructionsfrom
themanufacturer.Reactionswerestoppedafter30minwith3MHClandabsorbanee
readings(492nm)weretakenwithaspectrometer.Foralldeterminations,background
absorbancereadingsfrom(i)non-specificbinding
ofsecondaryAb,and(ii)non-specific
binding
ofprimaryAbtovector-transfectedcells,weresubtractedforeachsample.Cell
surfacereadingswerenormalizedtototalNramp2-HA’valuesforeachcellcloneand
wereexpressed
asapercentage.
159
Immunofluorescence
LLC-PK1cellsstablyexpressingNramp1,Nramp2-HAorchimericproteinswere
transientlytransfectedwithGFP-fusionproteinstolabelrecyc1ingendosomes(GFP
syntaxin13)orlateendosomesandlysosomes(GFP-Lamp1).Twentyfourhourslater,
censwerefixedwith4%paraformaldehyde(inPBS)for20minandwereblockedand
permeabilizedwithaPBSbuffercontaining0.2%saponinand5%non-fatmilk(30min).
Anantibodiesweredilutedinblockingsolutionandincubationswereperformedfor1hat
roomtemperature.censstablyexpressingN2HA,N2N,andNIC-HAproteinswere
labeledwithanti-Nramp2NTAb(1:200),whilecensexpressing
NI,NIN-HA,andN2C
proteinswerelabeledanti-Nramp1NTAb(1:400).
Ancensweresubsequentlyincubated
withagoatanti-rabbitsecondaryAbcoupledtoCy3.
censwerevisualizedusingan
Axiovert200Mepi-fluorescencemicroscopewitha
lOOxoilimmersionobjective.Digital
imageswereacquiredwithaZeissAxioCamHRmcameraoperatedwithAxioVision4.3.
Imageswerecropped,assembledandlabeledusingAdobePhotoshopandIllustrator
softwares.
160
A
N
terminus:
Nrarnp2
MVLDPKEKMPDDGASGDHGDSASLGA~NSSLPHSTGDSEEPFTTYFDEKIPIPEE~FRK~~
Nrarnpl
MISDKSPPRLSRP~SLPGPAPQPAPCRETYLSEKIPIPSADQGTFSLRKY~
F”
1Pd”tdtff”
k”
t”f
~
Igure
“re
IC
era
IC
mg
mo
1s
C
terminus:
and
design
of
Nramp1/Nramp2
chimeric
Nrarnp2
GBD-~LGLSFLDCGRSYRLGLTAQPElmnJmvDADSVVSR
proteins”
(A)Sequences
of
theamino(N)
N1
~
andcarboxyl(C)terminalregions
of
rarnp
GD-
HGATFLTHSSHKHFLYGLPNEEQGGVQGSG
N1dN2h.
1d·
rampanramparesown,
!nC
u!ng
~Anti-HAAb
y
Anti-Nramp2
Ab
y
Anti-Nrampl
Ab
y
Glycosylation
thelocations
of
predictedtargetingmotifs
(highlightedblack),transmembrane
domains1(TM1)and12(TM12),andthe
locations
of
thesequenceboundariesfor
thechimeras(scissors).
In
Nramp1,amine
acidresidues1-54and519-548were
considered
as
theNandC-terminal
regions,respectively.
In
Nramp2,residues
1-69and533-568wereconsideredasthe
NandC-terminalregions,respectively.
(8)
Nramp1/2chimeraswerecreatedby
exchangingtheaminoandcarboxyltermi
naisegments
of
Nramp1andNramp2.
Schematicrepresentationsareshown
of
wild-typeNramp2-HA(gray),wild-type
Nramp1(black)asweilasthefourchime
rascreated.Thepositions
of
antigenic
epitopesrecognizedbytheanti-HA,anti
Nramp1andanti-Nramp2antibodiesas
weilastheputativeN-linkedglycosylation
sites(Nramp1:N321,N335;Nramp2:
N336,N349)areindicated.
RESULTS
ConstructionandexpressionofNramplandNramp2chimericproteins
TobetterunderstandthetraffickingofNrampproteins,wecreatedchimeric
moleculesbyexchangingthepredictedcytosolicaminoandcarboxylterminalsegments
ofNramp1andNramp2.Nramp2chimerascontainingeithertheamino(NIN-HA)or
carboxyl(NIC-HA)predictedcytosolicsegments
ofNramp1wereconstructedusingthe
Nramp2isoform
II(non-IRE)backbonebearinganexofacialhemagglutinin(HA)tagthat
allowedforrecognition
ofcellsurfacemoleculesinintactcells(FigurelB).Wehave
previouslyshownthatinsertion
ofthisHAtaginthefourthpredictedextra-cytoplasmic
loop(EC4)
ofNramp2doesnotaffectitsexpression,activity,orsubcelIularlocalization
51.Nramp1chimerascontainingeithertheamino(N2N)orcarboxyl(N2C)predicted
cytosolicsegments
ofNramp2isoformII(non-IRE)wereintroducedintoanunmodified
Nramp1proteinbackbone(FigurelB).WehavepreviouslyshownthatanNramp1
moleculemodifiedbyinsertion
ofanHAtaginEC4ismis-targetedtotheplasma
membrane
oftransfectedCHOcelIs27,precludingtheuseofthatsiteforepitopetag
insertionforthepresentstudies.AlINramp1andNramp2chimeraswereintroducedinto
LLC-PK
1porcinekidneycellsandcellclonesstablyexpressingindividualvariantswere
selectedandexpandedforanalysis.Figure2Ashowsatypicalimmunoblot
ofextracts
preparedfromcellstransfectedwitheither
Nramp2,NramplorchimericcDNAs.
ImmunoblotswereprobedseparatelywithantibodiesagainsttheHAtag,theamino
terminus
ofNramp2,andtheaminoterminusofNramp1.Wild-type(WT)Nramp2was
detected
astwoimmunoreactivespecies,aminorspeciesat~60kDaandmajorspeciesat
~90-105kDa(Figure2A).Previousstudiesfromourgrouphaveshownthattheminor
speciescorrespondstotheprecursor”coreglycosylated”proteinwhilethemajorspecies
correspondstothemature”complexglycosylated”forms
oftheprotein51.Interestingly,
NIN-HAwasdetected
astwospeciesoffasterelectrophoreiticmobilitythanWT
Nramp2,indicatingasignificantdifferenceintheextent
ofcoreandcomplex
glycosylationinthischimericprotein(Figure2A).Thisresultsuggeststhatsequencesin
theaminoterminusofNramp2areimportantforproperglycosylation
ofthetransporter.
162
A
..-~0…
B«l,NE~z«l1Z,….z«l1UZ
,….
,….
~q..
CHX
treatment
(h)
~q..
CHX
treatment
(h)
u
~——~
:::l
ra
M
L.f)
~
~
~
–…._–”
…J
a
M
L.f)
r-…
Figure
2.
Expressionand
stability
of
Nramp1/2chimerasintransfectedLLC-PK1cells.
Totalcellextracts
trom
untransfectedcells(LLC-PK1)andcellclonesstably-‘expressingwildtypeNramp1(Nramp1),wildtypeNramp2
(Nramp2-HA)andtheNramp1/2chimeras(N1N-HA,N1C-HA,N2N,N2C)wereresolvedbySDS-PAGEandanalyzedby
immunoblotting.(A)ImmunoblotswereincubatedwithantibodiesrecognizingeithertheHAepitope(anti-HA),theamino
terminusofNramp2(anti-Nramp2),ortheaminoterminus
of
Nramp1(anti-Nramp1).Thesizes
of
molecularmassstan
dards(inkilodaltons)areindicated.Inpanel(8),transfectantsexpressingtheindicatedproteinsweretreatedwiththe
translationinhibitorcycloheximide(20Jlg/mL)for
0,
1,
3,
5,
or
7hours.ImmunoblottingoftotalcellIysateswascarried
outtoassessthestabilityofwild-typeandchimericproteins.Antibodiesusedforeachblotareindicated.
N1C-HAwasdetectedastwoimmunoreactivespeciesofelectrophoreticmobilitysimilar
tothat
ofWTNramp2.However,theproportionoftheimmatureprecursorNIC-HA
protein
(~60kDa)appearedsignificantlygreaterthanthatfoundforWTNramp2,
suggestingpossibleincompletematuration
ofthischimera(Figure2A).WTNramp1as
weIlaschimerasN2NandN2C,weredetectedassingleimmunoreactivespeciesof~70-
75kDa(Figure2A,bottompanel).LLC-PK1cellsexpressingthedifferentWTand
chimericproteinsweretreatedwiththetranslationinhibitorcycloheximidefordifferent
periods
oftimeandthefateofcoreglycosylatedandmatureproteinisoformswas
analyzedbyimmunoblotting(Figure2B).TheseexperimentsshowedthatWTNramp2
andNrampI,
aswellasthechimerasNIN-HA,N2N,andN2Cquicklymaturedfroma
coreglycosylatedprecursortoafullymatureformwhichremainedstablefora7-hour
period.Interestingly,theNIC-HAchimeradisplayedincreasedpersistence
ofprecursor
andreducedstability
ofthematureformcomparedtocontrolswithlittleprotein
detectableat7hours(Figure2B).Theseresultssuggestimpairedprocessing
oftheNIC
HAchimera.
Cel!surfaceexpressionofchimericproteins
Weusedacellsurfacebiotinylationtechniquetoquantifyplasmamembrane
expression
oftheNramp1l2chimerasatsteady-state184;262.LLC-PK1cellsstably
expressingeitherWTorchimericNrampconstructsweretreatedwithamembrane
impermeablebiotincompoundtolabelallcellsurfaceproteins.Aftersolubilization,cell
surfaceproteinswereisolatedwithimmobilizedstrepavidin,followedbyseparationby
SDS-PAGEandvisualizationbyimmunoblotting(Figure3A,
Surface).Cellsurface
expression
ofeachproteinwasthencomparedtototalproteinexpressionforeachcell
line(Figure3A,
Total).WTNramp2-HAandNIC-HAweredetectedatrobustlevelsat
theplasmamembranewhileWTNrampI,NIN-HA,N2N,andN2Cdisplayedlittleor
no
cellsurfaceexpression(Figure3A).Clearly,replacingeithertheamino(N2N)or
carboxyl(N2C)termini
ofNramp1withtheequivalentsegmentsofNramp2didnot
resultinplasmamembraneexpression.BecauseNramp2-HA,NIN-HA,andNIC-HA
wereconstructedwithexofacialHAtagsinpredictedEC4,thefraction
ofeachvariant
present
atthecellsurfaceatsteady-statecouldbedeterminedbyexposingfixedLLC-PKI
164
«I
A
..-
1
«
..-
«
~
N
~
..-
a.
Q.I
a.
Q.I
1
E
1
Z
1
E
1
()
ca
()
()
ca
Z
–l
….
..-
N
–l
….
..-
i)1
–l
z
Z
z
1ii)
–l
ZZ
B
.IF
«
50%,
:c1~40%EroZ
30%
Q)0-ê
20%
~Cf)Q)
10%
Ü
0%_
««ll11N
Z
Cl..
..-
E
z
~z
•«l1Ü..-z
C
0.0125
0.0100(IJ+-‘fi.
0.0075
..t::.uc:(IJ~0
…..
…..
z
z
Figure
3.
Cell
surface
expression
and
metal
transport
activity
of
the
Nramp1/2
chimeras.
(A)Cellsurfacebiotinylation
was
usedtoassessplasmamembraneexpression
of
wildtypeNramp1(Nramp1),wildtypeNramp2(Nramp2-HA)andtheNramp1’2
chimeras
(N1
N-HA,
N1
C-HA,N2N,N2C).Livecellswerelabeledwithamembraneimpermeablebiotincompound(see”Materials
andMethods”).Totalcellproteinextractswerepreparedandbiotinylatedproteins
were
isolatedbyaffinitycapturewithstreptavidin
agarosebeads.Equivalentamounts
of
totalcellextracts(Total)andcapturedbiotinylatedprotein(Surface)wereresolvedby
SDS
PAGEfollowedbyimmunoblottingwithanti-Nramp2(i)
or
anti-Nramp1(ii)polyclonalantibodies.(B)Thefraction
of
Nramp2-HA,
N1
N-HA,andN1C-HAexpressed
at
thecellsurface
was
quantifiedusingtheexofacial
HA
epitopetaginsertedinpredictedextra
cytoplasmicloop4
of
thoseproteins.Briefly,cellswerefixedandincubatedwithprimaryanti-HAantibodywith
or
withoutprior
detergentpermeabilizationfollowedbyincubationwithanHRP-coupledsecondaryantibodyandquantificationbyacolorimetrie
assay.Theamount
of
Nrampproteinsexpressedatthecellsurface(innon-permeabilizedcells)isshownasafraction(%)
of
total
proteinexpression(inpermeabilizedcells).Inpanel(C),metaltransportactivity
of
theNramp1/2chimeras
was
determinedusinga
fluorescencequenchingassay.Cellsloadedwithametal-sensitivefluorescentdyewereincubatedwithC02+inacidicbuffer.
Resultsareshownastheinitialrates
of
fluorescencequenching.Errorbarsrepresentstandarderror
of
themeans
of
three
or
more
independentdeterminations.
transfectantstoanti-HAantibody,with(totalexpression)orwithout(cellsurface)prior
permeabilizationwithdetergent.Theamount
ofboundanti-HAantibodywasquantified
usingasecondaryantibodycoupledtohorse-radishperoxidase.Wedeterminedthat35.9
±3.9%(mean±S.E.)ofWTNramp2-HAand21.3±1.3%ofNIC-HAwereexpressedat
thecellsurfacecomparedtoamodest
0.5±1.2%forNIN-HA(Figure3B).Thesevalues
areconsistentwithceIlsurfacebiotinylationresults(Figure3A)andsuggestthatthe
aminoterminus
ofNramplimpairsnormaltargetingofNramp2totheplasmamembrane.
Nramp2isoformIIisnormallyexpressedattheplasmamembraneandin
recyc1ingendosomesinLLC-PK
1ceIls.ReplacingthecarboxylterminusofNramp2-HA
withtheequivalentsegment
ofNrampl(NIC-HA)didnothaveamajoreffectontheceIl
surfaceexpression
ofthetransporter(Figure3A,3B).Interestingly,despitetheimpaired
processingandreducedstability,NIC-HAretainedsignificantmetaltransportactivity
(Figure3C).Thisactivitylikelystemsfromthesignificantfraction
ofmatureNIC-HA
proteinthatisexpressedatthecellsurface.Ontheotherhand,replacingtheamino
terminus
ofNramp2-HAwiththeequivalentsegmentfromNrampl(NIN-HA)
drasticallyreducedsurfaceexpression(Figure3A,3B)
asweIlasmetaltransportactivity
(Figure3C).Thisresultsuggeststhattheaminoterminus
ofNramplcontainstargeting
informationthataffectsthenormaltraffickingofNramp2totheplasmamembrane.
Subcellularlocalizationofchimericproteins
Immunofluorescencewasusedtocomparethesubcellularlocalizationofwild
typeandchimericproteinsatsteady-state.LLC-PKItransfectantswerefixed,
permeabilized,andchimericproteinswerelabeledwithpolyc1onalantibodiesrecognizing
eitherNramplorNramp2.Consistentwithourpreviouslypublisheddata
51;184,Nramp2-
HAdisplayedstrongcolocalizationwithGFP-syntaxin13,whileNramplshowedlittle
overlapwiththerecyc1ingendosomemarker(Figure4A).Interestingly,
aIlchimeras
showed
nosignificantcolocalizationwithGFP-syntaxin13(Figure4A).Theseresults
suggestthatsubstitution
ofeithertheaminoorcarboxylterminusofNramp2by
homologoussegments
ofNramp1impairstargetingtotherecyc1ingendosome
compartment.
166
A
GFP-Syn13Merge
B
Figure4.Subcellular
localization
of
Nramp1/2
chimeras.LLC-PK1cells
stablyexpressingwildtype
Nramp1(Nramp1).wild
typeNramp2(Nramp2-HA)
andtheNramp1/2chimeras
(N1N-HA.N1C-HA,N2N,
N2C)weretransiently
transfectedwitheither
GFP-syntaxin13
tolabel
recyclingendosomes(A)or
GFP-Lamp1tolabellate
endosomesandlysosomes
(8).Twenty-fourhourslater,
cellswerefixed,permeabi
lizedandstainedwitheither
anti-Nramp1.oranti
Nramp2polyclonalantibod
iesfollowedbyasecondary
antibodycoupledtofluores
centCy3.Imageswere
acquiredbyepifluores
cencemicroscopy.Insets
showmagnifications
of
the
areaboxedinthefigure.
Consistentwithpreviouslypublisheddata35,Nramp1showedstrong
colocalizationwiththelysosomalmarkerGFP-LamplwhileNramp2-HAdisplayedlittle
colocalizationwiththismarker(Figure4B).TheseresultsconfirmthatNramplis
properlytargetedtothelysosomalcompartmentinLLC-PK
1cells.Remarkably,all
chimerasdisplayedsignificantcolocalizationwithGFP-Lampl(Figure4B),indicating
theirpresenceinlateendosomesandlysosomes.Clearly,replacingtheaminoorcarboxyl
termini
ofNramplwiththeequivalentregionsofNramp2(N2N,N2C)didnot
significantlyaffectthesubcellulardistribution
ofNramp1atsteady-state(Table2).
PreviousworkbyusandothershavedemonstratedthattheCterminus
ofNramp2
isoform
IIcontainstargetinginformation(includingaYLLNTsignal)crucialfor
endocytosisandrecycling
ofthetransporterfromtheplasmamembrane155;184.Mutations
ordeletionsinthismotifresultinaproteinthathasimpairedrecyclingfollowing
intemalizationfromthecellsurfaceandaccumulatesinthelysosomalcompartment
184.
Consistentwiththesefindings,replacingtheCterminusofNramp2withtheequivalent
segmentfromNrampl(NIC-HA)resultedinplasmamembraneexpression(Figure3)
andlysosomaltargeting(Figure4B,Table2).Therefore,themistargetingdisplayedby
NIC-HAisprobablyduetoremovaloftheCterminusofNramp2ratherthanthe
presence
ofadominanttargetingsignalintheCterminusofNramp1.AfractionofNIC
HAshowedsornecolocalizationwiththeendoplasmicreticulummarkerGFP-Sec61(data
notshown),suggestingthatsorneNIC-HAmayberetainedintheER.Finally,
substitutingtheaminoterminus
ofNramp2withtheequivalentsegmentfromNramp1
(NIN-HA)yieldedthemostintriguingresult.
NIN-HAshowedsubcellularlocalization
indistinguishablefromWTNramp
l,withnosignificantsurfaceexpression(Table2)but
stronglysosomaltargeting(Figure4B).Theseresultssuggestthattheaminoterminus
of
Nramp1possesseslysosomaltargetinginformationthatcanactinadominantfashion
overtherecyclingendosomesignalpresentintheCterminusofNramp2isoform
II.
CharacterizationoftheYSGImotifintheNterminusofNrampl
CloserexaminationofthepredictedcytoplasmicNterminalregionofNramp1
revealedaYGSI
I5
-18motifthatfitstheYXX<1>consensussignature(FigurelA).YXX<1>
motifshavebeenimplicatedintargetingmembraneproteinssuchasLAMP-Iand
168
Table2:SubcellularlocalizationofNramp1/2chimeras
Nramp2-HA
N1N-HA
N1C-HANramp1
N2N
N2C
PM
+
+
169
RE
+
LE/Lys
+
+
+
+
+
ER
+
A..-«~a..I11()z…J..-…JZ
108
90
B30%
«I1Z
Z20%
~
-ê::J
CI)10%
ID()
,
«LO..->-
N1N-HA
ti§««………-..-ex>(!)en..-
C0.0150
0.0125
Q)+-‘
&0.0100..r:.u
~0.0075::Jo
0.0050
0.0025
o
Figure5.CharacterizationofN1N-HAtargetingmotifmutants.(A)TotalceU
extractswerepreparedfromtransfectantsstably-expressingindividualmutants
(Y15A,G16A,S17A,and118A)intheputative
YGSllysosomaltargetingmotif
presentintheN1N-HAbackbone.Equalamounts
ofceUextractwereresolvedby
SOS-PAGEandanalyzedbyimmunobloUingwithanti-HAantibody.Thesizes
of
molecularmassstandards(inkilodaltons)areindicated.Inpanel(8),thefraction
ofN1N-HAexpressedattheceUsurfacewasdeterminedasdescribedinthe
legend
ofFigure38.(C)MetaltransportactivityoftheN1N-HAtargetingmotif
mutants
wasmeasuredbyquenchingofcalceinfluorescence,asdescribedinthe
legend
ofFigure3C.Errorbarsrepresentstandarderrorofthemeansofthreeor
moreindependentdeterminations.
LAMP-2tolysosomesbyinteractingwiththeIlsubunitsofAP-IandAP-2complexes
183;331;332;351;352.TodetermineiftheYGSImotifintheNterminusofNramplwas
responsibleforthelysosomaltargeting
oftheNIN-HAchimera,wecreatedalanine
substitutionsateachposition
oftheYGSIsignalwithintheNIN-HAchimericconstruct.
ThemutantswerestablytransfectedintoLLC-PK
1cellsandclonespositivefor
expressionwereselectedforanalysis.Immunoblotanalysisperformedoncellextracts
showedstableexpressionofY15A,GI6A,S17Amutantsatlevelscomparable
toNIN
HA,althoughlowerexpressionwasdetectedfor
Il8A(Figure5A).Wedeterminedthe
fraction
ofeachNIN-HAexpressedatthecellsurface.Mutatinganyoftheresiduesin
theYGSImotif
toalanineincreasedthesurfaceexpressionofthechimera(Figure5B).
Y15Ademonstratedthehighestsurfaceexpression(19.2±2.0%)followedbyS17A(10.2
±1.2%),Il8A(7.4±2.1%),andG16A(4.4±0.3%).Thisincreaseinsurfaceexpression
wasconcomitanttoacommensurateincreaseinmetaltransportactivityforthemutants
(Figure5C),implyingthatNIN-HAisindeedproperlyfoldedinatransportcompetent
manneratthecellsurface.
WedeterminedthesubcellularlocalizationoftheNIN-HAmutantsatsteady-state
lUfixedandpermeabilizedcells.MutantsG16AandS17Adisplayedsubcellular
localizationsimilar
toNIN-HA,showinglittlecolocalizationwiththerecycling
endosomemarkerGFP-syntaxin
13(Figure6A)butstrongcolocalizationwiththe
lysosomalmarkerGFP-Lampl(Figure6B).TheseresultssuggestthatG16andS17are
notessential
forthelysosomaltargetingofNIN-HA.Strikingly,mutantsY15AandIl8A
displayedsubcellularlocalizationsimilartoWTNramp2,showingstrongcolocalization
withsyntaxin13-positiverecyclingendosomes(Figure6A)butlittlecolocalizationwith
Lampl-positivelysosomes(Figure6B;summarizedinTable
3).Theseresultssuggest
that
Y15and118arecriticalforthelysosomaltargetingofNIN-HAandthattheYGSI
motifintheNterminus
ofNramplfunctionsasaYXX<1>lysosomaltargetingsignal.
Furthermore,thisYGSIsignalappearstodominatetherecyclingendosomesignalpresent
intheCterminusofNramp2isoform
II.
171
A
B~..->-~..-<9«r-..-CI)«00
Figure6.Subcellularlocalization
of
N1N-HAmutants.
LLC-PK1cellsstablyexpressingN1N-HAYGSImutantswere
transientlytransfectedwitheithertherecyclingendosomemarkerGFP-syntaxin13(A)orthelateendosomalandIyso
somalmarkerGFP-Lamp1(8).Twenty-fourhourslater,cellswerefixed,permeabilizedandstainedwithananti-Nramp1
polyclonalantibodyfollowedbyasecondaryantibodycoupledtofluorescentCy3.Imageswereacquiredbyepifluores
cencemicroscopy.Insetsshowmagnifications
of
theareaboxedinthefigure.
Table3:SubcellularlocalizationofN1N-HAmutants
PMRELE/LysER
N1N-HA+
Y15A++
G16A++
S17A+
+
118A++
173
DISCUSSION
Inthisstudy,wesoughttoinvestigatethesubcellulartargetingandtrafficking
properties
ofNramp1,inc1udingtheidentificationofsortingsignaIsresponsibleforits
distinctlocalizationinlysosomes
ofmononuc1earphagocytes.Forthis,weconstructed
chimericproteinsinwhichhomologousdomains
ofNramp1andthec1osely-relatediron
transporterNramp2(isoform
II)wereexchanged.Replacingtheamino(N2N)orcarboxyl
(N2C)terminalcytoplasmicsegments
ofNramp1withtheequivalentsegmentsfrom
Nramp2didnotdrasticallyaffecttheexpressionorsubcellularlocalization
ofthe
transporter.Indeed,bothN2NandN2Cchimerasexhibitedlittlecellsurfaceexpression
butshowedstronglysosomaltargeting,typical
ofWTNramp1.Theseresultssuggested
thatsortingsignaIsintheaminoandcarboxylcytoplasmictermini
ofNramp2areunable
toconfercellsurfaceand/orrecyc1ingendosometargetingtotheNramplbackbone.
ResultswiththeN2Nchimeraaresupportedbyourpreviousworkshowingthatde1etion
mutationsatputativesortingsignaIsintheNterminus
ofNramp2donotsignificantly
affectthecellsurfaceorrecyc1ingendosometargeting
ofthetransporter184.Incontrast,
weandothergroupshaveshownthataYLLNTmotifintheCterminus
ofNramp2
isoform
IIcontrolstheprotein'sintemalizationandrecyc1ingfromplasmamembrane
155;184.However,resultswiththeN2CchimerasuggestedthatthisYLLNTmotifwas
somehowmaskedornon-functionalwhenplacedonthebackground
ofNramp1.
AnNramp2-HAchimerabearingtheCterminalcytoplasmicsegmentofNrampl
(NIC-HA)wasrobustlyexpressedinLLC-PK
1cellsbutdisplayedahigherfractionofa
~60kDaprecursorspeciesandalowerfractionofthe~90kDamatureprotein,suggesting
impairedprocessing
ofthischimera.NIC-HAalsodisplayedreducedstability.Surface
biotinylationexperimentsrevealedthatthemature,fullyglycosylated
NIC-HAwas
expressedatthecellsurface(Figure2A)andwaslikelyresponsiblefortheresidualmetal
transportactivityobservedforthisconstruct(Figure2C).Thisresultsuggeststhat
complexglycosylationacquiredbytheprecursorproteinintheGolgiapparatusiscritical
fortheplasmamembranetargeting
ofmatureNramp2.Strikingly,themultiple
biosyntheticandfunctionaldefects
oftheNIC-HAchimeravirtuallymirrorthose
observedfortheNramp2
G185RmutantproteinproducedinmkmiceandBelgraderats148.
174
WhileasrnallfractionofNIC-HAdisplayedsornecolocalizationwiththeERmarker
GFP-Sec61(datanotshown),amoresignificantfraction
ofthechimerawasexpressedin
lateendosomesandlysosomes.Wehavepreviouslyshownthatcriticalmutations
attheC
terminus
ofNramp2isoformII(includingatruncationoftheentireCterminalsegment)
resultsinaproteinthatisunabletorecycleafterinternalizationfromthecellsurfaceand
istargetedtothelysosomesbydefault
184.Therefore,wereasonedthatthelysosomal
targetingdisplayedbyNIC-HAislikelytheresultfromtheremoval
oftheCterminusof
Nramp2ratherthanthepresenceofadominanttargetingsignalintheCterminusof
Nrampl.
AnNramp2chimerabearingtheNterminusofNrampl(NIN-HA)yielded
perhapsthemostintriguingresults.NIN-HAwasstablyexpressedintransfectedLLC
PK1cellsasaproteinoflowerapparentmolecularweightcomparedtoNramp2-HA
(Figure2A),probablyduetoalteredglycosylation
ofthechimera.futerestingly,theextent
ofglycosylationofNIN-HAresembledmorec10selythatofNrampl(Figure2A)and
raisesthepossibilitythattheNterminus
ofNramp2containssequenceinformationthat
affectstheextent
ofglycosylationofthetransportereitherdirectlyorindirectlyby
mediatingitstraffickingandretentiontospecificsubcellularcompartments.Strikingly,
NIN-HAalsodisplayedsubcellulartargetingpropertiessirnilartoNrampI,withno
surfaceexpressionyetstronglysosomaltargeting(Figures3and4).Theseresultsstrongly
suggestedthattheNterminalcytoplasmicsegment
ofNramp1containedlysosomal
targetinginformationthatwasabletoactinadominantfashionoveraknownrecycling
endosometargetingsignal
(YLLNT555-559)intheCterminusofNramp2isoformIIpresent
inNIN-HA.Thelack
ofsignificanttransportactivityforNIN-HA,asforNrampI,likely
resultedfromthelack
ofexpressionatthesiteoftransportmeasurements(plasma
membrane)ratherthanexpression
ofanon-functionalprotein(Figure3).Closer
examination
oftheaminoacidsequencewithintheNterminusofNramp1revealeda
potentialtyrosine-basedsignal
oftheformYXX
substitutionmutationsat
Yl5andIl8withintheNIN-HAchimerawereabletorestore
cellsurfaceandrecyclingendosometargeting
oftheNramp2chimera(Figures5Band
6B).However,alaninemutationsat
GI6andSl7ofNIN-HAretainedtheirlysosomal
175
targeting(Figure6B).Theseresultsc1earlydemonstratethatYGSI1S-18functionsasa
lysosomaltargetingmotifthatfitstheconsensus
YXX
lysosomaltargetingwithoutsignificantlydisruptingintemalization
351;353;354.Anindirect
targeting
ofNIN-HAGl6A,S17Amutantstolysosomesviatheplasmamembranewould
explaintheincreasedcellsurfaceexpressionobservedforthesemutants(Figure5B).
Interestingly,theYGSIsignalinNrampldoesnotcompletelyconformtothe
YXX
YGSImayrepresentanovelsubset
ofYXX
signaIsinvolvedinlysosomaltargetingseemtobethepresenceofaglycineresidue
precedingtheeriticaltyrosine
asweIlastheprevalenceofthemotifsintheCterminusof
membraneproteins183.Nramp1possessesaserineinsteadofaglycineupstreamofits
YGSIsignal.Also,theYGSIsignalisfoundintheNterminus
oftheproteinasopposed
tothemoreeommonlyfoundCterminus.N-terminal
YXX
inproteinssuehasthetransferrinreeeptor(YTRF)butfunetiontypieallyasendoeytie
signaIs.Additionalexperimentsareneededtoidentifythespecifiebindingpartners
of
YGSIandbetterunderstandthemolecularmachineryinvolvedinthesortingofNrampl
tolateendosomesandlysosomes.HopefuIly,thecharacterization
ofothertyrosine-based
signaIsintheeytoplasmicdomains
oflysosomalproteinswillc1arifywhethertheYGSI
motifinNramp1representsanovelsubset
ofYXX
Clearly,properlysosomaltargeting
ofNramp1iseritiealforitsanti-microbial
aetivity
asametaleffluxpumpatthephagosomalmembraneofmacrophagesandother
phagocyticcells.Uponphagoeytosis
ofalivepathogen,NrampI-positivelysosomesfuse
withthemembranes
ofthepathogen-containingphagosomesandNramp1remains
associatedwiththephagosomesthroughtheirmaturationtofullyanti-microbial
phagolysosomes
35.Ourdiscoverythatasequencemotifintheamineterminusof
Nramp1issufficienttotargetNrampproteinstothelysosomessuggestamodelfor
funetionalevolution
ofNrampproteins.Inthismodel,Nramp1andNramp2were
generatedbygeneduplicationfromacommonancestor,followedbysubsequent
sequencedivergencetoretainfunctionwithrespecttomechanism
oftransportandion
selectivity.Furtherdivergence,inc1uding(a)restriction
ofNrampltranscriptionalactivity
toeells
ofthemononuc1earandpolymorphonuc1earphagocytelineages,and(b)
178
acquisitionofatargetingsignalintheaminotenninus(YGS!)activelydirectingNrampl
localizationtolysosomesfordeliverytopathogen-containingphagosomes,wouldhave
ensued
toenablemetaleffluxatthephagosomalmembrane.Thisactivityappearstohave
beenextremelybeneficialtothehostandhasbeenpreservedthroughoutevolutionfrom
lowereukaryotestohumans
1.
179
Chapter8:
SummaryandFuturePerspectives
180
8.1Summary
Theworkpresentedinthisthesisexaminedthestructure,function,andsubcellular
trafficking
ofNrampproteinsbycenteringonthetwomammalianorthologs,Nramp1and
Nramp2.Thefirstthreechapters
ofthisthesisexploredstructure-functionrelationshipsin
theNrampsuper-family.InChapter2,therole
ofchargedaminoacidswithinthe
membrane-spanningsegments
ofNramp2wereexaminedbysite-specifiemutagenesis
followed
byfunctionaicharacterizationinbothyeastandtransfectedCHOcells28.These
studiesidentifiedthreenegativelychargedandhighlyconservedresidues
inTM1,TM4,
andTM7asessentialformetaltransport
byNramp2.Thesestudiesalsoidentifiedapair
ofinvarianthistidinesinTM6thatplayanimportantroleinpHregulationofmetai
transport
byNrampproteins.InChapters3and4,theeffectsoftwoNRAMP2mutations
foundinhumanpatientssufferingfromseverecongenitalhypochromicmicrocytic
anemiaandironoverloadwerecharacterized.Themutationfoundinfirstpatienthadtwo
effects:itseverelyimpairedproper
NRAMP2pre-mRNAsplicing,andintroducedan
amineacidpolymorphism(E399D)intheproteinencoded
bytheremainingproperly
splicedtranscript
4.ThefunctionalpropertiestheE399Dmutationaswellasother
independentmutantsatthatposition(E399Q,E399A),werestudiedintransfectedLLC
PKj
kidneycells(Chapter3)262.AlImutationsatE399wereshowntobefullyfunctional
withrespecttostability,metaltransportactivity,andtargeting,indicatingthatthereduced
Nramp2function
inthepatientwaslikelycausedbyaquantitativereductioninNRAMP2
mRNAlevelsduetoimpropersplicing.Thesecondpatientwasfoundtobecompound
heterozygoteformutationsin
NRAMP2,inc1udinga3basepairdeletionthatpartially
impairssplicing,andanaminoacidsubstitution(R416C)ataconservedresidueinTM9
oftheprotein5.Thefunctionalpropertiesandpossiblecontributiontodiseaseofthe
R416Cvariantwerestudiedinindependentmutantsatthatposition(R416C,R416A,
R416K,R416E)expressedinLLC-PKjcells(Chapter4)
264.Non-conservative
substitutionsatR416(C,A,E)causedmultiplefunctionaldeficienciesconcomitantwith
retention
ofthetransporterintheendoplasmicreticulum.Conversely,aconservative
isoelectricsubstitution(R416K)waslessvulnerable,resulting
inafunctionaltransporter
thatwasproperlyprocessedandtargetedtothecellsurfaceandtorecyc1ingendosomes.
181
TheseresultsshowedthatR416Crepresentsacompleteloss-of-functionandthata
quantitativereductioninNramp2expressionisthecause
ofthemicrocyticanemiaand
ironoverloadinthispatient.
Thelastthreechapters
ofthisthesiswereaimedatbetterunderstandingthe
subcellulardistribution
ofNramp1andNramp2.AlternatesplicingofNramp2pre
mRNAat
3’exonsgeneratestwoproteinisoforms(1andn)differingonlyintheir
carboxylterminalsegments.
IthadbeenpreviouslyshownthatNramp2isoformnis
expressedattheplasmamembraneand
intransferrinreceptor-positiverecyc1ing
endosomesatsteady-state.TheworkdescribedinChapter
5focusedonidentifying
signal(s)inthecytoplasmicsegments
ofNramp2isoformnresponsibleforitssubcellular
targetingandinternalizationfromtheplasmamembraneintorecyc1ingendosomes
184.
Deletionmutationswereintroducedatseveralpredictedtyrosine(NPXY,YXX<1»and
dileucine-based(LL)motifsintheaminoandcarboxylterminalsegments
ofNramp2
isoformnandthemutantswereexpressedandstudiedintransfectedLLC-PK1cellsfor
transportactivity,subcellularlocalization,cellsurfaceandrecyc1ingpooldistribution,
andinternalizationfromtheplasmamembrane.Thisworkledtotheidentification
ofa
tyrosine-based
motifinthecarboxylterminusofNramp2isoformn(YLLNT555-559)that
iscriticalforthetransporter'sinternalizationfromthecellsurfaceanditsrecyc1ingback
totheplasmamembrane.Severalstudiessuggestedthatwhileisoform
nispredominantly
expressedinerythroidcells,isoform1ispredominantlyexpressedinepithelialcells
40;155;160.However,thesignificanceofthistissue-specificexpressionpattern,inc1udinga
possiblephysiologicaladvantage
ofsuchadistribution,wasunc1earandneededtobe
investigated.Therefore,thetargetingandtraffickingproperties
ofNramp2isoforms1and
nwerecomparedintransfectedLLC-PK1cells,withrespecttodifferencesinfunction,
subcellularlocalization,endocytosiskinetics,andfateuponinternalization(Chapter6)
182.Thesestudiesdemonstratedthatisoform1possessedhighersurfaceexpressionand
wasinternalizedfromtheplasmamembranewithslowerkineticsthanisoform
n.As
opposedtoisoform
n,whichisefficientlysortedtorecyc1ingendosomesupon
internalization,isoform1wasnotefficientlyrecycledandwastargetedtolysosomes.
182
Theseresultssuggestedamechanismwherebyisoform1wouldbeenrichedatthecell
surface,favoring
Fe2+uptakeacrosstheplasmamembraneinepithelialcells.
ItwasknownforsometimethatNramp1isexpressedinthelysosomesof
phagocyticcellsandmorerecentdatasupportamodelwherebyNramp1restrictsthe
replication
ofintracellularpathogensbyremovingdivalentmetals(Mn2+,Fe2+)fromthe
phagolysosome.InChapter7,thesubcellulartraffickingproperties
ofNramp1,including
cytoplasmicsequencesresponsiblefortargetingtolysosomes,wereinvestigated.
ChimericNramp1/Nramp2proteinswerecreated
byexchanginghomologousdomainsof
eachproteinandwerecharacterizedinstablytransfectedLLC-PKIcellswithrespectto
expression,maturation,stability,cellsurfacetargeting,transportactivity,andsubcellular
localization.
AnNramp2isoformIIchimerabearingtheNterminusofNramplwasnot
expressedatthecellsurfacebutwastargetedtolysosomes.Thislysosomaltargetingwas
abolished
bysinglealaninesubstitutionsatY15and118ofaYGSI15-18motifpresentin
theNterminus
ofNrampl.Together,theseresultsidentifiedYGSIasatyrosine-based
sortingsignal
oftheformYXX
providednovelinsightintothetransporter’ssubcellulartrafficking.
8.2FuturePerspectives
8.2.1Insightsintostructure/functionrelationships
inNrampproteins
Inthelasttwentyyears,theidentificationandcharacterizationofNrampfamily
proteinsfromawiderange
oforganismshasgreatlyfurtheredourunderstandingofa
number
ofcellularandphysiologicalprocesses.Inmammals,themostsignificant
advancementshavebeenmade
inthefieldsofhostresistancetoinfectionandthe
metabolism
ofiron,fromthecharacterizationofbothNramplandNramp2,respectively.
WenowknowthatNramp1isexpressedinphagocyticcellsandcountersintracellular
pathogenreplication
byfunctioningasapH-dependentdivalentmetaleffluxpumpatthe
phagosomalmembrane.
Ontheotherhand,Nramp2isexpressedattheduodenalbrush
borderwhere
itcontrolsuptakeofdietaryiron,andispresentattheplasma
membrane/recyclingendosomes
ofmostcells,whereitisnecessaryforacquisitionof
183
transferrin-associatediron.BecauseofthehighsequenceconservationamongNramp
proteins,eventhosefromphylogenicallydistantspecies,itislikelythataIlNramp
transportersshareasimilartransportmechanism.Structure/functionstudiescarriedout
in
variousNramporthologshavebeenhighlyinformativeandessentialtounderstandingthe
molecularbasis
ofmetaltransportandpHdependence.Thesestudieshavebeendifficult
tocarryoutinNramp1mostlybecause
ofitsstrictlysosomallocalizationatsteady-state.
Converse1y,theubiquitousplasmamembraneexpression
ofNramp2atsteady-statehas
madeitamuchmoreattractivetargetformutagenesisstudies.
Notsurprisingly,the
amount
ofdataconcemingstructure/functionrelationshipsforNramp2significantly
exceedsthat
ofNrampl.
8.2.2Evidenceforametalbindingsiteorpermeationpathway
TheworkdescribedinChapter2ofthisthesisexaminedthefunctionalroleof
highlyconservedchargedresiduesintheTMdomainsofNramp2onsubstratetransport
and
pHregulation.ThisworkidentifiedthreenegativelychargedresiduesinTM1(D86),
TM4(D192),andTM7(E299)thatarecriticalformetaltransport
byNramp228.Based
onourresults,wehypothesizedthatthenegativelychargedsidechains
oftheseamino
acidsmediatedeitherdirectorindirectinteractionwiththepositivelychargeddivalent
cationsubstrates
ofNramptransporters.Thenegativechargesmayformpartofmetal
bindingpocketorlinethewallsofahydrophiliciontransportpathwayacrossthe
membrane.Whilethistheoryseemsplausibleandwould
beeiectrostaticallyfavorableit
hasnotyetbeenproven.Additionalmutagenesisstudiesattheseconservedaminoacid
residueswouldhelpdeterminetheexactimportance
ofthesechargedresiduesinNramp2
function.ChargedresiduesshowntobeimportantforNramp2functioncouldbereplaced
withaminoacidsthataltersomephysicalproperties
oftheresidueswhileretaining
others.Forexample,aspartatecouldbereplacedwithglutamatetoconservechargeand
altersize,
orasparaginetoretainsizewhileeliminatingcharge.Arequirementofresidues
withnegativelychargedsidechainsatpositions86,192,and299wouldsupportan
e1ectrostaticrolefortheseresiduesinsubstrateinteraction.Interestingly,Suand
colleagueshavepreviouslycharacterizedtheeffects
ofconservativemutationsatD192
184
(D192N,D192E)onNramp2functionintransientlytransfectedHEK293Tcells146.At
thetime,theypostulatedthatsinceD192andG185resided
incloseproximityinTM4
fromhelicalwheelprojections,theloss-of-functionmutationfoundin
mkmiceand
Belgraderats(G185R)couldresultfromafunctionallyunfavorablenovelionic
interactionbetweenD192andR185.Whiletheirmutagenesisdatadidnotultimately
supporttheirhypothesis,theyshowedthatD192NandD192Emutantsretained
significantmetaltransportactivity,while
aD192Gmutantwasseverelyimpaired.
Althoughexactcomparisonsbetweenactivity
ofthemutantswasnotpossiblesincethey
didnotmeasureproteinexpression,theirresultssuggestedthatD192doesnotplaya
majorroleinsubstrateinteraction.OurobservationthatD192Aresultsinonlyapartial
butnotcompleteloss
ofactivitysupportsthisidea28.
SeveralotherstudieshavepointedtoTM4ofNramp2asaregioncriticalfor
metaltransportactivity.Work
byXuandcolleagueshassuggestedthatthemutation
causingmicrocyticanemia
inrodents(GI85R)inTM4ofNramp2exposesanovel
calciumchannelpathwaythatmayprovidesorneunknownselectiveadvantagetomice
bomwiththismutation149.Thisselectiveadvantagewouldhelpexplainthespontaneous
reoccurrence
oftheG185Rmutationonthreeseparateoccasionsinrodents.However,the
increasedcalciumpermeabilitywouldhavetofunctionincontextwiththeother
biosyntheticandfunctionaldefectsassociatedwiththeG185R.Touretandcolleagues
clearlydemonstratedthatNramp2
G185RislessstablethantheWTprotein,isnotproperly
glycosylated,andisnotefficientlytargetedtotheplasmamembrane
148.Rather,the
majorityofNramp2G185Rwasshownto
beretainedintheendoplasmicreticulumand
degraded
byaproteosome-dependentmechanism.
Independentstudies
byseveralothergroupshavesupportedaroleforD86in
substrateinteraction.Mutagenesisstudiescarriedout
byChaloupkaandcolleagues
examinedtherole
ofD86inthebacterialNramportholog,MntH156.Consistentwithour
results,theyshowedthataglycinemutationatD86(correspondingtoD34GinMntH)
resultsinasevereloss
ofmetaluptake(Mn2+,Fe2+,C02+).While
amoreconservative
asparaginesubstitution(D34N)didnotrescuemetaltransport,theydidnotcharacterizea
negativecharge-retainingD34Emutant.Suchamutant
mayindeedretainsomeactivity,
185
confinningtheneedforanegativechargeatthatpositionandsupportingaroleforD86in
substrateinteraction.Interestingly,D86residesattheend
ofTMlimmediatelyupstream
ofthefirstextra-cytoplasmicloop(ECl)andformspartofafouraminoacidsequence
(DPGN
86-89
)inNramp2thatisstrikinglyinvariantthroughoutaUmembersoftheNramp
family.Inaddition,studies
byCohenandcolleagueshavesuggestedthatcertainresidues
within
ECIofNramp2(G88,D93,Q95)arecriticalformetalionbindingandspecificity
oftransport
159.Theyusedelectrophysiologicalmeasurementsandradioisotopicmetal
transportassaystocharacterizetheeffects
ofECImutantsexpressedinXenopusoocytes.
G88AandQ95Dmutantsresulted
inacompleteinhibitionofmetaiuptakewhileaD93A
mutantretainedpartialactivityforFe
2+butnotMn2+.ThedoublemutantD93A1Q95D
retainedpartialactivityandshiftedsubstratespecificityinfavorofFe2+.However,the
triplemutant
G88A1D93A1Q95Dshowednoapparenttransportactivity.Pre-steady-state
CUITentmeasurementsatdifferentpHvaluesforthemutantssuggestedthatEC1isnot
onlyinvolved
inmetaibindingbutisaisocriticalforproton-binding.
Together,thesefindingssuggestamechanismwherebykeyresiduesin
ECIof
Nrampproteinsworkinconjunctionwithnegatively-chargedresiduesinTMI(D86),
TM7(E299),andpossiblyTM4(DI92)tobindandtranslocatedivalentmetalcations
acrossthemembrane.Theenergyforthistranslocationislikelyderivedfroma
simultaneousproton-couplingmechanism.
8.2.3AmolecularbasisforpHdependenceandproton-coupling
EarlyelectrophysiologicalstudiesbyGunshinandcolleaguesshowedthat
Nramp2usedaproton-coupledmechanismtotransportdivalentmetalsacrossthe
membrane
40.Theirinitialworksuggestedatransportstoichiometryof1:1(H+:divalent
metal)atphysiological
pH~6,yetanadditionalprotonslipmechanismcouldproceed
uncoupledfromdivalentmetaltransportatamoreacidic
pH(pH<5.5)40.These
observationswerelatersupported
instudiesbySacherandcolleagues,usingsimilar
electrophysiologicaltechniquestosimultaneouslycharacterizethetransportproperties
of
Nramp2andtheyeastorthologSmflp50.TheyproposedthatthisH+slipmechanism
mightbeadvantageoustotheorganismand
mayprotectagainstoverioadingofmetalions
186
inthepresenceofexcessacid,forexampleintheduodenum.Atthistime,however,a
molecularbasisfor
pHdependenceand/orprotoncouplingofmetaltransportbyNramp2
wasstillunknown.
Theworkdescribed
inChapter2ofthisthesisidentifiedtwohighlyconserved
histidines(H267,H272)thatarecriticalfor
pHdependenceofmetaltransportbyNramp2
28.Unchargedhydrophobicsubstitutions(alanine,cysteine)atbothhistidines,either
independently
ortogether,retainedlittleactivityatpH6.5butshowedsignificantactivity
atamuchlower
pH5.0.Interestingly,substitutingbothhistidinestopositivearginine,
independentlyortogether,resulted
inacompletelyinactiveproteinatanypHtested.
Theseresultsofferedone
ofthefirstattemptstoelucidatethemolecularbasisofpH
dependenceformetaltransportbyNrampproteins.Sorneofourresultswerelater
supported
inastudybyMackenzieandcolleagues,whoexpressedwild-typeandmutant
Nramp2inXenopusoocytesandusedelectrophysiologicaltechniquestostudythe
mechanismofNramp2-mediatedmetaltransport
355.Inagreementwithourfindings,they
showedthatmutationsatH267andH272affectoptimalactivity
ofNramp2,andthat
substitutionsatH272arelesstoleratedthansubstitutionsatH267.Theyalsofoundthat
anargininemutationateitherhistidinecompletelyabrogatesNramp2function.However,
thestudy
byMackenzieetalreportedsornecontradictoryfindingswithrespecttoour
study.TheyreportedthatH267Adisplayedpropertiessimilarto
WTNramp2butata
lowerlevel,andthatthislowerlevel
ofactivitywassimplyareflectionofalowerdensity
oftransporterspresentonthemembrane.TheyfoundmorestrikingeffectsforH272A,
reportingthatthismutationactuallyuncoupledFe
2+andH+fluxes,andshowedsorne
evidencethatmetaltransport
bythisvariantwasindependentofextracellularpH355.
TheseresultsclearlycontradictourfindingsthatinactiveH267andH272mutantspossess
increased
pHdependencethatcouldberescuedbyloweringtheextracellularpH28.Still,
bothourstudiesevidentlyshowthattheconservedhistidines
inTM6(particularlyH272)
playanimportantroleinregulatingthe
pHdependenceand/orprotoncouplingof
Nramp2activity.SincehistidineresiduescanbetitratablewithinthephysiologicalpH
range,itispossiblethattransientprotonation
ofH267and/orH272maybearequirement
forthe
WcouplingofNramp2.
187
TheimportanceoftheconservedTM6histidinesinthefunctionofNramp
proteinshasbeenverifiedinotherNramporthologs.Inarecentpublication,Chaloupka
andcolleaguesreportedthatcriticalmutationsateitherH211
orH216ofabacterial
Nramportholog(correspondingto
H2671H272ofNramp2),impairmetaltransport156.
Consistentwithourstudies,H211A1H216Amutationsdisplayedsignificantmetal
transportatlowpH(pH4.7)whileH211R1H272Rmutationsdisplayednosignificant
metaltransportactivity.Furthermore,mutationsatthesecondhistidine(H216)wereless
toleratedthanatthefirsthistidine(H211).
IncontrasttotheTM6histidinemutantsthatappeartouncouple
H+anddivalent
metaltransport,atleastoneothermutationinNramp2hasbeenshowntohavethe
oppositeeffect
oncoupling.In2004,NevoandNelsonusedyeastcomplementation
assaysandelectrophysiologicalmeasurementsonfrogoocytestoshowthataF196I
substitutioninNramp2(referredtoasF2271inthismanuscript),results
ina14-fold
increaseintheratiobetweenmetaltoprotonstransported
158.Ineffect,theF196I
mutationseemstoenhancethecoupling
ofmetaliontransportinNramp2.Interestingly,
F196residesinTM4,whichisalreadyknowntobecriticalforNramp2activitysincea
G185Rmutation
inthisregioncausesmicrocyticanemiainrodents.Thus,itappearsthat
keyresiduesinTM4(F196)andTM6(H267,H272)
ofNramp2playvitalrolesin
maintainingproperprotoncoupling
ofthetransporter.Hopefully,theidentificationof
additionalmutantsthataffectprotoncouplingand/orpHdependencewillfurtherour
understanding
ofmetaltransportbyNrampproteins.
8.2.4Structuraldeterminants
ofNrampproteins
Hydropathyplotspredictthepresence
ofbetweentenandtwelvemembrane
spanningalphahelicesforNrampproteins
1.Theproposedtopologyhasbeenverifiedin
differentregions
ofdifferentorthologs.ThemembranetopologyofthebacterialNramp
orthologMntHwasstudied
byCourvilleandcolleaguesusinggeneticfusionswith
cytoplasmicandperiplasmicreporters
356.Theirresultswereingeneralagreementwith
initialcomputer-assistedpredictions,withMntHpossessingelevenputative
transmembranedomains,anintra-cytoplasmicaminoterminus,andanextra-cytoplasmic
188
carboxylterminus.ThetopologyofMntHwasfoundtomorecloselyresemblethe
structure
oftheyeastorthologs(Smf)thanthemammalianproteins.Indeed,epitope
accessibilitystudieswiththeyeastNramporthologSmf3palsoplaceitsCterminusextra
cytoplasmically
357.However,themembranetopologiesofthebacterialandyeastproteins
maydifferfromthemammalianproteins,whichpossesstwelvetransmembranedomains
comparedtoeleven.Inthemammalianproteins,antibodyaccessibilitystudieswith
Nramp1andNramp2haveshownthatboththeNandCtermini
ofthesetwoproteinsface
thecytosolandthatthesequencebetweenTM7andTM8isindeedextra-cytoplasmic
26;27;148.However,thecompletemembranetopologyofNramp1andNramp2hasnotyet
beenverifiedexperimentallyandrequiresfurtherstudy.Onemethodtoverifythe
topology
ofNramp2wouldbetouseasystematicmutagenesisapproachindependently
insertingepitopetagswithineachintra-andextra-cytoplasmicloops
ofthetransporter.
Immunofluorescencelabelingexperiments
infixedintact(non-permeabilized)vs.non
intact(permeabilized)cellscouldthenbeusedtodetermineaccessibility
ofthevarious
epitopestospecificantibodies.Epitopesaccessibletoantibody-labelinginintactcells
wouldconfirmtheextracellularnature
ofthoseregionsoftheprotein.Theuseofepitope
tagssuchashemagluttinin(HA)wouldbefavoredoverthecreation
ofgeneticfusions
withreporterproteinsbecause
oftheircomparativelysmallersize,whichwouldminimize
possibledisruptionsintransporterfoldingandprocessing.
He1icalwheelprojections
oftheputativetransmembranedomainsofNramp
proteinsrevealstrongamphiphilicityforTM3,TM5,andTM9,c1earlysegregatinga
polarfacemadeup
ofcharged/polarresiduesfromanon-polarhydrophobieface1.The
polarfaces
ofthesedomainslikelyjuxtaposetheaqueoussolventwhilethehydrophobic
portions
ofthedomainsprobablyinteractwiththehydrophobiclipidsinthemembrane.
Interestingly,workdescribed
inChapter2revealedthatalaninesubstitutionsattwo
chargedresidueswithinamphipathictransmembranedomainsTM3(EI54)andTM9
(R416)
ofNramp2resultedinproteinsthatcouldnotbestablyexpressedinCHOcells28.
Theseresultssuggestedthatthesetwochargedresiduesareimportantforprocessingor
stabilityofthetransporter.WorkinChapter4showedthataconservativeaminoacid
substitutionatR416retainingapositivecharge(R416K)wasmoderatelytolerated.One
189
possibilityisthatanionicinteractionbetweenE154andR416isnecessaryforinter-helix
packingbetweenTM3andTM9,andthatmutatingeither
oftheseresiduestouncharged
alaninepreventsthisinteractionandcausesdecreasedstability
ormis-foldingofthe
protein.Indeed,conservedchargedresiduesresidingwithinthehydrophobic
transmembranedomains
ofpolytopicmembraneproteinshavebeenshowntobe
importantforthestructure
ofothertransporterssuchasCFTRandthelactosepermease
322;323.OnewaytoindirectlyverifyaninteractionbetweenE154andR416wouldbeto
exchangetheresiduesatbothpositionsandattempttoexpressthemutantproteinin
transfectedcells.Expression
ofanR1541E416mutantproteinthatshowednormal
processing,stability,andfunctionwouldsupportaninteractionbetweenbothcharged
residues.Relatingbiochemicaldatatothesecondaryandtertiarystructuresofproteinscan
bedifficultintheabsence
ofahighresolutioncrystalstructure.Severalattemptshave
beenmade
byLiandcolleaguestodeterminethestructureofa24aminoacidsynthetic
peptidecorrespondingtopredictedTM4
ofNramp2358-361.ThroughtheuseCDand
NMRspectroscopy,theyshowedevidencethattheTM4-correspondingpeptideadoptsan
alpha-helicalstructure
inseveralmembrane-mimickingenvironments359.Furthermore,
environmentalchangesin
pHseemedtobeassociatedwithstructural/conformational
changesinthe
TM4peptide360.Fromtheirdata,theyproposedamodelwherebyseveral
TM4heliceswouldself-assembleinthemembrane,delineatingahydrophilicchannel
throughwhichmetalionswouldbetransported.AnotherstudycomparingTM4peptides
from
WTandG185RmutantNramp2proteinssuggestedthatthemutationcausing
microcyticanemiainrodentsalterstheself-assembly
ofthehelicesanddisruptsthe
quatemarystructure
ofthetransporter361.Althoughthisisaninterestingtheory,therehas
notyetbeenanybiochemicalevidencesuggestingthatNrampproteinsfunctionas
multimericcomplexes.If,aspostulated,theG185Rmutationseverelydisruptedthe
quatemarystructure
oftheprotein,itwouldlikelyresultinacompletelossoftransport
activityandnottheattenuatedfunctionalactivityactuallydisplayed
bythismutant28;148.
Wemustbecautiousininterpretingthestructuralbehaviorofasinglesynthetic
hydrophobiepeptide(TM4)intheabsence
oftheremainingtransmembranedomainsand
190
othersegmentsofNramp2thatarepotentiallycriticalformaintainingthetransporter's
structure.Insornecases,theidentification
ofadominantnegativevariantofaproteinis
anindicationthattheproteinfunctionsasamultimericcomplexinvolvinginteraction
of
severalsubunits,wherethemutantproteinaffectsthefunctionofthewild-typeprotein.
Forexample,dominantnegativemutantshavebeenidentifiedforferroportinandsorne
experimentshaveshownthattheironexporterMayoligomerize
invitro295.Thefactthat
nodominantnegativemutationshavebeenidentifiedtodateforNramp2either
invitroor
invivo,suggeststhatthetransporterMayactuallyfunctionasamonomer.However,this
hasnotyetbeenconfirmedexperimentaIly.One
waytoanswerthisquestionwouldbeto
purifyandreconstituteNramp2inproteoliposomesunderconditionswherethe
transporterisfullyfunctionalandthenusefreeze-fractureelectronmicroscopytolookat
thedistributionoftransporters
inthemembrane.Inaddition,theinitialrateofmetal
transportintotheproteoliposomescanbecomparedwiththeratioofNramp2to
phospholipid.AlinearrelationshipwouldsuggestthatNramp2functionsasamonomer
whileasigmoidalrelationshipwouldindicatetheneedforoligomerization
ofthe
transportertoremainactive.Suchtechniqueswereused
byCostelloandcolleaguesto
verifythemonomericnature
ofthelacpermeaseandcytochrome0oxidase362.
Intheabsenceofahighresolutioncrystalstructure,severalmolecularbiology
approachescanbeemployedtocharacterizethesecondarystructure
ofmembrane
proteins.Thelactosepermease
ofEscherichiacoli(LacY)isoneofthemostweIl
understoodmembraneproteintransportersandseveralpioneeringtechniqueshavebeen
usedanddevelopedtostudyitsnativestructurewithinthebacterialmembrane.Many
of
thetechniquesusedtocharacterizeLacYcanbeusedtostudyothermembraneproteins
withsimilarcharacteristics.LikeNramp2,LacYisanintegralmembraneprotein
consisting
oftwelvemembrane-spanningalphahelicesandfunctionsasasymporterthat
usesaprotongradienttotransportitssubstrate(galactosides)acrossthemembrane.
Severalyearsago,thegroup
ofKabackandcolleaguesusedacysteine-scanning
mutagenesisapproachto:(a)determineresiduesthatplayed
anobligatoryroleinthe
function
ofLacY,and(b)createalibraryofsingleCysmutantsforfuture
structure/functionstudies(reviewed
in363).TheaminoacidCysisaverageinbulkand
191
relativelyhydrophobie.However,themajoradvantageofCysisitssulfhydrylsidechain,
whichisopentohighly-specificmodification
byvariouschemicalgroupssuchasN
ethylmaleimide.Furthermore,byintroducingsingleCysmutantsintoafunctionalmutant
backbonedevoid
ofendogenousCysresidues,theprecisespecificityofthechemical
modificationcanbecontrolled.Thegroup
ofKabackandcolleagueshaveusedthisCys
scanningmutagenesisapproachinconjunctionwithbiochemicalandbiophysical
techniquestoelucidatemembranetopology
364-366aswellasspatialproximitybetween
transmembranedomainsandperiplasmicloops
oflacpermease367-369.Thelatterstudies
utilizedsulfhydryl-specificbifunctionalchemicalcross-linkers,whichhavealsobeen
usedtostudythetransmembranedomainstructure
ofnumerousotherpolytopic
transmembraneproteinssuchastheTar
370andaspartatesensoryreceptors371ofE.coli.
Cys-scanningmutagenesishasalsobeenusedtoidentifyhydrophilicpathwaysformedby
membranetransporters.In1995,YanandcolleaguesusedCys-scanningmutagenesisto
demonstratethatTM7
ofthebacterialmembranetransporterUhpTlinesahydrophilic
transportpathwayforitschargedsubstrate(glucose-6-phosphate)
372.Beginningwitha
Cys-Iessversion
ofUhpT,theyintroducedCysresidues,oneatatime,ateachposition
alongTM7andthenprobedthesesingle-cysteinevariantswith
p
chloromercuribenzosulfonate(PCMBS),ahydrophilicandimpermeantsulfhydryl
reactiveagent.TheyfoundthatTM7
ofUhpTformsana-helixwhosecentralportionis
highlyaccessibletoPCMBSfrombothmembranesurfaces,andsubsequentlythey
conc1udedthatresidueswithinTM7lineahydrophilicpathwaythroughthetransporter.
Finally,aCys-scanningmutagenesisapproachcanbeusedtoidentifyspecifieamino
acidsthatresidein
ornearasubstratebindingsite.Insornecases,pre-incubationwith
substrate
ofasingle-cysteinemutantproteinisabletoprotectagainstchemical
modificationwithathiol-specificreagent.
Forexample,Wuandcolleaguesmadeuseof
thefluorescentcompoundMIANS[2-(4'-maleimidylanilino)naphthalene-6-sulfonic
acid],whosefluorescenceincreasesdramaticallyuponreactionwithathiol,to
demonstratethatCys148
ofthelacpermeaseisacomponentofthesubstratebindingsite
373
192
Intwosimultaneouslandmarkpublicationsin1995,Vossandcolleaguesmade
use
ofparamagneticmetal-nitroxylinteractionstoestimateintramoleculardistances
withinproteins.
Inthefirstpublication,theyvalidatedthetechniquebymeasuring
distancesinT4lysozyme,aprotein
ofknownstructure314.Thecalculateddistances
exhibitedstrongcorrespondencetothedistancesestimatedfrommodelingbasedonthex
raystructure
oftheprotein.Inthesecondpublication,theyappliedtheirmetal-nitroxide
approachtomeasuredistancesinthelacpennease,aparadigm
ofpolytopicmembrane
proteins
374.Site-directedspinlabeling(SDSL)isapowerfultoolforstudyingprotein
structure/dynamics,andhasbeensuccessfullyusedtoobtainstructuraldata
onanumber
ofproteins.However,athoroughdescription
ofSDSLtechniqueisbeyondthescopeof
thisthesisandhasbeenreviewedelsewhere375-377.Briefly,thetechniqueinvolves
introducingametal-ionbindingsite(e.g.sixcontiguoushistidineresidues)inafixed
region
oftheproteinandaspin-labeledsidechain(cysteines)atanotherregionof
interest.Ofcourse,othernativecysteinesmustfirstbereplaced.Afterpurificationofthe
protein,thespin-labelsidechains(Cys)aremodifiedwithaspecifieparamagnetic
nitroxidereagentandtheelectronparamagneticresonance(EPR)spectraisanalyzed
in
thepresenceofmetal(e.g.Cu2+).Generally,changesinthedistancebetweenthemetal
andnitroxidelabelreflectchangesinthe
EPRspectraofthespin-labe1edprotein.
SimilarCys-scanningmutagenesisandspin-Iabelingapproachesmayyield
valuableinfonnationaboutthetransmembranestructure
ofNrampproteins.However,
theseapproachesrequiresuccessfulpurificationandreconstitution
ofNrampproteinsina
functionalstateandloraCys-lessNrampproteinthatretainssignificantmetaltransport
activity.Todate,therehavebeennopublishedreportsdescribingthe
purification/reconstitution
offunctionalmammalianNramp112,norreportscharacterizing
theactivity
ofamammalianNrampproteindevoidofanendogenouscysteines.However,
Nramp2containstenendogenouscysteineresidues,sorne
ofwhichareconserved,anda
Cys-lessNramp2variantmaynotbestablyexpressed
orretainsignificanttransport
activity.
Anattempthasbeenmadetocarryoutsomeofthesestudiesinthe
mycobacterialortholog,MntH.
In2002,Reeveandcolleaguesreportedthepurification
andreconstitution
ofacysteine-lessvariantofMntHfromMycobacteriumleprae,and
193
usedsite-directedspinlabelingtogainstructuralinformationaboutthetransporter378.
However,theydidnottestthefunctionalityoftheCys-IessMntHtransporteranditisnot
knownwhetherthisvariantrepresentedaproperlyfoldedprotein.Perhapsanideal
candidateforfuturesite-directedspinlabelingstudieswould
betheMntHproteinfromE.
coli,whichdoesnotcontainanyendogenouscysteines.
8.2.5SubcellulartargetingandtraffickingofNrampproteins
Itisc1earthatpropersubcellulartargetingisessentialforthefunctionofNramp
proteins.TheworkdescribedinChapters5,6,and7ofthisthesisfocusedon
characterizingthesubcellulartargetingandtraffickingofbothNramplandNramp2.
TraffickingdifferenceswereidentifiednotonlybetweenNramp1andNramp2,
butalso
betweentwosplicingisoforms
ofNramp2.InNramp2,themostcriticaltargeting
informationappearstoresideinthecarboxylterminalsegment
oftheisoformII(B)
protein,inc1udingaputativeYLLNT
motif155;184.However,theroleoftheadditional29-
31N-terminalresiduesofthe(A)isoformsofNramp2insubcellulartargetingarenot
currentlyknownandneedto
befurtherstudied.Conversely,oneofthesignaIscriticalfor
properlysosomalsorting
ofNramplappearstoresideintheaminoterminalsegmentof
thetransporter,mostnotablyaYGSImotif.Inthepastfewyears,tremendousprogress
hasbeenmade
inthefieldoftransmembraneproteinsorting(reviewedin183).In
particular,numerouscytosolicmotifshavenowbeenidentified
invariousfunctionally
unrelatedtransmembraneproteinsandmany
oftheirbindingpartnershavealsobeen
characterized.
Animportantnextsteptobetterunderstandthesubcellulartraffickingof
Nrampproteinswillbetoidentifythespecifieadaptorproteincomplexesthatinteract
withthetargetingmotifspresent
intheaminoandcarboxylterminiofNramp1and
Nramp2.Thiscanbedoneseveralways.In1989,Glickmanandcolleaguesusedan
in
vitroaffinitybindingmethodtostudyinteractionsofthecytosolictailofmannose-6-
phosphatereceptor(M6PR)withthec1athrin-associatedplasmamembraneadaptor
proteinAP-2
379.TheyusedeitherintactM6PRorafusionproteincontainingthe
cytoplasmictail
ofM6PRforcolumnchromatographyexperimentstoidentifyadaptor
proteinsthatcouldbindM6PR.However,theapparentlowaffinity
oftheinteractionsin
194
vitromayprecludetheuseofthisbiochemicalapproachtostudyothertransmembrane
proteins.Amoresensitivetechniquemightbeayeasttwo-hybridscreen.
Inalandmark
publicationin1995,Ohnoandcolleaguessuccessfullyusedtheyeasttwo-hybridsystem
toidentifyproteinsthatbindtotyrosine-basedsignaIs
331.As"bait",theyusedatriple
repeat
ofthepeptideSDYQRLfromthecytoplasmictailoftheintegralmembrane
proteinTGN38,whichwasknowntopossesscharacteristics
ofaYXX
intemalizationandtargeting
oftheproteintothetrans-Golginetwork(TGN).After
screeningamousespleencDNAlibrary,theyisolatedclonescorrespondingtothe
mediumchain
().12)oftheplasmamembraneclathrin-associatedproteincomplexAP-2.
Theinteractionbetween).12andthe
YXX<1>motifwaslaterconfirmedthroughinvitro
bindingassays.Asimilarapproachcouldbeusedtoidentifyproteinsinvolvedin
traffickingofNramplandNramp2proteins,usingeithertheaminoorcarboxylterminal
segments
ofeachproteinas”bait”.
Currently,onestudyhassuggestedthatintemalization
ofNramp2isoformIIfrom
thecellsurfaceoccursviaaclathrinanddynamindependentmechanism
intransfected
LLC-PKIcells
51.Theseobservationsweremadebasedonsomecolocalizationbetween
Nramp2andGFP-clathrin,andtheapparentlack
ofintemalizationofNramp2incells
expressingadominantnegativevariant(K44A)
ofdynamin.Morestudiesareneededto
verifytheseresults.Traditionally,therequirement
ofclathrinfortheendocytosisofa
transmembraneproteinhasbeenverifiedseveralways.Overtwentyyearsago,Larkinand
colleaguesdescribedamethodforinhibitingreceptormediatedendocytosis
oflow
densitylipoprotein(LDL)receptor
380.Theyfoundthatwhenthelevelofintracellular
potassiumwasloweredinculturedhumanfibroblastsbelow40%
ofnormal,the
intemalization
ofLDLreceptorwasinhibited.Moreover,underconditionsofmaximal
inhibition,thesecellshadan80%reduction
inthenumberofclathrin-coatedpitsonthe
cellsurface
380.Inhibitionofendocytosisbypotassiumdepletionisreversibleandhas
beenfoundtobesurprisinglyspecifieforclathrin-mediatedintemalization,andtherefore
hassincebeenusedtostudyendocytosis
ofanumberofmembraneproteins334;380;381.A
reducedrate
ofintemalizationand/oranincreaseincellsurfaceaccumulationofNramp2
isoformIIinpotassiumdepletedcellswouldsupporttheroleofclathrininthe
195
intemalizationofNramp2fromtheplasmamembrane.Furthermore,Sandvigand
colleaguesfoundthatwhenintracellular
pHwasloweredbelowpH6.0,therewasa
dramaticandspecificinhibition
ofendocytosisthroughcoatedpits382.Unlikepotassium
depletion,acidifiedcellsappeartohavenormalnumbers
ofcoatedpitsbutthesepits
appeartobeparalyzedandunabletointemalizeligand.Again,reducedintemalization
and/orincreasedcellsurfaceaccumulation
ofNramp2uponcellularacidificationwould
supportarole
ofc1athrininNramp2intemalizationfromtheplasmamembrane.
Intriguingly,sinceNramp2functionsthroughaprotonco-transportmechanism,ititself
causescellularacidificationasitimportsdivalentmetals.Thisraisesthepossibility
ofa
positive-feedbackregulationmechanismwherebymetaluptake
byNramp2causes
accumulation
ofthetransporteratthecellsurfacethroughinhibitionofc1athrin-mediated
intemalizationviacellularacidification.
WorkdescribedinChapter7
ofthisthesisshowedthatatyrosine-basedmotif
(YGSI)intheaminoterminusofNramplcouldfunctionasalysosomaltargetingsignal
whenplacedonthebackgroundofNramp2.Ourresultsallowedustoproposeamodel
for
NrampltraffickingwherebyrecognitionoftheYGSIsignalbyspecificadaptor
proteinsmediatethesorting
ofNrampldirectlyfromthetrans-Golginetworktolate
endosomesandlysosomes.However,wecouldnotcompletelyexc1udethepossibilitythat
Nramp1isfirsttargetedtothecellsurface,thenrapidlyintemalizedandtargetedtothe
lysosomesviatheendocyticpathway.Therefore,thequestionremainswhetherNramp1is
targeteddirectly
orindirectlytothelysosomalcompartment.Onewayofansweringthis
questionwouldbetotreatcellsexpressingNramp1withaninhibitor
ofendocytosisand
lookforanaccumulation
ofNramp1atthecellsurfacethroughcellsurfacebiotinylation.
AnincreaseinNramp1atthecellsurfacecouldindicateanindirectsortingroute.Sorne
well-characterizedchemicalinhibitors
ofendocytosisaremonodansylcadaverine,
concanavalinA(conA)andphenylarsineoxide(PAO).AnotherwaytostudyNramp1
sortingwouldbetopulse-labelnewlysynthesized
Nrampltransporterswithradiolabeled
35S-methionineandperformsubcellularfractionationatdifferenttimeintervalsusing
differentialcentrifugationtoisolatefractionsfromvariousorganellarcompartments.
Nramp1couldbeisolatedfromthesefractions
byimmunoprecipitationanddetectedby
196
SDS-electrophoresisandautoradiography.AtransientaccumulationofNramplin
fractionsenrichedforplasmamembranecomponentswouldsupportanindirectsorting
route.
8.3FinalConclusions
FuturestudiesonNrampproteinsareessentialforabetterunderstandingofthe
role
ofdivalentmetalsinanumberofcellularandphysiologicalprocesses.Thenumerous
studiescharacterizing
Nramplhavegiveninsightintothecomplexnatureofhost
pathogeninteractionsandbroughttotheforefronttheimportanceofdivalentmetalsin
thesurvival/replication
ofawiderangeofintracellularparasites.Theseandfuture
findingsmayleadtonoveltherapeuticapproachesforcombatingthe
CUITentalarmingrise
inpathogenicinfections.Inparallel,theidentificationandcharacterization
oftheiron
transporter
Nramp2hashelpedsparkarecentgrowthinresearchinthefieldofiron
metabolism.Abetterunderstanding
ofthefunctionNramp2mayleadtonovelor
improvedtreatmentsforiron-relateddisorderssuchashereditaryhemochromatosis.
197
REFERENCES
1.CellierM,PriveG,BelouchiAetal.Nrampdefinesafamilyofmembrane
proteins.Proc.NatI.Acad.Sci.U.S.A1995;92:10089-10093.
2.RodriguesV,CheahPY,RayK,ChiaW.malvolio,theDrosophilahomologueof
mouseNRAMP-l(Bcg),isexpressedinmacrophagesandinthenervoussystem
andisrequiredfornormaltastebehaviour.EMBO
J.1995;14:3007-3020.
3.FortierA,Min-OoG,ForbesJ,Lam-Yuk-TseungS,GrosP.Singlegeneeffects
inmousemodelsofhost:pathogeninteractions.J.Leukoc.Biol.2005;77:868-877.
4.MimsMP,GuanY,PospisilovaDetal.Identificationofahumanmutationof
DMTlinapatientwithmicrocyticanemiaandironoverload.Blood
2005;105:1337-1342.
5.IolasconA,d’ApolitoM,ServedioVetal.Microcyticanemiaandhepaticiron
overloadinachildwithcompoundheterozygousmutationsin
DMTl(SCLI1A2).
Blood2006;107:349-354.
6.BeaumontC,DelaunayJ,HetetGetal.TwonewhumanDMTlgenemutations
inapatientwithmicrocyticanemia,lowferritinemia,andliverironoverload.
Blood2006;107:4168-4170.
7.VidalSM,MaloD,VoganK,SkameneE,GrosP.Naturalresistancetoinfection
withintracellularparasites:isolation
ofacandidateforBcg.CellI993;73:469-
485.
8.PlantJ,GlynnAA.GeneticsofresistancetoinfectionwithSalmonella
typhimuriuminmice.J.Infect.Dis.1976;133:72-78.
9.BradleyDJ.Letter:GeneticcontrolofnaturalresistancetoLeishmaniadonovani.
Nature1974;250:353-354.
10.LissnerCR,SwansonRN,O’BrienAD.Geneticcontroloftheinnateresistanceof
micetoSalmonellatyphimurium:expressionoftheItygeneinperitonealand
splenicmacrophagesisolatedinvitro.J.Immunol.1983;131:3006-3013.
Il.StachJL,GrosP,ForgetA,SkameneE.Phenotypicexpressionofgenetically
controllednaturalresistancetoMycobacteriumbovis(BCG).J.Immunol.
1984;132:888-892.
12.CrockerPR,BlackwellJM,BradleyDJ.Expression
ofthenaturalresistancegene
Lshinresidentlivermacrophages.Infect.Immun.1984;43:1033-1040.
13.VidalSM,PinnerE,LepageP,GauthierS,GrosP.Naturalresistanceto
intracellularinfections:Nramplencodesamembranephosphoglycoproteinabsent
198
inmacrophagesfromsusceptible(NramplD169)mousestrains.J.Immunol.
1996;157:3559-3568.
14.WhiteJK,StewartA,PopoffJF,WilsonS,BlackwellJM.Incomplete
glycosylationanddefectiveintracellulartargeting
ofmutantSlc11al.Biochem.J.
2004;Pt3:811-819.
15.VidalS,TremblayML,GovoniGetal.Thelty/LshIBcglocus:naturalresistance
toinfectionwithintracellularparasitesisabrogatedbydisruptionoftheNramp1
gene.J.Exp.Med.1995;182:655-666.
16.GovoniG,VidalS,GauthierSetal.TheBcg/Ity/Lshlocus:genetictransferof
resistancetoinfectionsinC57BL/6JmicetransgenicfortheNramp1Gly169
allele.Infect.Immun.1996;64:2923-2929.
17.GovoniG,VidalS,CellierMetal.Genomicstructure,promotersequence,and
induction
ofexpressionofthemouseNramp1geneinmacrophages.Genomics
1995;27:9-19.
18.GovoniG,GauthierS,BilliaF,IscoveNN,GrosP.Cell-specificandinducible
Nramp1geneexpressioninmousemacrophagesinvitroandinvivo.
J.Leukoc.Biol.1997;62:277-286.
19.ZhongW,LafuseWP,ZwillingBS.InfectionwithMycobacteriumavium
differentiallyregulatestheexpression
ofirontransportproteinmRNAinmurine
peritonealmacrophages.Infect.Immun.2001;69:6618-6624.
20.ter-KoltunoffM,EhrlichS,DrorNetal.Nrampl-mediatedinnateresistanceto
intraphagosomalpathogensisregulated
byIRF-8,PU.1,andMiz-l.J.Biol.Chem.
2003;278:44025-44032.
21.BowenH,BiggsTE,PhillipsEetal.c-MycrepressesandMiz-1activatesthe
murinenaturalresistance-associatedprotein1promoter.J.Biol.Chem.
2002;277:34997-35006.
22.BowenH,LaphamA,PhillipsEetal.Characterization
ofthemurineNramp1
promoter:requirementsfortransactivation
byMiz-1.J.Biol.Chem.
2003;278:36017-36026.
23.AtkinsonPG,BlackwellJM,BartonCH.Nramp110cusencodesa65kDa
interferon-gamma-inducibleproteininmurinemacrophages.Biochem.J.1997;325
(Pt3):779-786.
24.LafuseWP,AlvarezGR,ZwillingBS.Role
ofMAPkinaseactivationinNrampl
mRNAstabilityin
RAW264.7macrophagesexpressingNrampl(Gly169).Cell
Immunol.2002;215:195-206.
199
25.LafuseWP,AlvarezGR,ZwillingBS.RegulationofNramp1mRNAstabilityby
oxidantsandproteinkinaseCin
RAW264.7macrophagesexpressing
Nrampl(GlyI69).Biochem.J.2000;351Pt3:687-696.
26.PicardV,GovoniG,JabadoN,GrosP.Nramp2(DCT1/DMT1)expressedatthe
plasmamembranetransportsironandotherdivalentcationsintoacalcein
accessiblecytoplasmicpool.J.BioI.Chem.2000;275:35738-35745.
27.ForbesJR,GrosP.Iron,manganese,andcobalttransport
byNrampl(Slcllal)
andNramp2(Slclla2)expressedattheplasmamembrane.Blood2003;102:1884-
1892.
28.Lam-Yuk-TseungS,GovoniG,ForbesJ,GrosP.Irontransport
by
Nramp2/DMTl:pHregulationoftransportby2histidinesintransmembrane
domain
6.Blood2003;101:3699-3707.
29.MackenzieB,UjwalML,ChangMH,RomeroMF,HedigerMA.Divalentmetal
iontransporter
DMTlmediatesbothH(+)-coupledFe(2+)transportand
uncoupledfluxes.PflugersArch.2005
30.BartonCH,WhiteJK,RoachTI,BlackwellJM.NH2-terminalsequence
of
macrophage-expressednaturalresistance-assoCÎatedmacrophageprotein(Nramp)
encodesaproline/serine-richputativeSrchomology3-bindingdomain.
J.Exp.Med.1994;179:1683-1687.
31.DassaE,HofuungM.Sequence
ofgenemalGinE.coliK12:homologies
betweenintegralmembranecomponentsfrombindingprotein-dependenttransport
systems.EMBOJ.1985;4:2287-2293.
32.KerppolaRE,AmesGF.Topology
ofthehydrophobiemembrane-bound
components
ofthehistidineperlplasmicpermease.Comparlsonwithother
members
ofthefamily.J.BioI.Chem.1992;267:2329-2336.
33.JanLY,JanYN.Potassiumchannelsandtheirevolvinggates.Nature
1994;371:119-122.
34.PinnerE,GruenheidS,RaymondM,GrosP.Functionalcomplementation
ofthe
yeastdivalentcationtransporterfamilySMF
byNRAMP2,amemberofthe
mammaliannaturalresistance-associatedmacrophageproteinfamily.
J.BioI.Chem.1997;272:28933-28938.
35.GruenheidS,PinnerE,DesjardinsM,GrosP.Naturalresistancetoinfectionwith
intracellularpathogens:theNramp1proteinisrecruitedtothemembrane
ofthe
phagosome.J.Exp.Med.1997;185:717-730.
36.Canonne-HergauxF,CalafatJ,RicherEetal.Expressionandsubcellular
localization
ofNRAMPIinhumanneutrophilgranules.Blood2002;100:268-275.
200
37.Cuellar-MataP,JabadoN,LiuJetal.Nramp1modifiesthefusionofSalmonella
typhimurium-containingvacuoleswithcellularendomembranesinmacrophages.
J.Biol.Chem.2002;277:2258-2265.
38.GovoniG,Canonne-HergauxF,PfeiferCGet
al.Functiona1expressionof
Nramp1invitrointhemurinemacrophage1ineRAW264.7.Infect.hnmun.
1999;67:2225-2232.
39.SearleS,BrightNA,RoachTIetal.Localisation
ofNramp1inmacrophages:
modulationwithactivationandinfection.J.CellSei.1998;111
(Pt19):2855-
2866.
40.GunshinH,MackenzieB,BergerUVetal.Cloningandcharacterization
ofa
mammalianproton-coupledmetal-iontransporter.Nature1997;388:482-488.
41.ForbesJR,Gros
P.Diva1ent-metaltransportbyNRAMPproteinsattheinterface
ofhost-pathogeninteractions.TrendsMicrobiol.2001;9:397-403.
42.GruenheidS,Canonne-HergauxF,GauthierSetal.Theirontransportprotein
NRAMP2isanintegralmembraneglycoproteinthatcolocalizeswithtransferrin
inrecyc1ingendosomes.J.Exp.Med.1999;189:831-841.
43.KuhnDE,LafuseWP,ZwillingBS.Irontransportintomycobacteriumavium
containingphagosomesfromanNrampl(G1yI69)-transfectedRAW264.7
macrophagecellline.J.Leukoc.Bioi.2001;69:43-49.
44.KuhnDE,BakerBD,LafuseWP,ZwillingBS.DifferentiaIirontransportinto
phagosomesisolatedfromthe
RAW264.7macrophagecelllinestransfectedwith
Nramp1Gly169orNramplAspl69.J.Leukoc.Bioi.1999;66:113-119.
45.ZwillingBS,KuhnDE,WikoffL,BrownD,Lafuse
W.RoleofironinNrampl
mediatedinhibitionofmycobacterialgrowth.Infect.hnmun.1999;67:1386-1392.
46.GoswamiT,BhattachatjeeA,BabalPetal.Natural-resistance-associated
macrophageprotein1isanH+/bivalentcationantiporter.Biochem.J.
2001;354:511-519.
47.BlackwellJM,GoswamiT,EvansCAetal.SLC11Al(formerlyNRAMP1)and
diseaseresistance.CellMicrobiol.2001;3:773-784.
48.ChenXZ,Peng
lB,CohenAetaLYeastSMFImediatesH(+)-couplediron
uptakewithconcomitantuncoupledcationcurrents.J.Bio1.Chem.
1999;274:35089-35094.
49.KehresDG,ZaharikML,FinlayBB,MaguireME.TheNRAMPproteins
of
SalmonellatyphimuriumandEscherichiacoliareselectivemanganese
201
transportersinvolvedintheresponsetoreactiveoxygen.Mol.Microbiol.
2000;36:1085-1100.
50.SacherA,CohenA,NelsonN.Properties
ofthemammalianandyeastmetal-ion
transporters
DCTIandSmflpexpressedinXenopuslaevisoocytes.J.Exp.Biol.
2001;204:1053-1061.
51.TouretN,FuruyaW,ForbesJ,GrosP,Grinstein
S.Dynamictrafficthroughthe
recyc1ingcompartmentcouplesthemetaltransporterNramp2
(DMTl)withthe
transferrinreceptor.J.BioI.Chem.2003;278:25548-25557.
52.JabadoN,JankowskiA,DougaparsadSetal.Naturalresistancetointracellular
infections:naturalresistance-associatedmacrophageprotein1(Nramp1)functions
asa
pH-dependentmanganesetransporteratthephagosomalmembrane.
J.Exp.Med.2000;192:1237-1248.
53.SkameneE,GrosP,ForgetAetal.Geneticregulationofresistanceto
intracellularpathogens.Nature1982;297:506-509.
54.GotoY,IwakiriA,ShinjoT.[Pathogenicities
ofMycobacteriumintracellulare
andM.aviumstrainstothemicewhichwereisolatedfromnon-tuberculous
mycobactriosispatients].KansenshogakuZasshi2002;76:425-431.
55.SkameneE,GrosP,ForgetA,PatelPJ,NesbittMN.Regulationofresistanceto
leprosy
bychromosome1locusinthemouse.Irnmunogenetics1984;19:117-124.
56.GotoY,BuschmanE,SkameneE.Regulation
ofhostresistanceto
Mycobacteriumintracellulareinvivoandinvitro
bytheBcggene.
Irnmunogenetics1989;30:218-221.
57.GautierAV,Lantierl,Lantier
F.Mousesusceptibilitytoinfectionbythe
SalmonellaabortusovisvaccinestrainRv6iscontrolled
bytheItylNramp1gene
andinfluencestheantibodybutnotthecomplementresponses.Microb.Pathog.
1998;24:47-55.
58.HuJ,BumsteadN,BurkeDetal.Geneticandphysicalmapping
ofthenatural
resistance-associatedmacrophageprotein1(NRAMP1)
inchicken.
Mamm.Genome1995;6:809-815.
59.BarthelR,FengJ,Piedrahita
JAetal.StabletransfectionofthebovineNRAMPI
geneintomurine
RAW264.7cells:effectonBrucellaabortussurvival.
Infect.Irnmun.2001;69:3110-3119.
60.ChapesSK,MosierDA,WrightAD,HartML.MHCII,Tlr4and
Nramplgenes
controlhostpulmonaryresistanceagainsttheopportunisticbacteriumPasteurella
pneumotropica.J.Leukoc.Biol.2001;69:381-386.
202
61.McLeodR,BuschmanE,ArbuckleLD,SkameneE.Immunogeneticsinthe
analysisofresistancetointracellularpathogens.Curr.Opin.Immunol.1995;7:539-
552.
62.BlackwellJM,RobertsCW,RoachTI,Alexander
J.Influenceofmacrophage
resistancegeneLsh/ItylBcg(candidateNramp)onToxoplasmagondiiinfectionin
mice.Clin.Exp.Immunol.1994;97:107-112.
63.KovarovaH,Hemychova
L,HajduchM,SirovaM,MacelaA.Influenceofthe
bcglocusonnaturalresistancetoprimaryinfectionwiththefacultative
intracellularbacteriumFrancisellatularensisinmice.Infect.Immun.
2000;68:1480-1484.
64.KovarovaH,HaladaP,ManPetal.Proteomestudy
ofFrancisellatularensislive
vaccinestrain-containingphagosomeinBcg/Nramp1congenicmacrophages:
resistant
alle1econtributestopermissiveenvironmentandsusceptibilityto
infection.Proteomics.2002;2:85-93.
65.MedinaE,RogersonBJ,North
RJ.TheNramplantimicrobialresistancegene
segregatesindependentlyofresistancetovirulentMycobacteriumtuberculosis.
Immunology1996;88:479-481.
66.MedinaE,North
RJ.EvidenceinconsistentwitharolefortheBcggene(Nrampl)
inresistance
ofmicetoinfectionwithvirulentMycobacteriumtuberculosis.
J.Exp.Med.1996;183:1045-1051.
67.North
RJ,LaCourseR,RyanL,GrosP.ConsequenceofNrampldeletionto
Mycobacteriumtuberculosisinfectioninmice.Infect.Immun.1999;67:5811-5814.
68.
MaX,DouS,WrightJAetal.5’dinucleotiderepeatpolymorphismofNRAMPI
andsusceptibilitytotuberculosisamongCaucasianpatientsinHouston,Texas.
Int.J.Tuberc.LungDis.2002;6:818-823.
69.KimJH,LeeSV,LeeSHetal.NRAMPIgeneticpolymorphismsasariskfactor
oftuberculouspleurisy.Int.J.Tuberc.LungDis.2003;7:370-375.
70.AbeT,IinumaY,AndoMet
al.NRAMPIpolymorphisms,susceptibilityand
clinicalfeaturesoftuberculosis.J.Infect.2003;46:215-220.
71.LiuW,CaoWC,ZhangCYetal.VDRandNRAMPIgenepolymorphismsin
susceptibilitytopulmonarytuberculosisamongtheChineseHanpopulation:a
case-controlstudy.Int.J.Tuberc.LungDis.2004;8:428-434.
72.FitnessJ,FloydS,
WamdorffDKetal.Large-scalecandidategenestudyof
tuberculosissusceptibilityintheKarongadistrictofnorthemMalawi.
Am.J.Trop.Med.Hyg.2004;71:341-349.
203
73.HoalEG,LewisLA,JamiesonSEetal.SLCIIAI(NRAMPl)butnotSLCI1A2
(NRAMP2)polymorphismsareassociatedwithsusceptibilitytotuberculosisina
high-incidencecommunityinSouthAfrica.Int.J.Tuberc.LungDis.2004;8:1464-
1471.
74.Malik
S,AbelL,TookerHetal.AllelesoftheNRAMPIgeneareriskfactorsfor
pediatrietuberculosisdisease.Proc.NatI.Acad.Sci.U.S.A2005;102:12183-12188.
75.ClemensDL,HorwitzMA.Characterization
oftheMycobacteriumtuberculosis
phagosomeandevidencethatphagosomalmaturationisinhibited.J.Exp.Med.
1995;181:257-270.
76.ClemensDL,LeeBY,HorwitzMA.Deviantexpression
ofRab5onphagosomes
containingtheintracellularpathogensMycobacteriumtuberculosisandLegionella
pneumophilaisassociatedwithalteredphagosomalfate.Infect.hnmun.
2000;68:2671-2684.
77.ClemensDL,LeeBY,HorwitzMA.MycobacteriumtuberculosisandLegionella
pneumophilaphagosomesexhibitarrestedmaturationdespiteacquisition
ofRab7.
Infect.Immun.2000;68:5154-5166.
78.RussellDG,DantJ,Sturgill-Koszycki
S.Mycobacteriumavium-and
Mycobacteriumtuberculosis-containingvacuolesaredynamic,fusion-competent
vesiclesthatareaccessibletoglycosphingolipidsfromthehostcellplasmalemma.
J.Immunol.1996;156:4764-4773.
79.SchaibleUE,Sturgill-KoszyckiS,SchlesingerPH,RussellDG.Cytokine
activationleadstoacidificationandincreasesmaturationofMycobacterium
avium-containingphagosomes
inmurinemacrophages.J.Immunol.
1998;160:1290-1296.
80.Sturgill-KoszyckiS,SchlesingerPH,ChakrabortyPetal.Lack
ofacidificationin
Mycobacteriumphagosomesproduced
byexclusionofthevesicularproton
ATPase.Science1994;263:678-681.
81.Sturgill-KoszyckiS,SchaibleUE,RussellDG.Mycobacterium-containing
phagosomesareaccessibletoearlyendosomesandreflectatransitionalstate
in
normalphagosomebiogenesis.EMBOJ.1996;15:6960-6968.
82.FrehelC,Canonne-HergauxF,GrosP,
DeCC.EffectofNramplonbacterial
replicationand
onmaturationofMycobacteriumavium-containingphagosomesin
bonemarrow-derivedmousemacrophages.CellMicrobiol.2002;4:541-556.
83.HackamDJ,RotsteinOD,ZhangWetal.Hostresistancetointracellular
infection:mutation
ofnaturalresistance-associatedmacrophageprotein1
(Nrampl)impairsphagosomalacidification.J.Exp.Med.1998;188:351-364.
204
84.BarreraLF,Kramnikl,SkameneE,RadziochD.Nitriteproductionby
macrophagesderivedfromBCG-resistantand-susceptiblecongenicmousestrains
inresponsetoIFN-gammaandinfectionwithBCG.Immunology1994;82:457-
464.
85.BartonCH,WhiteheadSH,BlackwellJM.Nramptransfectiontransfers
Ity/LshIBcg-relatedpleiotropiceffectsonmacrophageactivation:influenceon
oxidativeburstandnitricoxidepathways.Mol.Med.1995;1:267-279.
86.FritscheG,DlaskaM,BartonHetal.Nramp1functionalityincreasesinducible
nitricoxidesynthasetranscriptionviastimulation
ofIFNregulatoryfactor1
expression.J.Immunol.2003;171:1994-1998.
87.WagnerD,MaserJ,LaiBetal.ElementalanalysisofMycobacteriumavium-,
Mycobacteriumtuberculosis-,andMycobacteriumsmegmatis-containing
phagosomesindicatespathogen-inducedmicroenvironmentswithinthehostcell’s
endosomalsystem.J.Immunol.2005;174:1491-1500.
88.KelleyVA,SchoreyJS.Mycobacterium’sarrestofphagosomematurationin
macrophagesrequiresRab5activityandaccessibilitytoiron.Mo1.Biol.Cell
2003;14:3366-3377.
89.SchnappingerD,EhrtS,VoskuilMIetal.TranscriptionalAdaptation
of
MycobacteriumtuberculosiswithinMacrophages:InsightsintothePhagosomal
Environment.J.Exp.Med.2003;198:693-704.
90.GomesMS,Appelberg
R.Evidenceforalinkbetweenironmetabolismand
NramplgenefunctionininnateresistanceagainstMycobacteriumavium.
Immunology1998;95:165-168.
91.Ratledge
C.Iron,mycobacteriaandtuberculosis.Tuberculosis(Edinb).
2004;84:110-130.
92.GomesMS,Appelberg
R.NRAMP1-orcytokine-inducedbacteriostasisof
Mycobacteriumaviumbymousemacrophagesisindependentoftherespiratory
burst.Microbiology2002;148:3155-3160.
93.KnodlerLA,Steele-Mortimer
O.Takingpossession:biogenesisofthe
Salmonella-containingvacuole.Traffic.2003;4:587-599.
94.JabadoN,Cuellar-MataP,GrinsteinS,GrosP.Ironchelatorsmodulatethe
fusogenicproperties
ofSalmonella-containingphagosomes.
Proc.Natl.Acad.Sci.U.S.A2003;100:6127-6132.
95.KehresDG,MaguireME.Emergingthemesinmanganesetransport,biochemistry
andpathogenesisinbacteria.FEMSMicrobiol.Rev.2003;27:263-290.
205
96.KammlerM.SchonC.HantkeK.Characterizationoftheferrousironuptake
system
ofEscherichiacoli.lBacteriol.1993;175:6212-6219.
97.KehresDG,JanakiramanA,SlauchJM,MaguireME.SitABCDisthealkaline
Mn(2+)transporter
ofSalmonellaentericaserovarTyphimurium.J.Bacteriol.
2002;184:3159-3166.
98.TsolisRM,BaumIerAJ,HeffronF,Stojiljkovic
1.ContributionofTonB-and
Feo-mediatedironuptaketogrowth
ofSalmonellatyphimuriurninthemouse.
Infect.Immun.1996;64:4549-4556.
99.ZhouD.HardtWD,GalanJE.Salmonellatyphimuriurnencodesaputativeiron
transportsystemwithinthecentisome63pathogenicityisland.Infect.Irnmun.
1999;67:1974-1981.
100.BeardenSW,PerryRD.TheYfesystem
ofYersiniapestistransportsironand
manganeseandisrequiredforfullvirulence
ofplague.Mol.Microbioi.
1999;32:403-414.
101.BoyerE.BergevinI,MaloD,GrosP,CellierMF.Acquisition
ofMn(1I)in
additiontoFe(II)isrequiredforfullvirulence
ofSalmonellaentericaserovar
Typhimurium.Infect.Immun.2002;70:6032-6042.
102.JanakiramanA,SlauchJM.TheputativeirontransportsystemSitABCDencoded
on
SPIlisrequiredforfullvirulenceofSalmonellatyphimurium.Mol.Microbioi.
2000;35:1146-1155.
103.ZaharikML,CullenVL.Fung
AMetal.TheSalmonellaentericaserovar
typhimuriurndivalentcationtransportsystemsMntHandSitABCDareessential
forvirulenceinanNramp1G169murinetyphoidmodel.Infect.Irnmun.
2004;72:5522-5525.
104.ZaharikML,VallanceBA,PuenteJL,GrosP,FinlayBB.Host-pathogen
interactions:HostresistancefactorNramp1up-regulatestheexpression
of
Salmonellapathogenicityisland-2virulencegenes.Proc.Natl.Acad.Sci.U.S.A
2002;99:15705-15710.
105.CellierM,GovoniG,VidalSetal.Hurnannaturalresistance-associated
macrophageprotein:cDNAc1oning,chromosomalmapping,genomic
organization,andtissue-specifieexpression.J.Exp.Med.1994;180:1741-1752.
106.BlackwellJM,BartonCH,White
JKetal.Genomicorganizationandsequenceof
thehurnanNRAMPgene:identificationandmappingofapromoterregion
polyrnorphism.Mol.Med.1995;1:194-205.
206
107.KishiF,TanizawaY,NobumotoM.Structuralanalysisofhumannatural
resistance-associatedmacrophageprotein1promoter.Mol.Immunol.
1996;33:265-268.
108.KishiF,TabuchiM.Completenucleotidesequence
ofhumanNRAMP2cDNA.
Mol.Immunol.1997;34:839-842.
109.NewportM,LevinM,BlackwellJetal.Evidenceforexclusion
ofamutationin
NRAMPasthecause
offamilialdisseminatedatypicalmycobacterialinfectionin
aMaltesekindred.J.Med.Genet.1995;32:904-906.
110.BlackwellJM.Genetics
ofhostresistanceandsusceptibilitytointramacrophage
pathogens:astudy
ofmulticasefamiliesoftuberculosis,leprosyandleishmaniasis
innorth-easternBrazil.InU.Parasitol.1998;28:21-28.
111.GreenwoodCM,FujiwaraTM,BoothroydLJetal.Linkage
oftuberculosisto
chromosome2q35loci,includingNRAMP1,inalargeaboriginalCanadian
family.Am.J.Hum.Genet.2000;67:405-416.
112.BellamyR,RuwendeC,CorrahTetal.Variations
intheNRAMP1geneand
susceptibilitytotuberculosis
inWestAfricans.N.Eng1.J.Med.1998;338:640-644.
113.GaoPS,FujishimaS,Mao
XQetal.GeneticvariantsofNRAMPlandactive
tuberculosisinJapanesepopulations.InternationalTuberculosisGeneticsTeam.
Clin.Genet.2000;58:74-76.
114.CervinoAC,LakissS,Sow
0,HillAV.Allelicassociationbetweenthe
NRAMP1geneandsusceptibilitytotuberculosis
inGuinea-Conakry.
Ann.Hum.Genet.2000;64:507-512.
115.TaypeCA,CastroJC,Accinelli
RAetal.AssociationbetweenSLC11A1
polymorphismsandsusceptibilitytodifferentclinicalforms
oftuberculosisinthe
Peruvianpopulation.Infect.Genet.Evol.2006
116.LiHT,ZhangTT,ZhouYQ,HuangQH,HuangJ.SLC11A1(formerly
NRAMP1)genepolymorphismsandtuberculosissusceptibility:ameta-analysis.
InU.Tuberc.LungDis.2006;10:3-12.
117.AbelL,SanchezFO,ObertiJetal.Susceptibilitytoleprosyislinkedtothe
human
NRAMPIgene.J.Infect.Dis.1998;177:133-145.
118.AlcaisA,SanchezFO,Thuc
NVetal.Granulomatousreactiontointradermal
injection
oflepromin(Mitsudareaction)islinkedtothehumanNRAMP1genein
Vietnameseleprosysibships.J.Infect.Dis.2000;181:302-308.
207
119.MeisnerSJ,MucklowS,WarnerGetal.AssociationofNRAMPlpolymorphism
withleprosytypebutnotsusceptibilitytoleprosyperseinwestAfricans.
Am.J.Trop.Med.Hyg.2001;65:733-735.
120.KohWJ,KwonOJ,KimEJetal.NRAMPIgenepolymorphismand
susceptibility
tonontuberculousmycobacteriallungdiseases.Chest2005;128:94-
101.
121.MarquetS,SanchezFO,AriasMetal.Variants
ofthehumanNRAMPIgeneand
alteredhumanimmunodeficiencyvirusinfectionsusceptibility.lInfect.Dis.
1999;180:1521-1525.
122.JohnS,MarlowA,HajeerAetal.Linkageandassociationstudies
ofthenatural
resistanceassociatedmacrophageprotein1(NRAMP
1)locusinrheumatoid
arthritis.J.Rheumatol.1997;24:452-457.
123.ShawMA,ClaytonD,AtkinsonSEetal.Linkageofrheumatoidarthritistothe
candidategeneNRAMPIon2q35.J.Med.Genet.1996;33:672-677.
124.RodriguezMR,Gonzalez-EscribanoMF,AguilarFetal.Association
of
NRAMP1promotergenepolymorphismwiththesusceptibilityandradiological
severityofrheumatoidarthritis.TissueAntigens2002;59:311-315.
125.SanjeeviCB,MillerEN,DabadghaoPetal.PolymorphismatNRAMP1and
D2S1471lociassociatedwithjuvenilerheumatoidarthritis.ArthritisRheum.
2000;43:1397-1404.
126.HofmeisterA,NeibergsHL,PokomyRM,Galandiuk
S.Thenaturalresistance
associatedmacrophageproteingeneisassociatedwithCrohn’sdisease.Surgery
1997;122:173-178.
127.KojimaY,KinouchiY,TakahashiSetal.Inflammatoryboweldiseaseis
associatedwitha
nove1promoterpolymorphismofnaturalresistance-associated
macrophageprotein1(NRAMP1)gene.TissueAntigens2001;58:379-384.
128.KotzeMJ,deVilliersJN,Rooney
RNetal.AnalysisoftheNRAMPIgene
implicatedinirontransport:associationwithmultiplesclerosisandageeffects.
BloodCellsMol.Dis.2001;27:44-53.
129.BassunyWM,IharaK,MatsuuraNetal.Associationstudy
oftheNRAMP1gene
promoterpolymorphismandearly-onsettype1diabetes.Immunogenetics
2002;54:282-285.
130.KisslerS,StemP,TakahashiKetal.InvivoRNAinterferencedemonstratesa
roleforNramplinmodifyingsusceptibilitytotype1diabetes.Nat.Genet.
2006;38:479-483.
208
131.GruenheidS,CellierM,VidalS,GrosP.Identificationandcharacterizationofa
secondmouseNrampgene.Genomics1995;25:514-525.
132.SupekF,SupekovaL,NelsonH,NelsonN.Ayeastmanganesetransporterrelated
tothemacrophageproteininvolvedinconferringresistancetomycobacteria.
Proc.NatI.Acad.Sci.U.S.A1996;93:5105-5110.
133.Hawlitschek
G,SchneiderH,SchmidtBetal.Mitochondrialproteinimport:
identificationofprocessingpeptidaseand
ofPEP,aprocessingenhancingprotein.
CeIl1988;53:795-806.
134.MuirWA,HopferU,KingM.Irontransportacrossbrush-bordermembranesfrom
normalandiron-deficientmouseuppersmallintestine.J.BioI.Chem.
1984;259:4896-4903.
135.MuirA,HopferU.Regionalspecificity
ofironuptakebysmallintestinalbrush
bordermembranesfromnormalandiron-deficientmice.Am.J.Physiol
1985;248:G376-G379.
136.FlemingMD,TrenorCC,
III,SuMAetal.Microcyticanaemiamicehavea
mutationinNramp2,acandidateirontransportergene.Nat.Genet.1997;16:383-
386.
137.RussellES,NashDJ,BernsteinSEetal.Characterizationandgeneticstudies
of
microcyticanemiainhousemouse.Blood1970;35:838-850.
138.EdwardsJA,HokeJE.Defect
ofintestinalmucosalironuptakeinmicewith
hereditarymicrocyticanemia.Proc.Soc.Exp.BioI.Med.1972;141:81-84.
139.HarrisonDE.Marrowtransplantationandirontherapyinmousehereditary
microcyticanemia.Blood1972;40:893-901.
140.EdwardsJA,HokeJE.Redcellironuptakeinhereditarymicrocyticanemia.
Blood1975;46:381-388.
141.FlemingMD,RomanoMA,Su
MAetal.Nramp2ismutatedintheanemic
Belgrade(b)rat:evidence
ofaroleforNramp2inendosomalirontransport.
Proc.NatI.Acad.Sci.U.S.A1998;95:1148-1153.
142.Sladic-SimicD,MartinovitchPN,ZivkovicNetal.Athalassemia-likedisorderin
Belgradelaboratoryrats.Ann.N.Y.Acad.Sci.1969;165:93-99.
143.EdwardsJA,GarrickLM,HokeJE.Defectiveironuptakeandglobinsynthesis
by
erythroidcellsintheanemiaoftheBelgradelaboratoryrat.Blood1978;51:347-
357.
209
144.EdwardsJA,SullivanAL,HokeJE.Defectivedeliveryofirontothedeveloping
redcell
oftheBelgradelaboratoryrat.Blood1980;55:645-648.
145.GarrickMD,Gniecko
K,LiuY,CohanDS,GarrickLM.Transferrinandthe
transferrincycleinBelgraderatreticulocytes.J.Biol.Chem.1993;268:14867-
14874.
146.SuMA,TrenorCC,FlemingJC,FlemingMD,AndrewsNC.TheG185R
mutationdisruptsfunction
oftheirontransporterNramp2.Blood1998;92:2157-
2163.
147.Canonne-HergauxF,FlemingMD,LevyJEetal.TheNramp2/DMTliron
transporterisinducedintheduodenum
ofmicrocyticanemiamkmicebutisnot
properlytargetedtotheintestinalbrushborder.Blood2000;96:3964-3970.
148.TouretN,Martin-OrozcoN,ParoutisPetal.Molecularandcellularmechanisms
underlyingirontransportdeficiencyinmicrocyticanemia.Blood2004;104:1526-
1533.
149.XuH,JinJ,DeFeliceLJ,AndrewsNC,ClaphamDE.Aspontaneous,recurrent
mutationindivalentmetaltransporter-lexposesacalciumentrypathway.
PLoS.Biol.2004;2:E50.
150.LevyJE,Jin
0,FujiwaraY,KuoF,AndrewsNe.Transferrinreceptoris
necessaryfordevelopment
oferythrocytesandthenervoussystem.Nat.Genet.
1999;21:396-399.
151.CiW,LiW,KeY,QianZM,Shen
X.IntracellularCa(2+)regulatesthecellular
ironuptakeinK562cells.CellCalcium2003;33:257-266.
152.VidalS,BelouchiAM,CellierM,BeattyB,GrosP.Cloningandcharacterization
ofasecondhumanNRAMPgeneonchromosome12q13.Mamm.Genome
1995;6:224-230.
153.LeePL,GelbartT,WestC,HalloranC,Beutler
E.ThehumanNramp2gene:
characterization
ofthegenestructure,alternativesplicing,promoterregionand
polymorphisms.BloodCellsMol.Dis.1998;24:199-215.
154.HubertN,HentzeMW.Previouslyuncharacterizedisoforms
ofdivalentmetal
transporter(DMT)-I:implicationsforregulationandcellularfunction.
Proc.Nat1.Acad.Sci.U.S.A2002;99:12345-12350.
155.TabuchiM,TanakaN,Nishida-KitayamaJ,OhnoH,KishiF.Alternativesplicing
regulatesthesubcellularlocalization
ofdivalentmetaltransporter1isoforms.
Mol.Biol.Ce1l2002;13:4371-4387.
210
156.ChaloupkaR,CourvilleP,VeyrierFetal.Identificationoffunctionalaminoacids
intheNrampfamily
byacombinationofevolutionaryanalysisandbiophysical
studies
ofmetalandprotoncotransportinvivo.Biochemistry2005;44:726-733.
157.MackenzieB,UjwalML,ChangMH,RomeroMF,HedigerMA.Divalentmetal
iontransporter
DMTlmediatesbothH(+)-coupledFe(2+)transportand
uncoupledfluxes.PflugersArch.2005
158.NevoY,NelsonN.ThemutationF227Iincreasesthecoupling
ofmetalion
transportinDCT1.J.BioI.Chem.2004;279:53056-53061.
159.CohenA,NevoY,NelsonN.Thefirstextemalloop
ofthemetaliontransporter
DCTIisinvolvedinmetalionbindingandspecificity.Proc.NatI.Acad.Sci.U.S.A
2003;100:10694-10699.
160.JohnsonDM,YamajiS,TennantJ,SraiSK,SharpPA.Regulationofdivalent
metaltransporterexpressioninhumanintestinalepithelialcellsfollowing
exposuretonon-haemiron.FEBSLett.2005;579:1923-1929.
161.Canonne-HergauxF,GruenheidS,PonkaP,GrosP.Cellularandsubcellular
localization
oftheNramp2irontransporterintheintestinalbrushborderand
regulation
bydietaryiron.Blood1999;93:4406-4417.
162.TrinderD,OatesPS,ThomasC,SadleirJ,MorganEH.Localisation
ofdivalent
metaltransporter1(DMT1)tothemicrovillusmembrane
ofratduodenal
enterocytesinirondeficiency,buttohepatocytesinironoverload.Gut
2000;46:270-276.
163.GriffithsWJ,KellyAL,SmithSJ,CoxTM.Localization
ofirontransportand
regulatoryproteinsinhumancells.QJM.2000;93:575-587.
164.YehKY,YehM,WatkinsJA,Rodriguez-ParisJ,Glass
J.Dietaryironinduces
rapidchangesinratintestinaldivalentmetaltransporterexpression.Am.J.Physiol
Gastrointest.LiverPhysiol2000;279:G1070-G1079.
165.ZollerH,KochRO,Theurl1et
al.Expressionoftheduodenalirontransporters
divalent-metaltransporter1andferroportin1inirondeficiencyandironoverload.
Gastroenterology2001;120:1412-1419.
166.
MaY,SpecianRD,YehKYetal.Thetranscytosisofdivalentmetaltransporter1
andapo-transferrinduringironuptakeinintestinalepithelium.Am.J.Physiol
Gastrointest.LiverPhysiol2002;283:G965-G97
4.
167.SharpP,TandyS,YamajiSetal.Rapidregulationofdivalentmetaltransporter
(DMT1)proteinbutnotmRNAexpressionbynon-haemironinhumanintestinal
Caco-2cells.FEBSLett.2002;510:71-76.
211
168.TandyS,WilliamsM,LeggettAetal.Nramp2expressionisassociatedwithpH
dependentironuptakeacrosstheapicalmembrane
ofhumanintestinalCaco-2
cells.
lBiol.Chem.2000;275:1023-1029.
169.Canonne-HergauxF,ZhangAS,PonkaP,GrosP.Characterization
oftheiron
transporterDMT1(NRAMP2/DCT1)
inredbloodcellsofnormalandanemic
mk/mkmice.Blood2001;98:3823-3830.
170.ChenL,BoadleRA,HarrisDC.Toxicityofholotransferrin
butnotalbuminin
proximaltubulecellsinprimaryculture.J.Am.Soc.Nephrol.1998;9:77-84.
171.JabadoN,Canonne-HergauxF,GruenheidS,PicardV,GrosP.Irontransporter
Nramp2/DMT
-1isassociatedwiththemembraneofphagosomesinmacrophages
andSertolicells.Blood2002;100:2617-2622.
172.Canonne-HergauxF,GrosP.Expression
oftheirontransporterDMT1inkidney
fromnormalandanemic
mkmice.KidneyInt.2002;62:147-156.
173.FergusonCJ,WareingM,Ward
DTetal.Cellularlocalizationofdivalentmetal
transporterDMT-1
inratkidney.Am.J.PhysiolRenalPhysioI2001;280:F803-
F814.
174.AbouhamedM,GburekJ,LiuWetal.Divalentmetaltransporter1inthekidney
proximaltubuleisexpressed
inlateendosomes/lysosomalmembranes:
Implicationsforrenalhandlingofprotein-metalcomplexes.Am.J.PhysiolRenal
Physiol2006
175.TchemitchkoD,BourgeoisM,MartinME,BeaumontC.Expression
ofthetwo
rnRNAisoforms
oftheirontransporterNrmap2/DMTIinmiceandfunctionofthe
ironresponsiveelement.Biochem.J.2002;363:449-455.
176.FergusonCJ,WareingM,DelannoyMetal.Ironhandlingandgeneexpression
of
thedivalentmetaltransporter,DMT1,inthekidneyoftheanemicBelgrade(b)
rat.KidneyInt.2003;64:1755-1764.
177.BurdoJR,MenziesSL,Simpson
lAetal.Distributionofdivalentmetal
transporter1andmetaltransportprotein1inthenormalandBelgraderat.
lNeurosci.Res.2001;66:1198-1207.
178.MoosT,MorganEH.Thesignificance
ofthemutateddivalentmetaltransporter
(DMTl)onirontransportintotheBelgraderatbrain.J.Neurochem.2004;88:233-
245.
179.LisA,ParadkarPN,SingletonSetal.Hypoxiainduceschangesinexpression
of
isoformsofthedivalentmetaltransporter(DMTl)inratpheochromocytoma
(PCI2)cells.Biochem.Pharmacol.2005;69:1647-1655.
212
180.ZhangAS,XiongS,TsukamotoH,EnnsCA.Localizationofironmetabolism
relatedmRNAsinratliverindicatethatRFEisexpressedpredominantlyin
hepatocytes.Blood2004;103:1509-1514.
181.GeorgieffMK,WobkenJK,WelleJ,BurdoJR,ConnorJR.Identificationand
localization
ofdivalentmetaltransporter-1(DMT-1)intennhumanplacenta.
Placenta2000;21:799-804.
182.Lam-Yuk-TseungS,GrosP.DistinctTargetingandRecyclingProperties
ofTwo
IsofonnsoftheIronTransporterDMT1(NRAMP2,Slc11A2).Biochemistry
2006;45:2294-2301.
183.BonifacinoJS,TraubLM.SignaIsforsortingoftransmembraneproteinsto
endosomesandlysosomes.Annu.Rev.Biochem.2003;72:395-447.
184.Lam-Yuk-TseungS,TouretN,GrinsteinS,GrosP.Carboxyl-Tenninus
Detenninants
oftheIronTransporterDMTlISLCIIA2IsofonnII(-IREIlB)
MediateIntemalizationfromthePlasmaMembraneintoRecyc1ingEndosomes.
Biochemistry2005;44:12149-12159.
185.OhgamiRS,CampagnaDR,GreerELet
al.Identificationofaferrireductase
requiredforefficienttransferrin-dependentironuptakeinerythroidcells.
Nat.Genet.2005;37:1264-1269.
186.OhgamiRS,CampagnaDR,AntiochosBetal.nml054:aspontaneous,recessive,
hypochromic,microcyticanemiamutationinthemouse.Blood2005;106:3625-
3631.
187.KawabataH,YangR,HiramaTetal.Molecularc10ningoftransferrinreceptor
2.
Anewmemberofthetransferrinreceptor-likefamily.J.Biol.Chem.
1999;274:20826-20832.
188.CamaschellaC,RoettoA,CaliAetal.ThegeneTFR2ismutatedinanewtype
ofhaemochromatosismappingto7q22.Nat.Genet.2000;25:14-15.
189.KozyrakiR,FyfeJ,VerroustPJetal.Megalin-dependentcubilin-mediated
endocytosisisamajorpathwayfortheapicaluptakeoftransferrininpolarized
epithelia.Proc.NatI.Acad.Sci.U.S.A2001;98:12491-12496.
190.McKieAT,BarrowD,Latunde-DadaGOet
al.Aniron-regulatedferricreductase
associatedwiththeabsorption
ofdietaryiron.Science2001;291:1755-1759.
191.GunshinH,StarrCN,DirenzoCetal.Cybrdl(duodenalcytochromeb)isnot
necessaryfordietaryironabsorptioninmice.Blood2005;106:2879-2883.
213
192.MoosT.Immunohistochemicallocalizationofintraneuronaltransferrinreceptor
immunoreactivityintheadultmousecentralnervoussystem.J.CompNeurol.
1996;375:675-692.
193.MoosT,TrinderD,MorganEH.Cellulardistributionofferriciron,ferritin,
transferrinanddivalentmetaltransporter1(DMT1)insubstantianigraandbasal
ganglia
ofnormalandbeta2-microglobulindeficientmousebrain.Cell
Mol.Biol.(Noisy.-le-grand)2000;46:549-561.
194.MoosT,MorganEH.Evidenceforlowmolecularweight,non-transferrin-bound
ironinratbrainandcerebrospinalfluid.J.Neurosci.Res.1998;54:486-494.
195.JeongSY,David
S.Glycosylphosphatidylinositol-anchoredceruloplasminis
requiredforironeffluxfrorncellsinthecentralnervoussystem.lBiol.Chem.
2003;278:27144-27148.
196.ChangYZ,KeY,DuJRetal.IncreasedDivalentMetalTransporter1Expression
MightBeAssociatedwiththeNeurotoxicity
ofL-DOPA.Mol.Pharmacol.
2006;69:968-974.
197.MoosT,MorganEH.Thernetabolism
ofneuronalironanditspathogenicrolein
neurologicaldisease:review.Ann.N.Y.Acad.Sci.2004;1012:14-26.
198.WareingM,FergusonCJ,DelannoyMetal.Altereddietaryironintakeisastrong
rnodulator
ofrenalDMTlexpression.Am.J.PhysiolRenalPhysiol
2003;285:FI050-FI059.
199.WeintraubLR,ConradME,CrosbyWH.Absorption
ofhernoglobinironbythe
rat.Proc.Soc.Exp.Biol.Med.1965;120:840-843.
200.ConradME,WeintraubLR,SearsDA,CrosbyWH.Absorptionofhernoglobin
iron.Am.J.PhysiolI966;211:1123-1130.
201.ShayeghiM,Latunde-DadaGO,OakhillJSetal.Identification
ofanintestinal
hernetransporter.CeIl2005;122:789-801.
202.WhebyMS,SuttleGE,FordKT,
III.Intestinalabsorptionofhernoglobiniron.
Gastroenterology1970;58:647-654.
203.ParmleyRT,BartonJC,ConradME,AustinRL,HollandRM.Ultrastructural
cytochernistryandradioautographyofhernoglobin–ironabsorption.
Exp.Mol.Pathol.1981;34:131-144.
204.RaffinSB,WooCH,RoostKT,PriceDC,Schmid
R.Intestinalabsorptionof
hernoglobiniron-hernec1eavagebyrnucosalherneoxygenase.J.Clin.Invest
1974;54:1344-1352.
214
205.YangJ,GoetzD,LiJYetal.Anirondeliverypathwaymediatedbyalipocalin.
Mo1.Ce1l2002;10:1045-1056.
206.Kaplan
J.Mechanismsofcellularironacquisition:anotherironinthefire.Cell
2002;
111:603-606.
207.TortiFM,TortiSV.Regulationofferritingenesandprotein.Blood
2002;99:3505-3516.
208.HarrisonPM.Ferritin:aniron-storagemolecule.Semin.Hematol.1977;14:55-70.
209.HarrisonPM,ArosioP.Theferritins:molecularproperties,ironstoragefunction
andcellularregulation.Biochirn.Biophys.Acta1996;1275:161-203.
210.TaketaniS.Aquisition,rnobilizationandutilization
ofcellularironandherne:
endlessfindingsandgrowingevidence
oftightregulation.TohokuJ.Exp.Med.
2005;205:297-318.
211.ZhangAS,SheftelAD,PonkaP.Intracellularkinetics
ofironinreticulocytes:
evidenceforendosomeinvolvementinirontargetingtomitochondria.Blood
2005;105:368-375.
212.TaketaniS,Tokunaga
R.Hernetransportfrornratliverrnitochondriatothe
microsomesinvitro.Biochem.Biophys.Res.Cornmun.1980;92:1343-1347.
213.SenjoM,IshibashiT,Imai
Y.Purificationandcharacterizationofcytosolicliver
proteinfacilitatinghemetransportintoapocytochromeb5frommitochondria.
Evidenceforidentifyingthehemetransferproteinasbelongingtoagroup
of
glutathioneS-transferases.J.BioI.Chem.1985;260:9191-9196.
214.JohnsonDC,DeanDR,SmithAD,JohnsonMK.Structure,function,and
formationofbiologicaliron-suIfurc1usters.Annu.Rev.Biochern.2005;74:247-
281.
215.MuhlenhoffU,GerberJ,RichhardtN,LillR.Componentsinvolvedinassembly
anddislocation
ofiron-suifurclustersonthescaffoldproteinIsulp.EMBOJ.
2003;22:4815-4825.
216.ChenOS,HernenwayS,Kaplan
J.InhibitionofFe-Sclusterbiosynthesis
decreasesmitochondrialironexport:evidencethat
YfhlpaffectsFe-Sc1uster
synthesis.Proc.Nat1.Acad.Sci.U.S.A2002;99:12321-12326.
217.AdamecJ,RusnakF,OwenWGet
al.Iron-dependentself-assemblyof
recombinantyeastfrataxin:implicationsforFriedreichataxia.Am.J.Hum.Genet.
2000;67:549-562.
215
218.RotigA,deLP,ChretienDetal.Aconitaseandmitochondrialiron~sulphur
proteindeficiencyinFriedreichataxia.Nat.Genet.1997;17:215-217.
219.AbboudS,HaileDJ.Anovelmammalianiron-regulatedproteininvolvedin
intracellularironmetabolism.J.Biol.Chem.
2000;275:19906~19912.
220.DonovanA,BrownlieA,ZhouYetal.Positionalc10ningofzebrafish
ferroportinlidentifiesaconservedvertebrateironexporter.Nature2000;403:776-
781.
221.McKieAT,MarcianiP,RolfsAetal.Anovelduodenaliron-regulated
transporter,IREG
1,implicatedinthebasolateraltransferofirontothecirculation.
Mo1.Ce1l2000;5:299~309.
222.VulpeCD,KuoYM,MurphyTLetal.Hephaestin,aceruloplasminhomologue
implicatedinintestinalirontransport,isdefectiveintheslamouse.Nat.Genet.
1999;21:
195~199.
223.HarrisZL,DurleyAP,ManTK,GitlinJD.Targetedgenedisruptionrevealsan
essentialroleforceruloplasminincellularironefflux.Proc.NatI.Acad.Sci.U.S.A
1999;96:10812-10817.
224.DelabyC,PilardN,GoncalvesAS,BeaumontC,Canonne-Hergaux
F.Presence
oftheironexporterferroportinattheplasmamembraneofmacrophagesis
enhanced
byiron10adinganddown-regulatedbyhepcidin.B100d2005;106:3979-
3984.
225.NemethE,TuttleMS,PowelsonJetal.Hepcidinregulatescellularironeffluxby
bindingtoferroportinandinducingitsintema1ization.Science2004;306:2090-
2093.
226.Pantopoulos
K.IronmetabolismandtheIRE/IRPregulatorysystem:anupdate.
Ann.N.Y.Acad.Sci.2004;1012:1-13.
227.HentzeMW,KuhnLC.Molecularcontrolofvertebrateironmetabolism:mRNA
basedregulatorycircuitsoperated
byiron,nitricoxide,andoxidativestress.
Proc.Natl.Acad.Sci.U.S.A1996;93:8175-8182.
228.Meyron-HoltzEG,GhoshMC,RouaultTA.Mammaliantissueoxygenlevels
modulateiron-regulatoryproteinactivitiesinvivo.Science2004;306:2087-2090.
229.WangJ,ChenG,PantopoulosK.NitricoxideinhibitsthedegradationofIRP2.
Mol.CellBiol.2005;25:1347-1353.
230.CoopermanSS,Meyron-HoltzEG,Olivierre-WilsonHetal.Microcyticanemia,
erythropoieticprotoporphyria,andneurodegenerationinmicewithtargeted
deletionofiron-regulatoryprotein
2.Blood2005;106:1084-1091.
216
231.Meyron-HoltzEG,GhoshMC,IwaiKetal.Geneticablationsofironregulatory
proteins1and2revealwhyironregulatoryprotein2dominatesironhomeostasis.
EMBO
J.2004;23:386-395.
232.EisensteinRS,RossKL.Novelrolesforironregulatoryproteinsintheadaptive
responsetoirondeficiency.J.Nutr.2003;133:1510S-1516S.
233.MuckenthalerM,GrayNK,HentzeMW.IRP-1bindingtoferritinmRNA
preventstherecruitment
ofthesmallribosomalsubunitbythecap-binding
complexeIF4F.Mol.Cell1998;2:383-388.
234.GunshinH,AllersonCR,Polycarpou-SchwarzMetal.Iron-dependentregulation
ofthedivalentmetaliontransporter.FEBSLett.2001;509:309-316.
235.LudwiczekS,AignerE,Theurll,Weiss
G.Cytokine-mediatedregulationofiron
transportinhumanmonocyticcells.Blood2003;101:4148-4154.
236.YangF,LiuXB,QuinonesMet
al.Regulationofreticuloendothelialiron
transporterMTPI
(Slclla3)byinflammation.J.Biol.Chem.2002;277:39786-
39791.
237.SayersMH,EnglishG,Finch
C.Capacityofthestore-regulatorinmaintaining
ironbalance.Am.J.Hematol.1994;47:194-197.
238.FinchC.Regulators
ofironbalanceinhumans.Blood1994;84:1697-1702.
239.TrenorCC,III,CampagnaDR,SellersVM,AndrewsNC,FlemingMD.The
moleculardefectinhypotransferrinemicmice.Blood2000;96:1113-1118.
240.LevyJE,MontrossLK,CohenDE,FlemingMD,Andrews
Ne.TheC282Y
mutationcausinghereditaryhemochromatosisdoesnotproduceanullallele.
Blood1999;94:9-11.
241.KollerBH,MarrackP,KapplerJW,Smithies
O.Normaldevelopmentofmice
deficientinbeta2M,MHCclass1proteins,andCD8+Tcells.Science
1990;248:1227-1230.
242.KrauseA,NeitzS,MagertHJetal.LEAP-1,anovelhighlydisulfide-bonded
humanpeptide,exhibitsantimicrobialactivity.FEBSLett.2000;480:147-150.
243.ParkCH,ValoreEV,WaringAJ,Ganz
T.Hepcidin,aurinaryantimicrobial
peptidesynthesizedintheliver.J.Biol.Chem.2001;276:7806-7810.
244.PigeonC,IlyinG,CourselaudBetal.Anewmouseliver-specificgene,encoding
aproteinhomologoustohumanantimicrobialpeptidehepcidin,isoverexpressed
duringironoverload.J.Biol.Chem.2001;276:7811-7819.
217
245.NicolasG,BennounM,Devaux1etal.Lackofhepcidingeneexpressionand
severetissueironoverloadinupstreamstimulatoryfactor2(USF2)knockout
mice.Proc.NatI.Acad.Sci.U.S.A2001;98:8780-8785.
246.NicolasG,BennounM,PorteuAetal.Severeirondeficiencyanemiain
transgenicmiceexpressingliverhepcidin.Proc.NatI.Acad.Sci.U.S.A
2002;99:4596-4601.
247.AndersonGJ,FrazerDM,WilkinsSJetal.Relationshipbetweenintestinaliron
transporterexpression,hepatichepcidinlevelsandthecontrol
ofironabsorption.
Biochem.Soc.Trans.2002;30:724-726.
248.NicolasG,ChauvetC,ViatteLetal.Thegeneencodingtheironregulatory
peptidehepcidinisregulated
byanemia,hypoxia,andinflammation.J.Clin.Invest
2002;110:1037-1044.
249.ShikeH,LauthX,Westerman
MEetal.Basshepcidinisanovelantimicrobial
peptideinduced
bybacterialchallenge.Eur.J.Biochem.2002;269:2232-2237.
250.YehKY,YehM,GlassJ.Hepcidinregulationofferroportin1expressioninthe
liverandintestine
oftherat.Am.J.PhysiolGastrointest.LiverPhysiol
2004;286:G385-G394.
251.HentzeMW,MuckenthalerMU,AndrewsNC.Balancingacts:molecularcontrol
ofmammalianironmetabolism.Ce1l2004;117:285-297.
252.LaftahAH,RameshB,SimpsonRJetal.Effect
ofhepcidinonintestinaliron
absorptioninmice.Blood2004;103:3940-3944.
253.FrazerDM,WilkinsSJ,Becker
EMetal.Hepcidinexpressioninverselycorrelates
withtheexpression
ofduodenalirontransportersandironabsorptioninrats.
Gastroenterology2002;123:835-844.
254.NemethE,ValoreEV,TerritoMetal.Hepcidin,aputativemediator
ofanemiaof
inflammation,isatypeIIacute-phaseprotein.Blood2003;101:2461-2463.
255.GehrkeSG,KulaksizH,HerrmannTetal.Expression
ofhepcidininhereditary
hemochromatosis:evidenceforaregulation
inresponsetotheserumtransferrin
saturationandtonon-transferrin-boundiron.Blood2003;102:371-376.
256.OatesPS,ThomasC.Augmentedinternalisation
offerroportinto1ateendosomes
impairsironuptake
byenterocyte-likeIEC-6cells.PflugersArch.2005;450:317-
325.
257.NemethE,PrezaGC,JungCLetal.TheN-terminus
ofhepcidinisessentialfor
itsinteractionwithferroportin:structure-functionstudy.Blood2006;107:328-333.
218
258.GunshinH,FujiwaraY,CustodioAOetal.Slclla2isrequiredforintestinaliron
absorptionanderythropoiesisbutdispensableinplacentaandliver.J.Clin.lnvest
2005;115:1258-1266.
259.PriwitzerovaM,PospisilovaD,PrchalJTetal.Severehypochromicmicrocytic
anemiacaused
byacongenitaldefectoftheirontransportpathwayinerythroid
cells.Blood2004;103:3991-3992.
260.PriwitzerovaM,NieG,Sheftel
ADetal.Functionalconsequencesofthehuman
DMTl(SLCIIA2)mutationonproteinexpressionandironuptake.Blood
2005;106:3985-3987.
261.GunshinH,JinJ,FujiwaraYet
al.AnalysisoftheE399DmutationinSLCIIA2.
Blood2005;106:2221-2222.
262.Lam-Yuk-TseungS,MathieuM,GrosP.Functionalcharacterization
ofthe
E399DDMTIINRAMP2/SLCIIA2proteinproducedbyanexon12mutationina
patientwithmicrocyticanemiaandironoverload.BloodCellsMol.Dis.
2005;35:212-216.
263.IolasconA,d’ApolitoM,ServedioVetal.Microcyticanemiaandhepaticiron
overloadinachildwithcompoundheterozygousmutationsin
DMTl.Blood2005
264.Lam-Yuk-TseungS,CamaschellaC,IolasconA,GrosP.AnovelR416C
mutationinhuman
DMTl(SLCIIA2)displayspleiotropiceffectsonfunctionand
causesmicrocyticanemiaandhepaticironoverload.BloodCellsMol.Dis.
2006;36:347-354.
265.BeaumontC,DelaunayJ,HetetGetal.Twonewhuman
DMTlmutationsina
compoundheterozygouspatientwithmircocyticanemiaandlowironstores
(Poster).Atlanta,GA,USA.December10-13,2005:
266.PietrangeloA.Hereditaryhemochromatosis–anewlookatanolddisease.
N.Engl.J.Med.2004;350:2383-2397.
267.FederJN,GnirkeA,Thomas
Wetal.AnovelMHCc1assI-likegeneismutated
inpatientswithhereditaryhaemochromatosis.Nat.Genet.1996;13:399-408.
268.LebronJA,BennettMJ,VaughnDEetal.Crystalstructure
ofthe
hemochromatosisproteinHFEandcharacterizationofitsinteractionwith
transferrinreceptor.Ce111998;93:111-123.
269.DrakesmithH,SweetlandE,SchimanskiLetal.Thehemochromatosisprotein
HFEinhibitsironexportfrommacrophages.Proc.NatI.Acad.Sci.U.S.A
2002;99:15602-15607.
219
270.WaheedA,GrubbJR,ZhouXYetal.Regulationoftransferrin-mediatediron
uptake
byHFE,theproteindefectiveinhereditaryhemochromatosis.
Proc.NatI.Acad.Sci.U.S.A2002;99:3117-3122.
271.GrossCN,IrrinkiA,FederJN,EnnsCA.Co-trafficking
ofHFE,anonc1assical
majorhistocompatibilitycomplex
c1ass1protein,withthetransferrinreceptor
impliesaroleinintracellularironregulation.J.BioI.Chem.1998;273:22068-
22074.
272.ParkkilaS,WaheedA,BrittonRSetal.Association
ofthetransferrinreceptorin
humanplacentawithHFE,theproteindefective
inhereditaryhemochromatosis.
Proc.NatI.Acad.Sci.U.S.A1997;94:13198-13202.
273.BennettMJ,LebronJA,BjorkmanPJ.Crystalstructure
ofthehereditary
haemochromatosisproteinHFEcomplexedwithtransferrinreceptor.Nature
2000;403:46-53.
274.AhmadKA,AhmannJR,MigasMCetal.Decreasedliverhepcidinexpressionin
theHfeknockoutmouse.BloodCellsMol.Dis.2002;29:361-366.
275.MuckenthalerM,RoyCN,CustodioAOetal.Regulatorydefects
inliverand
intestineimplicateabnormalhepcidinandCybrdlexpressioninmouse
hemochromatosis.Nat.Genet.2003;34:102-107.
276.Nicolas
G,ViatteL,LouDQetal.Constitutivehepcidinexpressionpreventsiron
overloadinamousemodelofhemochromatosis.Nat.Genet.2003;34:97-101.
277.HattoriA,WakusawaS,HayashiHetal.AVAQ594-597deletion
oftheTfR2
gene
inaJapanesefamilywithhemochromatosis.Hepatol.Res.2003;26:154-156.
278.KoyamaC,WakusawaS,HayashiHetal.Twonovelmutations,L490Rand
V561X,
ofthetransferrinreceptor2geneinJapanesepatientswith
hemochromatosis.Haematologica2005;90:302-307.
279.RoettoA,TotaroA,PipemoAetal.Newmutationsinactivatingtransferrin
receptor2inhemochromatosistype3.Blood2001;97:2555-2560.
280.GirelliD,BozziniC,RoettoAetal.Clinicalandpathologicfindings
in
hemochromatosistype3duetoanovelmutationintransferrinreceptor2gene.
Gastroenterology2002;122:1295-1302.
281.LeGG,MonsF,
JacolotSetal.Earlyonsethereditaryhemochromatosisresulting
fromanovelTFR2genenonsensemutation(RI05X)
intwosiblingsofnorth
Frenchdescent.BrJ.Haematol.2004;125:674-678.
282.PipemoA,RoettoA,MarianiRetal.Homozygosityfortransferrinreceptor-2
Y250Xmutationinducesearlyironoverload.Haematologica2004;89:359-360.
220
283.DeGM,RoettoA,PipemoAetal.Naturalhistoryofjuvenilehaemochromatosis.
Br.J.Haematol.2002;117:973-979.
284.RoettoA,Papanikolaou
G,PolitouMetal.Mutantantimicrobialpeptidehepcidin
isassociatedwithseverejuvenilehemochromatosis.Nat.Genet.2003;33:21-22.
285.CamaschellaC,RoettoA,DeGM.Juvenilehemochromatosis.Semin.Hematol.
2002;39:242-248.
286.RoettoA,DaraioF,PorporatoPetal.Screeninghepcidinformutationsin
juvenilehemochromatosis:identification
ofanewmutation(C70R).Blood
2004;103:2407-2409.
287.MajoreS,BinniF,PenneseAetal.HAMPgenemutationc.208T>C(p.C70R)
identifiedinanItalianpatientwithseverehereditaryhemochromatosis.
Hum.Mutat.2004;23:400.
288.DelatyckiMB,AllenKJ,GowPetal.AhomozygousHAMPmutationina
multiplyconsanguineousfamilywithpseudo-dominantjuvenile
hemochromatosis.Clin.Genet.2004;65:378-383.
289.PapanikolaouG,SamuelsME,LudwigEHetal.MutationsinHFE2causeiron
overload
inchromosome1q-linkedjuvenilehemochromatosis.Nat.Genet.
2004;36:77-82.
290.Camaschella
C.Understandingironhomeostasisthroughgeneticanalysisof
hemochromatosisandrelateddisorders.Blood2005;106:3710-3717.
291.MontosiG,DonovanA,TotaroAetal.Autosomal-dominanthemochromatosisis
associatedwithamutationintheferroportin(SLC11A3)gene.J.Clin.Invest
2001;108:619-623.
292.NjajouOT,VaessenN,JoosseMetal.Amutation
inSLCIIA3isassociatedwith
autosomaldominanthemochromatosis.Nat.Genet.2001;28:213-214.
293.SchimanskiLM,DrakesmithH,Merryweather-Clarke
ATetal.Invitrofunctional
analysis
ofhumanferroportin(FPN)andhemochromatosis-associatedFPN
mutations.Blood2005;105:4096-4102.
294.DrakesmithH,SchimanskiLM,OrmerodEetal.Resistancetohepcidinis
conferred
byhemochromatosis-associatedmutationsofferroportin.Blood
2005;106:1092-1097.
295.DeDomenicol,WardDM,NemethEetal.Themolecularbasisofferroportin
linkedhemochromatosis.Proc.NatI.Acad.Sci.U.S.A2005;102:8955-8960.
221
296.GoncalvesAS,MuzeauF,BlaybelRetal.Wildtypeandmutantferroportinsdo
notfonnoligomersintransfectedcells.Biochem.J.2006
297.AgranoffD,MonahanlM,ManganJA,ButcherPD,KrishnaS.Mycobacterium
tuberculosisexpressesanovelpH-dependentdivalentcationtransporterbelonging
totheNrampfamily.
lExp.Med.1999;190:717-724.
298.BelouchiA,CellierM,KwanTetal.Themacrophage-specificmembraneprotein
Nrampcontrollingnaturalresistancetoinfections
inmicehashomologues
expressedintherootsystemofplants.PlantMol.Biol.1995;29:1181-1196.
299.HaemigHA,BrookerRJ.Importance
ofconservedacidicresiduesinmntH,the
Nramphomolog
ofEscherichiacoli.J.Membr.Biol.2004;201:97-107.
300.MackenzieB,UjwalML,ChangMH,RomeroMF,HedigerMA.Divalentmetal
iontransporter
DMTlmediatesbothH(+)-coupledFe(2+)transportand
uncoupledfluxes.PflugersArch.2005
301.TabuchiM,YoshimoriT,YamaguchiK,YoshidaT,KishiF.Human
NRAMP2IDMT1,whichmediatesirontransportacrossendosomalmembranes,is
localizedtolateendosomesandlysosomes
inHEp-2cells.J.Biol.Chem.
2000;275:22220-22228.
302.FlemingMD,AndrewsNC.Mammalianirontransport:anunexpectedlink
betweenmetalhomeostasisandhostdefense.J.LabClin.Med.1998;132:464-468.
303.ChungJ,Wessling-ResnickM.Molecularmechanismsandregulation
ofiron
transport.CritRev.Clin.LabSci.2003;40:151-182.
304.CellierM,ShustikC,DaltonWetal.Expression
ofthehumanNRAMPIgenein
professionalprimaryphagocytes:studiesinbloodcellsandinHL-60
promyelocyticleukemia.J.Leukoc.Biol.1997;61:96-105.
305.GruenheidS,GrosP.Geneticsusceptibilitytointracellularinfections:
Nrampl,
macrophagefunctionanddivalentcationstransport.Curr.Opin.Microbiol.
2000;3:43-48.
306.SupekF,SupekovaL,NelsonH,NelsonN.Function
ofmetal-ionhomeostasisin
thecelldivisioncycle,mitochondrialproteinprocessing,sensitivityto
mycobacterialinfectionandbrainfunction.J.Exp.Biol.1997;200
(Pt2):321-330.
307.OrgadS,NelsonH,SegalD,NelsonN.Metalionssuppresstheabnonnaltaste
behavior
oftheDrosophilamutantmalvolio.J.Exp.Biol.1998;201:115-120.
308.UrbatschIL,JulienM,Carrier1etal.Mutationalanalysis
ofconserved
carboxylateresidues
inthenucleotidebindingsitesofP-glycoprotein.
Biochemistry2000;39:14138-14149.
222
309.Sherman,F,Fink,G.R.,andHicks,J.B.MethodsofYeastGenetics.1982.NY,
Co
IdSpringsHarbor.
RefType:Generic
310.KwanT,GrosP.Mutationalanalysis
oftheP-glycoproteinfirstintracellularloop
andflankingtransmembranedomains.Biochemistry1998;37:3337-3350.
311.KastC,CanfieldV,LevensonR,GrosP.MembranetopologyofP-glycoprotein
asdeterminedbyepitopeinsertion:transmembraneorganizationoftheN-terminal
domainofmdr3.Biochemistry1995;34:4402-4411.
312.NakagamaH,HeinrichG,PelletierJ,HousmanDE.Sequenceandstructural
requirementsforhigh-affinityDNAbinding
bytheWTlgeneproduct.Mol.Cell
Biol.1995;15:1489-1498.
313.EngBH,GuerinotML,EideD,SaierMH,Jr.Sequenceanalysesandphylogenetic
characterization
oftheZIPfamilyofmetaliontransportproteins.J.Membr.Biol.
1998;166:1-7.
314.VossJ,Salwinski
L,KabackHR,HubbellWL.Amethodfordistance
determinationinproteinsusingadesignedmetalionbindingsiteandsite-directed
spinlabeling:evaluationwithT4lysozyme.Proc.NatI.Acad.ScLU.S.A
1995;92:12295-12299.
315.OmichinskiJG,TrainorC,EvansTetal.Asmallsingle-“finger”peptidefromthe
erythroidtranscriptionfactorGATA-lbindsspecificallytoDNAasazincoriron
complex.Proc.Nat1.Acad.Sci.U.S.A1993;90:1676-1680.
316.GhazalehFA,OmburoGA,ColmanRW.Evidenceforthepresenceofessential
histidineandcysteineresiduesinplateletcGMP-inhibitedphosphodiesterase.
Biochem.J.1996;317
(Pt2):495-501.
317.WorthingtonMT,AmannBT,NathansD,BergJM.Metalbindingpropertiesand
secondarystructure
ofthezinc-bindingdomainofNup475.
Proc.NatI.Acad.Sci.U.S.A1996;93:13754-13759.
318.WorthingtonMT,Browne
L,BattleEH,LuoRQ.Functionalpropertiesof
transfectedhumanDMT1irontransporter.Am.J.PhysiolGastrointest.Liver
Physio12000;279:GI265-GI273.
319.KabackHR.Thelacpermease
ofEscherichiacoli:aprototypicenergy
transducingmembraneprotein.Biochim.Biophys.Acta1990;1018:160-162.
320.Muller-BergerS,KarbachD,KangDetal.Roles
ofhistidine752andglutamate
699inthepHdependence
ofmouseband3protein-mediatedaniontransport.
Biochemistry1995;34:9325-9332.
223
321.Muller-BergerS,KarbachD,KonigJetal.Inhibitionofmouseerythroidband3-
mediatedchloridetransport
bysite-directedmutagenesisofhistidineresiduesand
itsreversaI
bysecondsitemutationofLys558,thelocusofcovalentH2DIDS
binding.Biochemistry1995;34:9315-9324.
322.CottenJF,WelshMJ.Cysticfibrosis-associatedmutationsatarginine347alter
theporearchitecture
ofCFTR.Evidencefordisruptionofasaltbridge.
J.BioI.Chem.1999;274:5429-5435.
323.GuanL,KabackHR.Lessonsfromlactosepermease.
Annu.Rev.Biophys.BiomoI.Struct.2006;35:67-91.
324.PonkaP,BeaumontC,RichardsonDR.Functionandregulationoftransferrinand
ferritin.Semin.Hematol.1998;35:35-54.
325.TouretN,Martin-OrozcoN,ParoutisPetal.MolecularandCellularMechanisms
UnderlyingIronTransportDeficiencyinMicrocyticAnemia.Blood2004
326.GoldsteinJL,BasuSK,BrunschedeGY,BrownMS.Release
oflowdensity
lipoproteinfromitscellsurfacereceptor
bysulfatedglycosaminoglycans.Cell
1976;7:85-95.
327.ChenWJ,GoldsteinJL,BrownMS.NPXY,asequenceoftenfoundin
cytoplasmictails,isrequiredforcoatedpit-mediatedinternalization
ofthelow
densitylipoproteinreceptor.J.Biol.Chem.1990;265:3116-3123.
328.PietrzikCU,BusseT,MerriamDE,WeggenS,KooEH.Thecytoplasmicdomain
oftheLDLreceptor-relatedproteinregulatesmultiplestepsinAPPprocessing.
EMBO
J.2002;21:5691-5700.
329.PrigentSA,GullickWJ.Identification
ofc-erbB-3bindingsitesfor
phosphatidylinositol3′-kinaseandSHCusinganEGFreceptor/c-erbB-3chimera.
EMBO
J.1994;13:2831-2841.
330.TakedaT,YamazakiH,FarquharMG.Identification
ofanapicalsorting
determinantinthecytoplasmictailofmegalin.Am.J.PhysiolCellPhysiol
2003;284:CII05-Cll13.
331.OhnoH,StewartJ,FournierMCetal.Interactionoftyrosine-basedsorting
signaIswithclathrin-associatedproteins.Science1995;269:1872-1875.
332.BoIlW,OhnoH,SongyangZetal.Sequencerequirementsfortherecognition
of
tyrosine-basedendocyticsignaIsbyclathrinAP-2complexes.EMBOJ.
1996;15:5789-5795.
224
333.HoningS,GriffithJ,GeuzeHJ,HunzikerW.Thetyrosine-basedlysosomal
targetingsignalinlamp-lmediatessortingintoGolgi-derivedclathrin-coated
vesicles.EMBOJ.1996;15:5230-5239.
334.LukacsGL,SegalG,KartnerN,GrinsteinS,ZhangF.Constitutiveintemalization
ofcysticfibrosistransmembraneconductanceregulatoroccursviaclathrin
dependentendocytosisandisregulated
byproteinphosphorylation.Biochem.J.
1997;328
(Pt2):353-361.
335.DuffieldA,KamsteegEJ,BrownAN,PagelP,CapIanMJ.ThetetraspaninCD63
enhancestheintemalization
oftheH,K-ATPasebeta-subunit.
Proc.NatI.Acad.Sci.U.S.A2003;100:15560-15565.
336.NesterovA,CarterRE,SorkinaT,GillGN,SorkinA.Inhibition
ofthereceptor
bindingfunction
ofclathrinadaptorproteinAP-2bydominant-negativemutant
mu2subunitanditseffects
onendocytosis.EMBOJ.1999;18:2489-2499.
337.van
DamEM,StoorvogelW.Dynamin-dependenttransferrinreceptorrecycling
byendosome-derivedclathrin-coatedvesicles.Mo1.Bio1.CelI2002;13:169-182.
338.GanY,McGrawTE,Rodriguez-BoulanE.Theepithelial-specifieadaptor
APIB
mediatespost-endocyticrecyclingtothebasolateralmembrane.Nat.CelIBiol.
2002;4:605-609.
339.YehJI,VerheyKJ,Bimbaum
MlKineticanalysisofglucosetransporter
traffickinginfibroblastsandadipoeytes.Biochemistry1995;34:15523-15531.
340.PetrisMJ,MercerJF.TheMenkesprotein(ATP7A;MNK)cyclesviatheplasma
membranebothinbasalandelevatedextraeellulareopperusingaC-terminaldi
leucineendoeytiesignal.Hum.MoI.Genet.1999;8:2107-2115.
341.PetrisMJ,CamakarisJ,GreenoughM,LaFontaineS,MercerJF.
AC-terminaldi
leucineisrequiredforlocalization
oftheMenkesproteininthetrans-Golgi
network.Hum.MoI.Genet.1998;7:2063-2071.
342.FrancisMJ,JonesEE,Levy
ERetal.Identificationofadi-leucinemotifwithin
theCterminusdomainoftheMenkesdiseaseproteinthatmediatesendocytosis
fromtheplasmamembrane.J.CelISei.1999;112
(Pt11):1721-1732.
343.JohnsonKF,Komfe1dS.Theeytoplasmictail
ofthemannose6-
phosphate/insulin-likegrowthfactor-IIreceptorhastwosignaIsforlysosomal
enzymesorting
intheGolgi.J.CelIBiol.1992;119:249-257.
344.ChenHJ,YuanJ,LobelP.Systematicmutationalanalysis
oftheeation
independentmannose6-phosphate/insulin-likegrowthfactorIIreeeptor
cytoplasmiedomain.
Anacidieclustercontainingakeyaspartateisimportantfor
functioninlysosomalenzymesorting.J.BioI.Chem.1997;272:7003-7012.
225
345.PriwitzerovaM,NieG,SheftelADetal.Functionalconsequencesofthehuman
DMT1mutationonproteinexpressionandironuptake.Blood2005
346.BanerjeeD,FlanaganPR,CluettJ,ValbergLS.Transferrinreceptorsinthe
humangastrointestinaltract.Relationshiptobodyironstores.Gastroenterology
1986;91:861-869.
347.AndersonGJ,PowellLW,HallidayJW.Transferrinreceptordistributionand
regulationintheratsmallintestine.Effect
ofironstoresanderythropoiesis.
Gastroenterology1990;98:576-585.
348.MaloD,VoganK,VidalSet
al.Haplotypemappingandsequenceanalysisofthe
mouseNrampgenepredictsusceptibilitytoinfectionwithintracellularparasites.
Genomics1994;23:51-61.
349.VidalS,GrosP,SkameneE.Naturalresistancetoinfectionwithintracellular
parasites:moleculargeneticsidentifiesNramp1astheBcglIty/Lshlocus.
J.Leukoc.Biol.1995;58:382-390.
350.AwomoyiAA,MarchantA,HowsonJMetal.Interleukin-lO,polymorphismin
SLCl1A1(formerlyNRAMP1),andsusceptibilitytotuberculosis.J.Infect.Dis.
2002;186:1808-1814.
351.RohrerJ,SchweizerA,RussellD,Komfeld
S.ThetargetingofLamp1to
lysosomesisdependentonthespacing
ofitscytoplasmictailtyrosinesorting
motifrelativetothemembrane.J.CellBiol.1996;132:565-576.
352.GuamieriFG,ArterbumLM,PennoMB,ChaY,AugustJT.ThemotifTyr-X-X
hydrophobieresiduemediateslysosomalmembranetargeting
oflysosome
associatedmembraneprotein
1.J.Biol.Chem.1993;268:1941-1946.
353.OhnoH,FournierMC,PoyG,BonifacinoJS.Structuraldeterminants
of
interactionoftyrosine-basedsortingsignaIswiththeadaptormediumchains.
J.Biol.Chem.1996;271:29009-29015.
354.HarterC,Mellman
1.TransportofthelysosomalmembraneglycoproteinIgp120
(lgp-A)tolysosomesdoesnotrequireappearanceontheplasmamembrane.J.Cell
Biol.1992;117:311-325.
355.MackenzieB,UjwalML,ChangMH,RomeroMF,HedigerMA.Divalentmetal
iontransporter
DMTlmediatesbothH+-coupledFe2+transportanduncoupled
fluxes.PflugersArch.2006;451:544-558.
356.CourvilleP,ChaloupkaR,VeyrierF,CellierMF.Determination
of
transmembranetopologyoftheEscherichiacolinaturalresistance-associated
macrophageprotein(Nramp)ortholog.J.Biol.Chem.2004;279:3318-3326.
226
357.PortnoyME,LiuXF,CulottaVC.Saccharomycescerevisiaeexpressesthree
functionallydistincthomologues
ofthenrampfamilyofmetaltransporters.
Mol.CellBiol.2000;20:7893-7902.
358.LiH,LiF,KwanM,HeQY,SunH.
NMRstructuresandorientationofthefourth
transmembranedomain
oftheratdivalentmetaltransporter(DMTl)withG185D
mutationinSDSmicelles.Biopolymers2005;77:173-183.
359.LiH,LiF,SunH,QianZM.Membrane-insertedconformationoftransmembrane
domain4
ofdivalent-metaltransporter.Biochem.J.2003;372:757-766.
360.LiH,LiF,QianZM,Sun
H.Structureandtopologyofthetransmembrane
domain4
ofthedivalentmetaltransporterinmembrane-mimeticenvironments.
Eur.J.Biochem.2004;271:1938-1951.
361.
LiF,LiH,HuLetal.Structure,assembly,andtopologyoftheG185Rmutantof
thefourthtransmembranedomainofdivalentmetaltransporter.lAm.Chem.Soc.
2005;127:1414-1423.
362.CostelloMJ,EscaigJ,MatsushitaKetal.Purifiedlacpermeaseandcytochrome
0
oxidasearefunctionalasmonomers.J.Biol.Chem.1987;262:17072-17082.
363.FrillingosS,Sahin-TothM,WuJ,KabackHR.Cys-scanningmutagenesis:a
novelapproachtostructurefunctionrelationshipsinpolytopicmembrane
proteins.FASEBl1998;12:1281-1299.
364.DuntenRL,Sahin-TothM,KabackHR.Cysteinescanningmutagenesis
of
putativehelixXIinthelactosepermeaseofEscherichiacoli.Biochemistry
1993;32:12644-12650.
365.SunJ,WuJ,CarrascoN,KabackHR.Identification
oftheepitopeformonoclonal
antibody
4Blwhichuncoupleslactoseandprotontranslocationinthelactose
permease
ofEscherichiacoli.Biochemistry1996;35:990-998.
366.SunJ,Li
J,CarrascoN,KabackHR.Thelasttwocytoplasmicloopsinthelactose
permease
ofEscherichiacolicompriseadiscontinuousepitopeforamonoclonal
antibody.Biochemistry1997;36:274-280.
367.WuJ,KabackHR.Ageneralmethodfordetermininghelixpackinginmembrane
proteinsinsitu:helices1andIIareclosetohelix
VITinthelactosepermeaseof
Escherichiacoli.Proc.Natl.Acad.Sci.U.S.A1996;93:14498-14502.
368.WuJ,KabackHR.Helixproximityandligand-inducedconformationalchangesin
thelactosepermease
ofEscherichiacolideterminedbysite-directedchemical
crosslinking.J.Mol.Bioi.1997;270:285-293.
227
369.SunJ,KabackHR.Proximityofperiplasmicloopsinthelactosepermeaseof
Escherichiacolideterminedbysite-directedcross-linking.Biochemistry
1997;36:11959-11965.
370.PakulaAA,SimonMI.Determination
oftransmembraneproteinstructureby
disulfidecross-linking:theEscherichiacoliTarreceptor.
Proc.Natl.Acad.Sci.U.S.A1992;89:4144-4148.
371.LynchBA,KoshlandDE,Jr.Disulfidecross-linkingstudies
ofthetransmembrane
regions
oftheaspartatesensoryreceptorofEscherichiacoli.
Proc.Natl.Acad.Sci.U.S.A1991;88:10402-10406.
372.
YanRT,MaloneyPC.Residuesinthepathwaythroughamembranetransporter.
Proc.Natl.Acad.Sci.U.S.A1995;92:5973-5976.
373.
WuJ,KabackHR.Cysteine148inthelactosepermeaseofEscherichiacoliisa
component
ofasubstratebindingsite.2.Site-directedfluorescencestudies.
Biochemistry1994;33:12166-12171.
374.VossJ,HubbellWL,KabackHR.Distancedetermination
inproteinsusing
designedmetalionbindingsitesandsite-directedspinlabeling:applicationtothe
lactosepermease
ofEscherichiacoli.Proc.Natl.Acad.Sci.U.S.A1995;92:12300-
12303.
375.
SteinhoffHJ.Inter-andintra-moleculardistancesdeterminedbyEPR
spectroscopyandsite-directedspinlabelingrevealprotein-proteinandprotein
oligonuc1eotideinteraction.Biol.Chem.2004;385:913-920.
376.HustedtEJ,
BethAH.Nitroxidespin-spininteractions:applicationstoprotein
structureanddynamics.Annu.Rev.Biophys.Biomol.Struct.1999;28:129-153.
377.HubbellWL,GrossA,LangenR,LietzowMA.Recentadvances
insite-directed
spinlabeling
ofproteins.Curr.Opin.Struct.Biol.1998;8:649-656.
378.ReeveI,HummelD,NelsonN,VossJ,HummellD.Overexpression,purification,
andsite-directedspinlabeling
oftheNrampmetaltransporterfrom
Mycobacteriumleprae.Proc.Natl.Acad.Sci.U.S.A2002;99:8608-8613.
379.GlickmanJN,ConibearE,PearseBM.Specificity
ofbindingofc1athrinadaptors
tosignaIs
onthemannose-6-phosphate/insulin-likegrowthfactorIIreceptor.
EMBOJ.1989;8:1041-1047.
380.LarkinJM,
BrownMS,GoldsteinJL,AndersonRG.Depletionofintracellular
potassiumarrestscoatedpitformationandreceptor-mediatedendocytosis
in
fibroblasts.Ce1l1983;33:273-285.
228
381.AltankovG,GrinnellF.Depletionofintracellularpotassiumdisruptscoatedpits
andreversiblyinhibitscellpolarizationduringfibroblastspreading.J.CellBiol.
1993;120:1449-1459.
382.SandvigK,OlsnesS,PetersenOW,van
DRAcidificationofthecytosolinhibits
endocytosisfromcoatedpits.J.CellBiol.1987;105:679-689.
229
OriginalContributionstoKnowledge
1.Identificationoffiveconservedchargedresidues(D86,E154,D192,E299,R416)
residinginthemembrane-spanningsegments
ofNramp2thatareessentialformetal
transport.
2.IdentificationandcharacterizationoftwoinvarianthistidinesinNramp2(H267,
H272)thatarecriticalforpH-dependence
ofNramp2activity.Proposedamodel
wherebyprotonation
ofthesehistidineswouldcontrolpH-dependenceofmetal
transport.
3.Characterization
ofthefunctionalpropertiesandpossiblecontributiontodiseaseof
thefirsttwohumanNRAMP2mutations(E399D,R416C)identifiedinpatients
sufferingfrommicrocyticanemiaandironoverload.
4.EstablishedanELISA-basedtechniquetobeabletoquantifytheproportion
of
Nramp2-HAexpressedatthecellsurfacewithrespecttoeithertotalcellular
expressionatsteady-stateorthedynamic”recyc1ingpool”expression.
5.EstablishedacellsurfacebiotinylationtechniquetomeaSuretherateofintemalization
ofNramp2fromthecellsurfaceinstablytransfectedLLC-PKIcells.
6.Exploredtherolesofthreeputativecytoplasmicmotifsinthesubcellulartargeting
andtraffickingofNramp2,andidentifiedatyrosine-basedsignal
(YLLNT555-559)in
thecarboxylterminus
ofNramp2responsibleforthetransporter’sintemalizationfrom
thecellsurfaceandrecyc1ingbacktotheplasmamembrane.
7.DemonstratedthattwosplicingisoformsofNramp2(isoform1andII)exhibitdistinct
targetingandrecyc1ingpropertiesinstablytransfectedLLC-PK
1•Proposed
a
traffickingmodelwherebyisoform1wouldbecomeenrichedatthecellsurfacein
epithelialcellstofacilitateironuptakeacrosstheplasmamembraneinthesecells.
8.Identificationofatyrosine-basedmotifoftheformYXXintheaminoterminusof
Nrampl(YGSI)thatfunctionsasalysosomaltargetingsignal.Proposedamodelfor
Nramp1traffickingtolateendosomesandlysosomes.
230