pull down buffer经典配方

ArabidopsisEPSIN1PlaysanImportantRoleinVacuolarTraffickingofSolubleCargoProteinsinPlantCellsviaInteractionswithClathrin,AP-1,VTI11,andVSR1

WJinheeSong,MyoungHuiLee,Gil-JeLee,CheolMinYoo,andInhwanHwang1DivisionofMolecularandLifeSciencesandCenterforPlantIntracellularTrafficking,PohangUniversityofScienceandTechnology,Pohang790-784,Korea

Epsinandrelatedproteinsplayimportantrolesinvariousstepsofproteintraffickinginanimalandyeastcells.Manyepsinhomologshavebeenidentifiedinplantcellsfromanalysisofgenomesequences.However,theirroleshavenotbeenelucidated.Here,weinvestigatetheexpression,localization,andbiologicalroleinproteintraffickingofanepsinhomolog,ArabidopsisthalianaEPSIN1,whichisexpressedinmosttissuesweexamined.Inthecell,onepoolofEPSIN1isassociatedwithactinfilaments,producinganetworkpattern,andasecondpoollocalizesprimarilytotheGolgicomplexwithaminorportiontotheprevacuolarcompartment,producingapunctatestainingpattern.Proteinpull-downandcoimmunoprecipitationexperimentsrevealthatArabidopsisEPSIN1interactswithclathrin,VTI11,g-adaptin-relatedprotein(g-ADR),andvacuolarsortingreceptor1(VSR1).Inaddition,EPSIN1colocalizeswithclathrinandVTI11.Theepsin1mutant,whichhasaT-DNAinsertioninEPSIN1,displaysadefectinthevacuolartraffickingofsporamin:greenfluorescentprotein(GFP),butnotinthesecretionofinvertase:GFPintothemedium.StablyexpressedHA:EPSIN1complementsthistraffickingdefect.Basedonthesedata,weproposethatEPSIN1playsanimportantroleinthevacuolartraffickingofsolubleproteinsatthetrans-Golginetworkviaitsinteractionwithg-ADR,VTI11,VSR1,andclathrin.

INTRODUCTION

Aftertranslationineukaryoticcells,alargenumberofproteinsaretransportedtosubcellularcompartmentsbyavarietyofdifferentmechanisms.Newlysynthesizedvacuolarproteinsthataredeliveredtotheendoplasmicreticulum(ER)bythecotrans-lationaltranslocationmechanismaretransportedtothevacuolefromtheERbyaprocesscalledintracellulartrafficking.Traffick-ingofaproteintothevacuolefromtheERoccursthroughtwoorganelles,theGolgicomplexandtheprevacuolarcompartment(PVC)(Rothman,1994;Hawesetal.,1999;BasshamandRaikhel,2000;Griffiths,2000).TransportofaproteinfromtheERtotheGolgicomplexisperformedbycoatproteincomplexIIvesicles.Transportfromthetrans-Golginetwork(TGN)tothePVCoccursviaclathrin-coatedvesicles(CCVs)(Robinsonetal.,1998;Tangetal.,2005;Yangetal.,2005).

TransportofaproteinfromtheERtothevacuole/lysosomerequiresalargenumberofproteins,includingcomponentsofvesicles,factorsinvolvedinvesiclegenerationandfusion,reg-ulatorsofintracellulartrafficking,adaptorsforthecargoproteins,andotheraccessoryproteins(RobinsonandKreis,1992;Bennett,

˜oetal.,1997;1995;SchekmanandOrci,1996;daSilvaConceic¸a

whomcorrespondenceshouldbeaddressed.E-mailihhwang@

postech.ac.kr;fax82-54-279-8159.

TheauthorresponsiblefordistributionofmaterialsintegraltothefindingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(www.plantcell.org)is:InhwanHwang(ihhwang@postech.ac.kr).WOnlineversioncontainsWeb-onlydata.

www.plantcell.org/cgi/doi/10.1105/tpc.105.039123

1ToKirchhausen,1999;Severetal.,1999;BasshamandRaikhel,2000;Griffiths,2000;Jinetal.,2001;RobinsonandBonifacino,2001).Mostoftheseproteinsarefoundinalleukaryoticcellsfromyeast,animals,andplants,suggestingthatproteintraffick-ingmechanismsfromtheERtothevacuole/lysosomemaybehighlyconservedinalleukaryoticcells.

Ofthelargenumberofproteinsinvolvedinintracellulartraf-ficking,agroupofproteinsthathavethehighlyconservedepsinN-terminalhomology(ENTH)domainhavebeenidentifiedasplayingacriticalroleatvarioustraffickingstepsinanimalandyeastcells(Chenetal.,1998;DeCamillietal.,2002;Wendland,2002;Overstreetetal.,2003;Legendre-Guilleminetal.,2004).TheENTHdomainbindstophosphatidylinositols(PtdIns),althoughthelipidbindingspecificitydifferswithindividualmembersoftheepsinfamily.Forexample,epsin1bindstoPtdIns(4,5)P2,whereasEpsinRandEnt3pbindtoPtdIns(4)PandPdtIns(3,5)P2,respectively(Itohetal.,2001).TheENTHdomainisthoughttoberesponsiblefortargetingtheseproteinstospecificcompartmentsandalsoforintroducingcurvaturetotheboundmembranestoassistinthegenerationofCCVs(Legendre-Guilleminetal.,2004).However,theexactstepsofintracellulartraffickinginwhichENTH-containingproteinsplayarolearecomplex.Epsinhomologscanbedividedintotwogroupsbasedonthepathwayinwhichtheyplayarole.Onegroup,whichincludesepsin1inanimalcellsandEnt1pandEnt2pinyeastcells,isinvolvedinendocytosisfromtheplasmamembrane(Chenetal.,1998;DeCamillietal.,2002;Wendland,2002).Theothergroup,whichincludesEpsinR/clint/enthoprotininanimalcellsandEnt3pandEnt4pinyeastcells,isinvolvedinproteintraffickingfromtheTGNtothelysosome/vacuoleaswellas

retrogradetraffickingfromtheearlyendosomestotheTGN(Kalthoffetal.,2002;Wasiaketal.,2002;Hirstetal.,2003;Chidambarametal.,2004;Eugsteretal.,2004;Saint-Poletal.,2004).

Anothercommonfeatureofepsin-relatedproteinsisthattheyplayaroleinCCV-mediatedproteintraffickingatboththeTGNandtheplasmamembrane.Theseproteinscanbinddirectlytoclathrinthroughtheirmultipleclathrinbindingmotifs;thus,theymayrecruitclathrintotheplasmamembraneortheTGNtogenerateCCVs(Rosenthaletal.,1999;Wendlandetal.,1999;Drakeetal.,2000).Inaddition,theseproteinsinteractwithmanyotherproteins,suchasheterotetramericclathrinadaptorcomplexes(APs),monomericadaptorGolgi-localized,g-ear–containingArfbindingproteins(GGAs),andsolubleNSFattach-mentproteinreceptors(SNAREs).Epsin1interactswithAP-2,Epsin15,andintersectin(Chenetal.,1998;Legendre-Guilleminetal.,2004),whereasEpsinR/enthoprotin/clintandEnt3pinteractwithSNAREssuchasvti1bandvti1p,respectively(Chidambarametal.,2004)andwithadaptorproteinssuchasGGAsandAP-1(Duncanetal.,2003;Millsetal.,2003).Inaddition,epsinhomologshaveubiquitin-interactingmotifsandareubiquitinated(Oldhametal.,2002;Shihetal.,2002).ProteinubiquitinationactsasasignalforendocytosisfromtheplasmamembraneandtraffickingfromtheTGNthroughtheendosome/PVCtothelysosome/vacuole(Poloetal.,2002;Horak,2003;Raiborgetal.,2003;Scottetal.,2004).Thebindingofepsinhomologstoubiquitinraisesthepossibilitythatepsinhomologsmaybinddirectlytocargoproteinsthataredestinedforthevacuole/lysosomefromeithertheplasmamembraneortheTGN(ChenandDeCamilli,2005;Sigismundetal.,2005).

Inplantcells,sequenceanalysisoftheentireArabidopsisthalianagenomerevealsseveralproteinswiththehighlycon-servedENTHdomains(HolsteinandOliviusson,2005).However,theirbiologicalroleshavenotbeenaddressed.Inthisstudy,weinvestigatethefunctionalroleofEPSIN1,anArabidopsisepsinhomolog,atthemolecularlevel.Inparticular,wefocusonitspossibleroleinproteintraffickinginplantcells.WedemonstratethatEPSIN1interactswithclathrin,AP-1,VSR1,andVTI11andplaysanimportantroleinthevacuolartraffickingofasolubleproteinfromtheGolgicomplextothecentralvacuole.RESULTS

EPSIN1,aMemberoftheEpsinFamily,IsUbiquitouslyExpressedinArabidopsis

TheArabidopsisgenomeencodesthreehighlysimilarepsin-relatedproteins,EPSIN1,EPSIN2,andEPSIN3(HolsteinandOliviusson,2005).Inthisstudy,weinvestigatedthebiologicalroleofEPSIN1.EPSIN1hasthehighlyconservedENTHdomainattheNterminus.However,therestofthemoleculeislesssimilartootherepsin-relatedproteins,althoughithasmotifs,suchasLIDLandDPF,thatmayfunctionasclathrinandAP-1bindingmotifs,respectively.

TounderstandthebiologicalroleofEPSIN1,itsexpressioninvariousplanttissueswasexamined.AnantibodywasraisedagainstthemiddledomainofEPSIN1(aminoacidresidues153to337).Theantibodyrecognizedaproteinbandat90kD,which

EPSIN1inVacuolarTrafficking2259

wasmuchlargerthantheexpectedsize,60kD,ofEPSIN1(Figure1A).Itwasshownpreviouslythatepsin-relatedproteinsmigrateslowerthanexpectedinSDS-PAGE(Chenetal.,1998).Thecontrolserumdidnotrecognizeanyproteinbands.Thisre-sultsuggestedthattheantibodyspecificallyrecognizedEPSIN1.Toconfirmthis,protoplastsweretransformedwithEPSIN1taggedwithHAattheNterminus(HA:EPSIN1)andproteinextractsfromthetransformedprotoplastswereanalyzedbyproteingelblottingusinganti-HAandanti-EPSIN1antibodies.Theanti-HAantibodyspecificallyrecognizedaproteinbandfromthetransformedprotoplasts,butnotfromtheuntransformedprotoplasts,at90kD(Figure1B).Inaddition,the90-kDproteinspecieswasrecognizedbytheanti-EPSIN1antibody,confirmingthatthe90-kDbandwasEPSIN1.TheexpressionofEPSIN1invarioustissueswasexaminedusingtheanti-EPSIN1antibody.Proteinextractswerepreparedfromvarioustissuesatdifferentstagesofplantgrowthandusedforproteingelblotanalysis.EPSIN1wasexpressedinallofthetissuesexamined,withthehighestexpressionincotyledonsandflowers(Figure1C).EPSIN1ProducesBothNetworkandPunctateStainingPatterns

ToexaminethesubcellulardistributionofEPSIN1,totalproteinextractsfromleaftissueswereseparatedintosolubleandmembranefractionsandanalyzedbyproteingelblottingusinganti-EPSIN1antibody.EPSIN1wasdetectedinbothmembrane(pellet)andsolublefractions(Figure2A).Ascontrolsforthefractionation,Arabidopsisaleurain-likeprotease(AALP)andArabi-dopsisvacuolarsortingreceptor(VSR)weredetectedwithanti-AALPandanti-VSRantibodies,respectively(Sohnetal.,2003).AALPisasolubleproteinpresentinthevacuolarlumen,andVSRisamembraneproteinthatislocalizedprimarilytothePVCwitha

minorportiontotheGolgicomplex(daSilvaConceic¸a

˜oetal.,1997;Ahmedetal.,2000).Asexpected,AALPandVSRweredetectedinthesupernatantandpelletfractions,respectively.TheseresultsindicatedthatEPSIN1localizedtomultipleloca-tions,consistentwiththebehaviorofotherepsin-relatedproteins(Legendre-Guilleminetal.,2004).

Next,wedefinedthesubcellularlocalizationofEPSIN1.OurinitialattemptstolocalizetheendogenousEPSIN1withtheanti-EPSIN1antibodyfailed.Thus,wedeterminedthelocalizationofEPSIN1proteintransientlyexpressedinprotoplasts.EPSIN1wastaggedwiththeHAepitope,greenfluorescentprotein(GFP),orredfluorescentprotein(RFP).TheamountoftotalEPSIN1proteinwasdeterminedusingvariousamountsofHA:EPSIN1plasmidDNAbyproteingelblotanalysiswithanti-EPSIN1an-tibodyandwasfoundtobeproportionaltotheamountofplasmidused(Figure2B).Forthelocalization,weusedaminimalamount(5to10mg)ofEPSIN1plasmidDNAs.ProtoplastsweretransformedwithHA:EPSIN1,andlocalizationofEPSIN1wasdeterminedbyimmunostainingwithanti-HAantibody.HA:EPSIN1producedprimarilyapunctatestainingpattern(Figure2Ca).Inadditiontopunctatestains,weoccasionallyobservedweaklystainedstringsthatconnectedpunctatestains(Figure2Cc,arrowheads).Bycontrast,thenontransformedcontrolsdidnotproduceanypatterns(Figure2Ce).Inprotoplaststrans-formedwithEPSIN1:GFPandEPSIN1:RFP,bothEPSIN1fusion

2260ThePlantCell

Figure1.EPSIN1IsExpressedinVariousArabidopsisTissues.

(A)Generationofanti-EPSIN1antibody.Themiddledomain,correspondingtoaminoacidresidues153to337,wasexpressedastheHisx6-taggedforminE.coliandusedtoraiseantibodyinarabbit.Controlserumwasobtainedfromtherabbitbeforeimmunization.Totalproteinextractswereobtainedfromleaftissuesandusedtotesttheanti-EPSIN1antibody.

(B)Specificityoftheanti-EPSIN1antibody.ProteinextractswereobtainedfromprotoplastsexpressingEPSIN1taggedwiththeHAepitopeattheNterminusandusedforproteingelblotanalysisusinganti-HAandanti-EPSIN1antibodies.

(C)ExpressionofEPSIN1invarioustissues.Totalproteinextractsfromtheindicatedtissueswereanalyzedbyproteingelblottingusinganti-EPSIN1antibody.Leaftissueswereharvested11and20daftergermination.Cotyledonswereobtainedfrom5-d-oldplants.ThemembraneswerestainedwithCoomassiebluetocontrolforproteinloading.RbcL,largesubunitoftheribulose-1,5-bis-phosphatecarboxylase/oxygenase(Rubisco)complex.

proteinsproducedanetworkpatternwithpunctatestains(Fig-ures2Cgand2Ch),whereasGFPandRFPaloneproduceddiffusepatterns(Figures2Dhand2Di),indicatingthatEPSIN1producesthenetworkpatternwithpunctatestains.TheseresultswerefurtherconfirmedbycotransformingtheprotoplastswitheitherEPSIN1:GFPandHA:EPSIN1orEPSIN1:GFPandEPSIN1:RFP.ThepunctatestainingpatternofEPSIN1:GFPcloselyover-lappedthatofHA:EPSIN1(Figures2Dato2Dc).Inaddition,thenetworkandpunctatestainingpatternsofEPSIN1:GFPcloselyoverlappedthoseofEPSIN1:RFP(Figures2Deto2Dg).However,thefinenetworksrevealedbyEPSIN1:GFPintheliveprotoplastswerenearlyabsentinthefixedprotoplasts.Thus,thedifferencesinthestainingpatternsbetweenfixedandliveprotoplastsmaybeattributabletothefactthatthenetworkpatternofliveprotoplastsarenotwellpreservedunderthefixingconditionsused.Inaddi-tion,thestringsoccasionallyobservedinthefixedprotoplastsmayrepresenttheremnantsofthenetworkpatternrevealedbyHA:EPSIN1.TheseresultsstronglysuggestthatEPSIN1isre-sponsibleforthenetworkpatternaswellasthepunctatestains.ThenetworkpatternwasreminiscentoftheERoractinpatterninplantcells(Boevinketal.,1998;Jinetal.,2001;Kimetal.,2005),whereasthepunctatestainingpatternsuggestedthatEPSIN1maylocalizetotheGolgicomplexorendosomes,asobservedpreviouslywithepsinhomologsinanimalandyeastcells(Wasiaketal.,2002;Chidambarametal.,2004;Saint-Poletal.,2004).Therefore,protoplastswerecotransformedwithEPSIN1:RFPandGFP:talin,amarkerforactinfilamentsconsist-ingofGFPandtheactinbindingdomainofmousetalin(Kostetal.,1998;Kimetal.,2005).Asexpected,GFP:talinproducedthenetworkpattern(Figure3A)(Kostetal.,1998;Kimetal.,2005).Furthermore,theredfluorescentnetworkpatternofEPSIN1:RFPcloselyoverlappedthegreenfluorescentnetworkpatternofGFP:talin(Figure3A),raisingthepossibilitythatEPSIN1:GFPboundtotheactinfilamentsratherthantotheER.Toconfirmthis,theEPSIN1:RFPpatternwasexaminedaftertreatmentwithlatrunculinB(LatB),achemicalagentknowntodisruptactinfilaments(Spectoretal.,1983).LatB–treatedprotoplastsproducedthediffusegreenfluorescentpatternofGFP:talin(Figure3A),anindicationofsolubilizedactinfilaments,asobservedpreviously(Kimetal.,2005).Inaddition,theLatB–treatedprotoplastsdisplayedadiffuseredfluorescentpatternofEPSIN1:RFP(Figure3A),indicatingthatEPSIN1isassociatedwithactinfilamentsbutnotwiththeER.Furthermore,thepunc-tatestainingpatternofEPSIN1:RFPalsowasnotobservedinthepresenceofLatB,indicatingthatactinfilamentsplayedaroleinyieldingthepunctatestainingpatternofEPSIN1.Inthesameconditions,BiP:GFP,anERmarker(Leeetal.,2002),producedanetworkpattern,indicatingthatLatBdoesnotdisrupttheERnetworkpatterns(Figure3Ai).

ToidentifytheorganelleresponsibleforthepunctatestainingpatternofEPSIN1,itslocalizationwascomparedwiththatofST:GFPandPEP12p/SYP21.ST:GFP,achimericprotein

EPSIN1inVacuolarTrafficking2261

Figure2.EPSIN1ProducesBothNetworkandPunctateStainingPatterns.

(A)SubcellularfractionationofEPSIN1.Total(T)proteinextractsofleaftissueswereseparatedintosoluble(S)andpellet(P)fractionsandanalyzedbyproteingelblottingusinganti-EPSIN1,anti-AALP,andanti-VSRantibodies.

(B)ExpressionlevelofEPSIN1intransformedprotoplasts.ProtoplastsweretransformedwithvariousamountsofHA:EPSIN1DNA,andthelevelofEPSIN1wasdeterminedbyproteingelblottingwithanti-EPSIN1antibody.Proteinextractsfromuntransformedprotoplastswereusedasacontrol.ThemembranewasalsostainedwithCoomassiebluetocontrolforloading.

(C)LocalizationofEPSIN1.Protoplastsweretransformedwiththeindicatedconstructs(5to10mg),andthelocalizationofEPSIN1wasexaminedeitherbyimmunostainingwithanti-HAantibodyorbydirectdetectionoftheGFPorRFPsignal.Untransformedprotoplastswereimmunostainedwithanti-HAantibodyasacontrol.Bars¼20mm.

(D)ColocalizationofEPSIN1proteins.ThelocalizationofEPSIN1proteinwasexaminedinprotoplaststransformedwithHA:EPSIN1andEPSIN1:GFPorwithEPSIN1:GFPandEPSIN1:RFP.Ascontrols,GFPandRFPaloneweretransformedintoprotoplasts.Bars¼20mm.

亚细胞定位可以荧光观察也可以做western 检测

2262ThePlantCell

Figure3.LocalizationofEPSIN1inProtoplasts.

EPSIN1inVacuolarTrafficking2263

betweenratsialyltransferaseandGFP,localizestotheGolgicomplex,andPEP12p,at-SNARE,localizestothePVC(daSilva

˜oetal.,1997;Boevinketal.,1998;Jinetal.,2001).Conceic¸a

ProtoplastswerecotransformedwithHA:EPSIN1andST:GFP.Thelocalizationoftheseproteinswasexaminedafterstainingwithanti-HAantibody.ST:GFPwasobserveddirectlywiththegreenfluorescentsignals.AmajorportionoftheHA:EPSIN1-positivepunctatestainscloselyoverlappedwiththoseofST:GFP(Figures3Bato3Bc).TofurtherconfirmtheGolgilocalizationofHA:EPSIN1,protoplaststransformedwithHA:EPSIN1weretreatedwithbrefeldinA(BFA),achemicalknowntodisrupttheGolgicomplex(Driouichetal.,1993),andthelocalizationofHA:EPSIN1wasexamined.InthepresenceofBFA,HA:EPSIN1yieldedalargelydiffusepatternwithaggregates,butnotthepunctatestainingpattern,indicatingthatBFAaffectsEPSIN1localization(Figure3Be).Inthesameconditions,ST:GFPpro-ducedanetworkpatternwithlargeaggregates(Figure3Bg),confirmingthattheGolgicomplexwasdisrupted.TheseresultssupportthenotionthatEPSIN1localizestotheGolgicomplex.Next,weexaminedthepossibilityofEPSIN1localizingtothePVC.ProtoplastswerecotransformedwithEPSIN1:GFPandPEP12p:HA.ThelocalizationofPEP12p:HAwasexaminedafterstainingwithanti-HAantibody.EPSIN1:GFPwasobserveddi-rectlywiththegreenfluorescentsignals.OnlyaminorportionoftheEPSIN1:GFP-positivepunctatestainsoverlappedwiththePEP12p:HA-positivepunctatestains(Figures3Bito3Bk,ar-rows).TheseresultsindicatedthatEPSIN1localizedprimarilytotheGolgicomplexwithaminorportiontothePVC.

Toobtainindependentevidenceforthelocalization,weex-aminedthecolocalizationofEPSIN1withVTI11,av-SNAREthatisdistributedequallytoboththeTGNandthePVC(Zhengetal.,1999;Basshametal.,2000;Kimetal.,2005).ProtoplastswerecotransformedwithEPSIN1:GFPandVTI11:HA,andthelocal-izationoftheseproteinswasexaminedbyimmunostainingwithanti-HAantibody.EPSIN1-positivepunctatestainslargelycolo-calizedwiththoseofVTI11:HA(Figures3Bmto3Bo),confirmingthatEPSIN1localizestoboththeGolgicomplexandthePVC.EPSIN1BindstoandColocalizeswithClathrin

Themembersoftheepsinfamilyhavetwoclathrinbindingmotifs(Rosenthaletal.,1999;Wendlandetal.,1999;Drakeetal.,2000).SequenceanalysisindicatedthatEPSIN1hasapotentialclathrinbindingmotif.ToexplorethepossibilitythatEPSIN1bindstoclathrin,glutathioneS-transferase–fusedEPSIN1(GST:EPSIN1)wasconstructedforaproteinpull-downassay(Figure4A).GST:EPSIN1wasexpressedinEscherichiacoliandpurifiedfromE.coliextracts(Figure4B).ThepurifiedGST:EPSIN1wasmixedwithproteinextractsobtainedfromleaftissues.Proteinspelletedwithglutathione–agarosewereanalyzedbyproteingelblottingusinganti-clathrinantibody.GST:EPSIN1,butnotGSTalone,precipitatedfromtheplantextractsa180-kDproteinspeciesthatwasrecognizedbyanti-clathrinantibody(Figure4C),indi-catingthatEPSIN1boundtoclathrin.

Tofurtherexamineitsbindingtoclathrin,EPSIN1wasdividedintotworegions,theENTHandtheremainderofthemolecule(EPSIN1DN)(Figure4A).TheseregionswereexpressedinE.coliasGSTfusionproteins,GST:ENTHandGST:EPSIN1DN,re-spectively(Figure4B).Proteinpull-downexperimentsusingleafcellextractswereperformedwithpurifiedGST:ENTHandGST:EPSIN1DN.GST:EPSIN1DN,butnotGST:ENTH,precipitatedclathrinfromtheplantextracts(Figure4C).Toidentifytheclathrinbindingmotif,theC-terminalregioncontainingtheputativeclathrinbindingmotif,LIDL(Lafer,2002),aswellasGST:RIDL,whichcontainedanArgsubstitutionofthefirstLeuresidueinthemotif,wereexpressedasGSTfusionproteinsinE.coli(Figures4Aand4B).GST:LIDL,butnotGST:RIDL,precipitatedclathrinfromproteinextracts(Figure4C),indicatingthattheLIDLmotiffunctionedasaclathrinbindingmotif.

TheinvitrobindingofEPSIN1withclathrinstronglysuggestedthatEPSIN1waslikelytocolocalizewithclathrin.Therefore,immunohistochemistryforthelocalizationofEPSIN1andclathrinwasperformed.ProtoplastsweretransformedwithHA:EPSIN1,andthelocalizationofHA:EPSIN1andclathrinwasexaminedbystainingwithanti-HAandanti-clathrinantibodies,respectively.Theanti-clathrinantibodyproducedapunctatestainingpattern(Figure4D).Amajority(60to70%)oftheHA:EPSIN1-positivepunctatestainscloselyoverlappedwithapool(40to50%)ofclathrin-positivepunctatestains(Figure4D),consistentwithaninteractionbetweenEPSIN1andclathrin.Therewasalsoapoolofclathrin-positivepunctatestainsthatlackedtheHA:EPSIN1signal,suggestingthatclathrinalsowasinvolvedinanEPSIN1-independentprocess.

TofurthercharacterizetheinteractionbetweenEPSIN1andclathrin,weexaminedwhetherornotEPSIN1ispermanentlyassociatedwithCCVs.Proteinextractsfromleaftissueswerefirstseparatedintosolubleandpelletfractionsbyultracentrifu-gation.ThepelletfractionwastreatedwithTritonX-100andfurtherfractionatedbygelfiltration,andthefractionswereana-lyzedbyproteingelblottingusinganti-clathrin,anti-EPSIN,andanti-VSRantibodies.Clathrinwasdetectedinapeakbetween443and669kD(seeSupplementalFigure1online).Interestingly,VSR,thevacuolarcargoreceptor,waselutedatthesameposi-tionwithclathrin.Bycontrast,EPSIN1waselutedat90kD.TheseresultssuggestthatEPSIN1isnotpermanentlyassoci-atedwithCCVs.

Figure3.(continued).

(A)ColocalizationofEPSIN1withactinfilaments.Protoplastsweretransformedwiththeindicatedconstructs,andthelocalizationoftheseproteinswasexaminedinthepresence(þLatB)andabsence(ÿLatB)ofLatB(10mM).Bars¼20mm.

(B)LocalizationofEPSIN1totheGolgicomplexandthePVC.Protoplastsweretransformedwiththeindicatedconstructs,andlocalizationoftheproteinswasexaminedafterimmunostainingwithanti-HA.TheGFPsignalswereobserveddirectlyinthefixedprotoplasts.ForBFAtreatment,BFA(30mg/mL)wasaddedtothetransformedprotoplastsat24haftertransformationandincubatedfor3h.ArrowsindicatetheoverlapbetweenEPSIN1:GFPandPEP12p:HA.Bars¼20mm.

22ThePlantCell

Figure4.EPSIN1BindstoandColocalizeswithClathrin.

(A)Constructs.GSTwasfusedtotheNterminus.ENTH,theepsinN-terminalhomologydomain.DLFandDPFmotifsaresimilartoAP-1andAP-3bindingmotifs,respectively.Q11indicatesastretchof11Gluresidues.Theclathrinbindingmotif(LIDL)andtheLeu-to-Argsubstitutionintheclathrinbindingmotif(RIDL)areshownintheC-terminalregion.Thenumbersindicateaminoacidpositions.

(B)ExpressionofGST-fusedEPSIN1proteins.ConstructswereintroducedintoE.coli,andtheirexpressionwasinducedbyisopropylthio-b-galactoside.GSTfusionproteinswerepurifiedfromE.coliextractswithglutathione–agarosebeads.PurifiedproteinswerestainedwithCoomassieblue.

(C)InteractionofEPSIN1withclathrin.GST-fusedEPSIN1proteinsweremixedwithproteinextractsfromleaftissues.EPSIN1bindingproteinswereprecipitatedusingglutathione–agarosebeadsandanalyzedbyproteingelblottingusinganti-clathrinantibody.Supernatantsalsowereincludedintheproteingelblotanalysis.Subsequently,themembraneswerestainedwithCoomassieblue.Bead,glutathione–agarosebeadsalone;P,pellet;S,supernatant(10%oftotal).

(D)ColocalizationofEPSIN1withclathrin.ProtoplaststransformedwithHA:EPSIN1werefixedwithparaglutaraldehyde,andthelocalizationofHA:EPSIN1andclathrinwasexaminedbyimmunostainingwithanti-HAandanti-clathrinantibodies,respectively.Bar¼20mm.

EPSIN1InteractswithVTI11

Epsin-relatedproteinsinanimalandyeastcellsareinvolvedineitherendocytosisorvacuolar/lysosomalproteintrafficking(Chenetal.,1998;DeCamillietal.,2002;Wendland,2002;Overstreetetal.,2003;Legendre-Guilleminetal.,2004).ToelucidatethepathwayofEPSIN1involvement,bindingpartnersofEPSIN1wereexamined.Inanimalandyeastcells,epsin-likeproteinshavebeenshowntointeractwithSNAREs(Chenetal.,1998;Chidambarametal.,2004).BecauseEPSIN1localizedtotheGolgicomplexandthePVC,EPSIN1interactionswithArabidop-sisVTI11andVTI12(formerlyAtVTI1aandAtVTI1b,respectively)wereexamined.VTI11isav-SNAREthatlocalizestotheTGNandtravelstothePVC(Zhengetal.,1999;Basshametal.,2000).VTI11andVTI12weretaggedwithHAattheCterminusandintroducedintoprotoplasts.TheexpressionofVTI11:HAandVTI12:HAinprotoplastswasconfirmedbyproteingelblotanalysisusinganti-HAantibody.Theanti-HAantibodydetectedproteinbandsat33and35kD(Figure5A),theexpectedpositionsofVTI11:HAandVTI12:HA,respectively.PurifiedGST:EPSIN1fromE.coliextractswasmixedwithplantextractsfromtheVTI11:HA-orVTI12:HA-transformedprotoplasts,andGST:EPSIN1-boundproteinswereprecipitatedfromthemixtureusingglutathione–agarosebeads.Thepelletfractionwasanalyzedbyproteingelblottingusinganti-HAantibody.VTI11:HA,butnotVTI12:HA,wasdetectedfromthepellet(Figure5A).GSTalonedidnotprecipitateVTI11:HAfromtheplantextracts.TheseresultsindicatedthatalthoughVTI11andVTI12arehighlysimilartoeachother,EPSIN1specificallybindstoVTI11:HA.Tofurtherconfirmthisinteraction,weperformedareciprocalproteinpull-downexperiment(i.e.,pull-downofEPSIN1withVTI11)usingproteinextractsobtainedfromprotoplaststransformedwithVTI11:HAandEPSIN1:GFP.VTI11:HA-boundproteinswereimmunoprecipitatedwithanti-HAantibody,andtheimmunopre-cipitateswereanalyzedbyproteingelblottingusinganti-HA,anti-GFP,andanti-calreticulinantibodies.Anti-calreticulinanti-bodywasusedasanegativecontrol.InadditiontoVTI11:HA,EPSIN1:GFPwasdetectedintheimmunoprecipitates(Figure5B).However,calreticulinwasnotdetectedinthepellet.TheseresultsfurtherconfirmtheinteractionbetweenVTI11andEPSIN1.TodeterminetheVTI11bindingdomainofEPSIN1,proteinpull-downexperimentswereperformedusingGST:ENTHandGST:EPSIN1DN.GST:ENTH,butnotGST:EPSIN1DN,precipi-tatedVTI11:HAfromtheplantextracts(Figure5C),indicatingthattheENTHdomaincontainedtheVTI11bindingmotif.Similarly,inanimalandyeastcells,EpsinRandEnt3phavebeenshowntobindtovti1bandvti1p,respectively(Chidambarametal.,2004).EPSIN1BindstotheArabidopsisHomologofg-AdaptinofAP-1

Epsinhomologsbindtoadaptorproteins(APs)(Duncanetal.,2003;Millsetal.,2003).Inanimalcells,EPSIN1bindstothea-adaptinofAP-2viatheDFF/W(whereFindicatesahydro-phobicaminoacid)andFXDXFmotifs(Figure4A)(Brettetal.,2002).ArabidopsisEPSIN1hasthreeDPFmotifstowhicha-adaptinofAP-2couldbind.Inaddition,EPSIN1hastworegionswithmotifssimilartotheacidicPhemotifforbinding

EPSIN1inVacuolarTrafficking2265

Figure5.EPSIN1BindstoVTI11.

(A)ProteinextractswerepreparedfromVTI11:HA-andVTI12:HA-transformedprotoplastsandmixedwithGSTaloneorGST:EPSIN1.EPSIN1-boundproteinswereprecipitatedfromthemixturewithgluta-thione–agarosebeadsandanalyzedbyproteingelblottingusinganti-HAantibody.

(B)CoimmunoprecipitationofEPSIN1:GFPwithVTI11:HA.Proteinex-tractsfromprotoplastscotransformedwithVTI11:HAandEPSIN1:GFPwereusedforimmunoprecipitationwithanti-HAantibody.Theimmuno-precipitateswereanalyzedbyproteingelblottingwithanti-HA,anti-GFP,andanti-calreticulinantibodies.P,immunoprecipitate;S,supernatant;T,totalproteinextracts(5%oftheinput).

(C)Forbindingexperiments,proteinextractsfromprotoplaststrans-formedwithVTI11:HAweremixedwithGSTalone,GST:ENTH,andGST:EPSIN1DN.Proteinswereprecipitatedwithglutathione-agarosebeadsandanalyzedbyproteingelblottingusinganti-HAantibody.Theamountoftheinputproteinsisindicated.

AP-1andGGAs(Duncanetal.,2003).Therefore,theinteractionsofEPSIN1withAPcomplexeswereexamined.WeisolatedtheArabidopsisproteinsg-adaptinrelatedprotein(g-ADR),a-ADR,andd-ADR,whichweremostcloselyrelatedtog-adaptin,a-adaptin,andd-adaptinofAP-1,AP-2,andAP-3,respectively.TheseArabidopsisproteinsweretaggedwithGFPandex-pressedtransientlyinprotoplasts.ProteinextractsfromthetransformedprotoplastsweremixedwithpurifiedGST:EPSIN1,andtheGST:EPSIN1-boundproteinswereprecipitated.Thepelletwasanalyzedbyproteingelblottingusinganti-GFPantibody.GFP:g-ADR,butnota-ADR:GFPord-ADR:GFP,wasdetectedinthepellet(Figure6A).Thecontrolfortheproteinpull-downassay,GSTalone,didnotprecipitateanyoftheseproteins.TheseresultsstronglysuggestedthatEPSIN1interactswithg-ADRspecifically.TofurtherconfirmtheinteractionbetweenEPSIN1andg-ADR,weperformedareciprocalproteinpull-downexperiment(i.e.,pulldownofEPSIN1proteinswith

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Figure6.EPSIN1Bindstog-ADR.

(A)GFP-fusedg-ADR,a-ADR,andd-ADRwereconstructedandtransientlyexpressedinprotoplasts.ProteinextractswerepreparedfromthetransformedprotoplastsandusedforbindingassayswithGST:EPSIN1.TheEPSIN1bindingproteinswereprecipitatedfromthereactionmixtureandanalyzedbyproteingelblottingusinganti-GFPantibody.P,pellet;S,supernatant(20%oftotal).

(B)Pull-downofEPSIN1proteinswithMBP:g-ADR(ear).MBP:g-ADR(ear)wasmixedwithproteinextractsfromprotoplaststransformedwiththeindicatedconstructs.MBP:g-ADR(ear)bindingproteinswereprecipitatedwithamyloseresin,andthepelletswereanalyzedbyproteingelblottingusinganti-HAantibody.

(C)Theeardomainsofg-ADRanda-ADRwereexpressedasMBPfusionproteinsinE.coliandusedforbindingexperiments.Proteinsweredetectedbyproteingelblottingusinganti-MBPantibody.

(D)GST:EPSIN1wasmixedwithMBPalone,MBP:g-ADR(ear),orMBP:a-ADR(ear).TheEPSIN1bindingproteinswereprecipitatedfromthereactionmixtureandanalyzedbyproteingelblottingusinganti-MBPantibody.

g-ADR).Theeardomainofg-ADRwasexpressedasamaltosebindingprotein(MBP)fusionprotein,MBP:g-ADR(ear),inE.coli.PurifiedMBP:g-ADR(ear)wasmixedwithproteinextractsob-tainedfromprotoplaststransformedwithfull-lengthEPSIN1:HAoritsdeletionmutants,andMBP:g-ADR(ear)-boundproteinswereprecipitatedwithamyloseresin.Theprecipitateswereanalyzedbyproteingelblottingusinganti-HAantibody.Full-lengthHA:EPSIN1andHA:EPSIN1DN,butnotHA:ENTH,weredetectedinthepellets(Figure6B),furtherconfirmingtheinter-actionbetweeng-ADRandEPSIN1.Furthermore,theseresultsindicatedthattheC-terminalregionofEPSIN1wasresponsiblefortheinteractionwithg-ADR.

ToconfirmtheinteractionbetweenEPSIN1andg-ADRandthatEPSIN1bindstog-ADRdirectly,thebindingofEPSIN1wasexaminedwithMBP-fusedeardomainsofg-ADRora-ADRex-pressedinE.coli,respectively(Figure6C).GST:EPSIN1wasin-cubatedwithMBP:g-ADR(ear),MBP:a-ADR(ear),orMBPalone.GST:EPSIN1-boundproteinswereprecipitatedfromthereactionmixtureandanalyzedbyproteingelblottingusinganti-MBPantibody.MBP:g-ADR(ear)boundstronglytoGST:EPSIN1(Fig-ure6D).Interestingly,MBP:a-ADR(ear)alsoboundweaklytoGST:EPSIN1,possiblybecauseofthethreeDPFmotifsinthemiddledomainofEPSIN1.DPForDPWmotifsareknowntobindtothea-eardomainofAP-2(Owenetal.,1999;Brettetal.,2002).However,thebindingaffinityofEPSIN1fortheg-ADReardomainwas>10-foldhigherthanthatforthea-ADReardomain.Theseresultsstronglysuggestthatg-ADRanda-ADRareArabi-dopsisg-adaptinofAP-1anda-adaptinofAP-2,respectively,

andthatEPSIN1bindsprimarilytog-ADR.Itisnotclearwhetherthea-ADRbindingofEPSIN1hasaphysiologicalroleinvivo.EPSIN1InteractswithVSR1

TofurthercharacterizethebindingpartnersofEPSIN1,EPSIN1bindingtoVSR—theArabidopsisvacuolarsortingreceptorthatlocalizesprimarilytothePVCbuttravelstotheTGNforcargobinding—wasexamined(Ahmedetal.,1997,2000;Tseetal.,2004).VSR1/ATELP1taggedwithHAattheCterminuswasexpressedtransientlyinprotoplasts.WeshowedpreviouslythattransientlyexpressedVSR1:HAlocalizedprimarilytothePVCwithaminorportiontotheGolgicomplex(Leeetal.,2004).ProteinextractswerepreparedfromthetransformedprotoplastsandincubatedwithGST:EPSIN1.Asnegativeandpositivecon-trols,proteinextractswerepreparedfromprotoplastsexpress-ingPEP12p:HAandVTI11:HA,respectively(daSilvaConceic¸a

˜oetal.,1997).EPSIN1-boundproteinswereprecipitatedfromthereactionmixtureandanalyzedbyproteingelblottingusinganti-HAantibody.VSR1:HA,butnotPEP12p:HA,wasdetectedinthepellet(Figure7A),indicatingthatEPSIN1boundspecificallytoVSR1.Tofurtherconfirmtheinteraction,weperformedaproteinpull-downexperimentofEPSIN1:GFPwithVSR1:HAfromtheextractsofprotoplastscotransformedwithEPSIN1:GFPandVSR1:HA.VSR1:HAwasimmunoprecipitatedwithanti-HAanti-body,andtheimmunoprecipitateswereanalyzedbyproteingelblottingusinganti-HA,anti-GFP,andanti-PEP12pantibodies.Anti-PEP12pantibodywasusedasanegativecontrol.Inaddi-tiontoVSR1:HA,EPSIN1:GFPwasdetectedintheimmunopre-cipitates(Figure7B).However,PEP12pwasnotdetectedinthepellet.TheseresultsfurtherconfirmtheinteractionbetweenEPSIN1andVSR1.TodefinetheVSR1bindingregionofEPSIN1,GST:ENTHandGST:EPSIN1DNwereusedinproteinpull-downexperiments.GST:ENTH,butnotGST:EPSIN1DN,broughtdownVSR1:HAfromthereactionmixture(Figure7C),indicatingthattheENTHdomaincontainedtheVSR1bindingdomain.EPSIN1IsInvolvedinVacuolarTraffickingofSolubleCargoProteins

Theprotein–proteininteractiondataofEPSIN1stronglysug-gestedthatEPSIN1wasinvolvedintraffickingsolubleproteinstothelytic(central)vacuole.ToexaminetheroleofEPSIN1,theT-DNAinsertionmutant,SALK_049204,fromtheSALKT-DNAinsertionmutantlibrarywasused.PCRwasusedtoconfirmthatthismutant,epsin1,hadaT-DNAinsertioninthepromoterregionofEPSIN1(Figures8Aand8B).HomozygotemutantplantswerescreenedbyPCR,becausetheepsin1mutantdidnotdisplayanymorphologicalalterations.Becausetheepsin1mutanthadaT-DNAinsertioninthepromoterregionofEPSIN1,proteingelblotanalysiswithanti-EPSIN1antibodywasusedtodeterminetheEPSIN1level.Inthehomozygoteepsin1plants,theEPSIN1levelwasreducedto10to20%ofthewild-typelevel(Figure8C).Themutantplantswereexaminedfordefectsinproteintrafficking.Previously,weshowedthatsporamin:GFP(Spo:GFP),achimericproteinconsistingofGFPandtheN-terminalregionofsporamin,wastargetedtothecentralvacuoleinproto-plasts(Kimetal.,2001).Ithadbeenshownpreviouslythatwhen

EPSIN1inVacuolarTrafficking2267

Figure7.EPSIN1BindstoVSR1viatheENTHDomain.

(A)ProteinextractswerepreparedfromprotoplaststransformedwiththeindicatedconstructsandincubatedwithGST-fusedEPSIN1.EPSIN1-boundproteinswereprecipitatedwithglutathione–agarosebeads,andthepelletedproteinswereanalyzedbyproteingelblottingusinganti-HAantibody.P,pellet;S,supernatant(10%oftotal).

(B)ProteinextractswerepreparedfromprotoplastscotransformedwithVSR1:HAandEPSIN1:GFPandusedforimmunoprecipitationwithanti-HAantibody.Theimmunoprecipitateswereanalyzedbyproteingelblottingusinganti-HA,anti-GFP,andanti-PEP12antibodies.P,immuno-precipitate;S,supernatant;T,totalproteinextracts(10%ofinput).

(C)ProteinextractsfromprotoplaststransformedwithVSR1:HAwereincubatedwithGSTaloneorGST-fusedEPSIN1proteins.EPSIN1bindingproteinswereprecipitatedwithglutathione–agarosebeads,andthepelletedproteinswereanalyzedbyproteingelblottingusinganti-HAantibody.

Spo:GFPwastransportedtothecentralvacuoleinprotoplasts,itwasprocessedfroma40-kDprecursortoa30-kDform(Sohnetal.,2003).Therefore,theamountoftheprocessedformcouldbeusedtomeasuretraffickingefficiency.Protoplastsobtainedfromtheepsin1mutantweretransformedwithSpo:GFP,andtraffickingofthereporterproteintothecentralvacuolewasexaminedbyproteingelblotanalysisusinganti-GFPantibody(Figure8D).Proteinextractswereobtainedfromprotoplastsatvarioustimepointsaftertransformation.Withwild-typeproto-plastsat36and48haftertransformation,;45and70%oftheSpo:GFP,respectively,wasintheprocessedform(Figure8D).

Figure8.EPSIN1IsNecessaryfortheEfficientVacuolarTraffickingofSpo:GFP.

(A)Screenofanepsin1mutant.T-DNAinsertioninEPSIN1wasconfirmedbyPCR.HE,heterozygote;HO,homozygote;LB,leftborderprimer;LP,leftprimer;M,sizemarker;RP,rightprimer.

(B)Schemeoftheepsin1mutant.ThepositionoftheT-DNAinsertionsiteisindicated.Thenumbersindicatenucleotidepositions.Shadedboxes,exons;darkboxes,promotersor39untranslatedregions.

(C)Totalproteinswerepreparedfromwild-typeplantsandheterozygotesandhomozygotesofepsin1plantsandanalyzedbyproteingelblottingusinganti-EPSIN1antibody.ThelargesubunitoftheRubiscocomplex(RbcL)stainedwithCoomassiebluewasusedasaloadingcontrol.

(D)TraffickingassayofSpo:GFPinepsin1protoplasts.Protoplastsfromwild-typeandepsin1plantsweretransformedwithSpo:GFP.Proteinextractspreparedfromprotoplastsatvarioustimepointsaftertransformationwereanalyzedbyproteingelblottingusinganti-GFPantibody.

(E)Proteingelblotanalysisofepsin1mutantsharboringHA:EPSIN1.ProteinextractswerepreparedfromtheT2generationofepsin1transgenicplantsharboringHA:EPSIN1andexaminedbyproteingelblotanalysisusinganti-HA,anti-EPSIN1,andanti-BiPantibodies.C,epsin1mutant;T1toT3,independenttransgenicplantsharboringHA:EPSIN1ontheepsin1background.

(F)and(G)Protoplastsobtainedfromwild-type,epsin1,andepsin1complementedwithHA:EPSIN1(EPSIN1/epsin1;T3)plantsweretransformedwithSpo:GFP.

(F)Proteinextractswereobtainedfromprotoplastsandanalyzedbyproteingelblottingusinganti-GFPantibody.

(G)TraffickingefficiencywasdeterminedastheratiooftheamountoftheprocessedformtothetotalamountofSpo:GFP(processedplusprecursorforms).ErrorbarsindicateSD(n¼3).

(H)Invertase:GFPsecretionwasnotinhibited.Protoplastsobtainedfromepsin1plantsweretransformedwithinvertase:GFP,andthesecretionofinvertase:GFPwasexaminedatvarioustimepointsaftertransformation.Proteinextractswerepreparedfromtheprotoplasts,aswellasfromthemedia,andanalyzedbyproteingelblottingusinganti-GFPantibody.

However,inprotoplastsfromepsin1mutants,theamountof30-kDprocessedproteinwasreducedsignificantly:at36and48haftertransformation,theamountoftheprocessedformwas;20and40%,respectively(Figure8D).TheseresultsclearlyindicatedthatEPSIN1wasinvolvedinthevacuolartraffickingofSpo:GFP.Toconfirmthattheinhibitionofvacuolartraffickingwasattributabletothemutation,transgenicplantsweregener-atedwithHA:EPSIN1ontheepsin1background.TheexpressionofHA:EPSIN1intransgenicplants(EPSIN1/epsin1)wasexam-inedbyproteingelblotanalysisusingproteinextractsobtainedfromthreeindependentlinesoftheT2generation.Theanti-HAantibodydetectedaspecificbandfromallthreeindependentlines(Figure8E).Furthermore,thelevelsofEPSIN1proteinsweresignificantlyincreasedinthetransgenicplantscomparedwithepsin1plants(Figure8E).Next,protoplastswereobtainedfromtheleaftissuesofwild-typeepsin1plantsandEPSIN1/epsin1plantsandtransformedwithSpo:GFP.Thetraffickingefficiencywasrecoveredtothewild-typelevelintheEPSIN1/epsin1plants(Figures8Fand8G),confirmingthatthedefectinthevacuolartraffickinginepsin1mutantplantsisattributabletothemutationinEPSIN1.

ToexaminethespecificityofEPSIN1invacuolartrafficking,thesecretionintothemediumofinvertase:GFP,achimericpro-teinconsistingofsecretoryinvertaseandGFP(Kimetal.,2005),wasexamined.Protoplastsobtainedfromepsin1andwild-typeplantsweretransformedwithinvertase:GFP.Proteinextractswerepreparedfromthetransformedprotoplastsandfromtheincubationmediumandanalyzedbyproteingelblottingusinganti-GFPantibody.Asexpected,wild-typeprotoplastsefficientlysecretedinvertase:GFPintothemedium.Norwasthesecretionofinvertase:GFPaffectedinprotoplastsfromtheepsin1mutants(Figure8I),indicatingthatEPSIN1isnotinvolvedinthesecretionoftheprotein.DISCUSSION

Inthisstudy,wedemonstratethatEPSIN1isinvolvedinthetraffickingofsolubleproteinstothecentral(lytic)vacuole.Thisconclusionisbasedonthreelinesofevidence.First,EPSIN1localizesprimarilytotheGolgicomplex(possiblytotheTGN)withaminorportiontothePVC.Second,EPSIN1interactsspe-cificallywithVTI11,VSR1,clathrin,andg-ADR,allofwhichplayimportantrolesinvacuolartraffickingfromtheTGN(Ahmedetal.,1997,2000;Basshametal.,2000;Surpinetal.,2003).Finally,thevacuolartraffickingofSpo:GFPisinhibitedinepsin1mutantprotoplasts,andthisdefectcanbecomplementedbystablyexpressedHA:EPSIN1.

LocalizationofEPSIN1inArabidopsis

Alargenumberofepsinhomologshavebeenidentified(Chenetal.,1998;DeCamillietal.,2002;Wendland,2002;Overstreetetal.,2003;Legendre-Guilleminetal.,2004).Onegroupoftheseproteins,whichincludesepsin1,hasbeenshowntobeinvolvedinendocytosis(Chenetal.,1998;DeCamillietal.,2002;Wendland,2002).Theothergroup,whichincludesEpsinR,hasbeenshowntoplayanimportantroleinlysosomal/vacuolartraffickinginanimalandyeastcells(Kalthoffetal.,2002;Wasiak

EPSIN1inVacuolarTrafficking2269

etal.,2002;Hirstetal.,2003;Chidambarametal.,2004;Eugsteretal.,2004;Saint-Poletal.,2004).Thus,aminoacidsequencehomologybetweenEPSIN1andotherepsinhomologscannotbeusedtodefinethepathwaysinwhichEPSIN1playsarole.Therefore,weexaminedthelocalizationoftransientlyexpressedHA-,GFP-,andRFP-taggedEPSIN1s.Regardlessofthetag,EPSIN1displaysbothnetworkandpunctatestainingpatterns.ThenetworkpatternresultsfromEPSIN1’sassociationwithactinfilaments.Thepunctatestainingpatternisattributabletotheprotein’slocalizationlargelytotheGolgicomplexwithaminorportiontothePVC.ThiswasdemonstratedbycolocalizationofEPSIN1withST:GFP,PEP12p,andVTI11.Atpresent,EPSIN1’sinteractionwithactinfilamentsisnotunderstoodclearly.ActinfilamentsrecentlywereshowntoplayacriticalroleinvacuolartraffickingfromtheGolgicomplexinplantcells(Kimetal.,2005).Thus,itispossiblethatactinfilamentsguideEPSIN1totheTGNandthePVC.ConsistentwiththisnotionistheobservationthatinthepresenceofLatBthepunctatestainingpatternofEPSIN1wasalsonotobserved.ItslocalizationsuggeststhatEPSIN1ismorecloselyrelatedfunctionallytoEpsinRinanimalcells.EpsinRhasbeenshowntolocalizetotheTGNaswellasendosomes(Kalthoffetal.,2002;Saint-Poletal.,2004).InhibitionoftheVacuolarTraffickingofSpo:GFPinepsin1Mutants

OneofthemostcompellingpiecesofevidencefortheroleofEPSIN1istheresultoftraffickingassaysinprotoplastsfromepsin1mutants.Anepsin1mutantwithaT-DNAinsertioninthepromoterregionhasgreatlyreducedEPSIN1proteinlevels,althoughthemutantsdisplaynovisiblephenotype.ItispossiblethatthelowlevelofEPSIN1inthemutantsisenoughtosupportvacuolartrafficking.AnotherpossibilityisthatAP-1cansupportvacuolartraffickingatlowlevelsofEPSIN1.However,despitethelackofavisiblephenotype,traffickingofSpo:GFPtothecentralvacuoleinprotoplastsisinhibitedsignificantly.Thedefectsinvacuolartraffickinginepsin1mutantsarerescuedbyHA:EPSIN1expressedstablyintransgenicplantswiththeepsin1back-ground,furthersupportingthehypothesisthatthetraffickinginhibitionisattributabletothemutationinEPSIN1.Inepsin1mutants,thesecretionintothemediumofthereporterproteininvertase:GFP(Kimetal.,2005)isnormal,indicatingthattheeffectsofthemutationsarespecificforthevacuolartrafficking.EPSIN1InteractionPartners

Alargenumbersofproteinsareinvolvedinlysosomal/vacuolartraffickingineukaryoticcells.ClathrinisbelievedtofunctionasacoatproteinforvesiclesderivedfromtheTGNinArabidopsis(Hohletal.,1996;Ahmedetal.,1997;Robinsonetal.,1998),ashasbeenobservedinothereukaryoticcells(Dahmsetal.,19;Pryeretal.,1992).EPSIN1stronglyinteractswithclathrin.Ithasatypicalclathrinbindingmotif,LIDL(Lafer,2002),andcolocalizeswithapoolofclathrin.Aswithmostepsinhomologs(Rosenthaletal.,1999;Wendlandetal.,1999;Drakeetal.,2000),EPSIN1appearstohaveatleasttwodifferentclathrinbindingmotifs,andthisstudydemonstratesthatLIDLisoneofthem.However,atthemoment,theothermotifisnotapparentanddoesnotshowany

2270ThePlantCell

sequencehomologywithknownclathrinbindingmotifs.ThefactthatEPSIN1bindstoandcolocalizeswithclathrinsuggeststhatEPSIN1mayrecruitclathrintotheTGNforvesicleformation(Rosenthaletal.,1999;Wendlandetal.,1999;Drakeetal.,2000).EPSIN1alsobindstoVTI11.EpsinRandEnt3pinteractwithvti1bandvti1pinanimalandyeastcells,respectively(Chidambarametal.,2004).However,EPSIN1doesnotbindtoVTI12,whichalsolocalizestotheTGNbutatadifferentregionthanVTI11(Basshametal.,2000;Surpinetal.,2003).ThetwoisoformsofVTIarethoughttobeinvolvedindifferentpathways:thezigmutantthathasamutationinVTI11displaysadefectinthegravitropicresponse(Katoetal.,2002),whereasatvti12mu-tantsappearnormalundernormalgrowthconditions(Surpinetal.,2003).Howevertheseisoformsappeartobesomewhatfunctionallyredundant,becauseplantscarryingeithermutationareviablebutmutationsinbothgenesarelethal(Surpinetal.,2003).ThefactthatdespitethehighdegreeofsimilaritybetweenVTI11andVTI12EPSIN1bindsspecificallytoVTI11butnotVTI12stronglysuggeststhatEPSIN1maycontributetothefunctionaldiversificationofthesetwov-SNAREsinthevacuolartraffickingattheTGN.

EPSIN1alsointeractswithVSR1.VSR1anditshomologsthatlocalizetotheTGNandthePVCarethebestcharacterizedsortingreceptorsinplants(Ahmedetal.,1997,2000;Sanderfootetal.,1998;ParisandNeuhaus,2002;Tseetal.,2004;daSilvaetal.,2005).VSR1hasbeenshowntointeractspecificallywiththeN-terminalpropeptidemotifthatispresentinmanysolubleproteinsdestinedforthecentralvacuole(Ahmedetal.,2000)andplaysaroleinthesortingofN-terminalpropeptidemotif–containingproteins.However,itsrolehasbeencomplicatedbythemutantphenotypeofatvsr1,whichdisplaysadefectinproteintraffickingtotheproteinstoragevacuoleinseedcells(Shimadaetal.,2003).TheENTHdomainofEPSIN1interactsspecificallywithVSR1,althoughtheexactsitesofinteractioninthetwomoleculesareunknown.ThisfindingisrathersurprisingbecauseithadbeenthoughtthatVSR1interactswithAP-1inplantcells(Sanderfootetal.,1998;Happeletal.,2004).Inaddition,inanimalcells,ENTH-containingproteinsarethoughttoassistinvesicleformationbyrecruitingclathrinforadaptors,APs,orGGAs(Legendre-Guilleminetal.,2004).However,theinteractionbetweenEPSIN1andVSR1stronglysuggeststhatEPSIN1isinvolveddirectlyinrecruitingclathrinforthesortingreceptorofvacuolartrafficking.

InadditiontotheinteractionbetweenEPSIN1andVSR1,EPSIN1alsointeractsdirectlywithg-ADR,aclosehomologofg-adaptinofAP-1invitro,butnotwitha-ADR,ahomologofa-adaptinofAP-2,ord-ADR,ahomologofd-adaptinofAP-3.EPSIN1hasaputativeacidicPhemotifthathasbeenshowntobethebindingsiteoftheheterotetramericadaptorcomplexAP-1andmonomericadaptorGGAs(Duncanetal.,2003;DuncanandPayne,2003).Proteinpull-downexperimentsclearlydemonstratethatg-ADRbindstotheEPSIN1fragmentcontain-ingtheacidicPhemotif.ThefactthatEPSIN1interactswithg-ADR,aclosehomologofg-adaptinofAP-1,placesitclearlyinthevacuolarpathwayratherthantheendocytoticpathway.Asinanimalcells,plantcellsarethoughttohavefourdifferenttypesofAPcomplexes,AP-1toAP-4,althoughtheirpreciseroleshavenotbeendemonstrateddirectly(Kirchhausen,1999;Robinson

andBonifacino,2001).Inanimalandyeastcells,AP-1functionsastheadaptorcomplexforCCVsandbindstothecargoreceptorattheTGN(Kirchhausen,1999;RobinsonandBonifacino,2001).ItislikelythatAP-1playsasimilarroleinplantcells(Ahmedetal.,1997).ConsistentwiththisnotionarethefindingsthatVSR1/ATELP1interactswithanimalAP-1andthatthemAsubunitofAP-1interactsinvitrowiththeYXXFmotif(whereFisabulky,hydrophobicresidue)thatispresentinthecytoplasmicC-terminaldomainofVSR-PS1,apea(Pisumsativum)VSR1(Sanderfootetal.,1998;Happeletal.,2004).AP-1islikelytoplayaroleinrecruitingclathrintotheTGNthroughitsinteractionwithVSR1(Ahmedetal.,1997).TheinteractionbetweenEPSIN1andg-ADRisquitesimilartothatbetweenEpsinRandg-adaptinofAP-1inanimalcells(Millsetal.,2003;Legendre-Guilleminetal.,2004).Thus,EPSIN1mayrecruitclathrinforAP-1–mediatedvesicleformationattheTGN.

WedemonstrateherethatEPSIN1isinvolvedinthetraffickingofSpo:GFPtothecentralvacuole.epsin1mutantsdisplaysignificantinhibitionofthevacuolartraffickingofSpo:GFPbutdonotshowacompleteblockinthevacuolartrafficking.ItispossiblethatbothAP-1andEPSIN1functionindependentlytorecruitclathrintotheTGNforthecargoesthataresortedbyVSR1,buttheymayrecruitclathrintotheTGNmuchmoreefficientlywhentheyactcooperatively.Inaddition,theinterac-tionbetweenEPSIN1andVTI11suggeststhatEPSIN1mayincorporatethev-SNAREVTI11intothevesicleattheTGN.

METHODS

GrowthofPlantsandScreeningofaT-DNAInsertionMutantArabidopsisthaliana(ecotypeColumbia)plantsweregrownonMurashigeandSkoogplatesinagrowthchamber.Leaftissueswereharvestedfrom10-to14-d-oldplantsandusedimmediatelyforprotoplastisolationasdescribedpreviously(Jinetal.,2001).Inaddition,plantsweregrownonsoilat20to258Cinagreenhousewitha16/8-hlight/darkcycle.

AT-DNAinsertionmutantofEPSIN1,theSALK_049204line,wasobtainedfromtheABRC(OhioStateUniversity).Toidentifyhomozygoticplants,genomicDNAfrom20individualplantswasusedforPCRwithleftborderprimer59-GATGCAATCGATATCAGCCAATTTTAGAC-39andthegene-specificprimer59-TCGATAGAGAAAACGGAAAAC-39.ConstructionofPlasmids

TheconstructionofVTI11:HAandGFP:talinwasdescribedpreviously(Kimetal.,2005).EPSIN1(At5g11710)wasisolatedbyPCRfromanArabidopsiscDNAlibraryusingthespecificprimersEpsin-5(59-AAG-AATTCAAATGGATTTCATGAAG-39)andEpsin-3(59-TTCTCGAGTCACT-GCTTAAAGCCACC-39).TotagtheNterminusofEPSIN1withtheHAepitope,thePCRproductwasreamplifiedwiththeprimers59-TCT-ATGGCTTACCCATACGATGTTCCAGATTACGCTATGGATTTCATGAA-GGTCTTC-39andEpsin-3.ToconstructEPSIN1:GFPandEPSIN1:RFP,theEPSIN1cDNAwithouttheterminationcodonwasamplifiedbyPCRusingtheprimers59-AAAGAGTACCGCCGAACAATGGATTTCATGAAG-GTC-39and59-GTCCCGGGACTGCTTAAAGCCACCAGATTGGTACC-39andligatedin-frametothe59endsoftheGFPandmRFPcodingregions,respectively(Campbelletal.,2002).EPSIN1:GFPandEPSIN1:RFPwereplacedbetweenthecauliflowermosaicvirus35SpromoterandthenosterminatorinapUCvector.

TogeneratetheGST:EPSIN1fusionconstruct,full-lengthEPSIN1wasligatedin-frametopGEX-5X-2usingEcoRIandXhoIsites.TheEPSIN1

deletionorsubstitutionmutants,ENTH,EPSIN1DN,LIDL(theC-terminalregionfromaminoacidposition461),andRIDL(identicaltoLIDLexceptfortheL-to-Rsubstitutionatposition470)wereamplifiedbyPCRusingthefollowingprimers:59-AAGAATTCAAATGGATTTCATGAAGGTC-39andEpsin-3forENTH,59-ATGAATTCATGGGTTCCTCAGCTTCA-39andEpsin-3forEPSIN1DN,59-GAATTCATTTGGGCAGATTCGCTG-39andEpsin-3forLIDL,and59-GAATTCATTTGGGCAGATTCGCTGAGCCGTG-GAAGGATTGATCTCAATATAACT-39andEpsin-3forRIDL.ThesePCRproductswereligatedtopGEX-5X-2usingEcoRIandXhoIsites.AllofthePCRproductsweresequenced.

a-ADR(At5g22770),g-ADR(At1g23900),andd-ADR(At1g48760)wereamplifiedbyPCRfromanArabidopsiscDNAlibraryusinggene-specificprimers.Theprimerswere59-AAGCTTATGAATCCCTTTTCTTCT-39and59-CTCGAGTCACAACCCGCGAGGGAA-39forg-ADR,59-GAATTCATG-ACCGGAATGAGAGGTCTCTCCGTA-39and59-GAATTCTTAAAGTAAG-CCAGCGAGCATAGCTCC-39fora-ADR,and59-GGTACCATGTCGTCG-TCTTCCACTTCTATAATG-39and59-GGTACCTCACAAGAGAAAATCT-GGAATTATAAC-39ford-ADR.ToconstructGFP:g-ADR,g-ADRwasligatedtotheCterminusoftheGFPcodingregionwithouttheterminationcodon.Toconstructa-ADR:GFPandd-ADR:GFP,a-ADRandd-ADRwereligatedtotheNterminusoftheGFPcodingregion.Theg-ADR-earanda-ADR-eardomainswereamplifiedbyPCR.Thegene-specificprimerswere59-ACCGAATTCCCAGCTTATGCACCTATTGTT-39and59-TCA-TCACAAAAGCTTAGGGAAGTTGCTGACTTG-39fortheg-ADR-eardo-mainand59-TTAGAATTCTTTCTTCAACCTCTACAATTG-39and59-TCA-AAGCTTTTGTTCCTTGATAAATTCTTCCAA-39forthea-ADR-eardomain.ThePCRproductswereligatedtopMAL-c2(NewEnglandBiolabs)togenerateMBP:g-ADR(ear)andMBP:a-ADR(ear).

TransientExpressionandinVivoTargetingofReporterCargoProteins

PlasmidswereintroducedintoArabidopsisprotoplastsbypolyethyleneglycol–mediatedtransformation(Jinetal.,2001;Leeetal.,2002).Theexpressionofthefusionconstructswasmonitoredatvarioustimepointsaftertransformation.ImageswerecapturedwithacooledCCDcameraattachedtoaZeissAxioplanfluorescencemicroscope.ThefiltersetsusedwereXF116(exciter,474AF20;dichroic,500DRLP;emitter,510AF23)andXF33/E(exciter,535DF35;dichroic,570DRLP;emitter,605DF50)(Omega)forGFP/fluoresceinisothiocyanateandRFP/tetrame-thylrhodamineisothiocyanate,respectively.ThedatawereprocessedusingAdobePhotoshopsoftware,andtheimageswererenderedinpseudocolor(Jinetal.,2001).

GenerationofAntibody

ToraiseanantibodytoEPSIN1,theDNAfragmentencodingthemiddledomain,aminoacidresidues153to337,wasamplifiedbyPCRus-ingspecificprimers(59-GGATCCGACAAAGCAGTAGCAAATCGT-39and59-GCTCGAGGTCCACTTGGTTGGCACTGCC-39)andligatedin-framewiththeHistagtopRSET-A(Invitrogen).Toraiseanantibodytocalreticulin(At1g56340),theC-terminalregioncontainingaminoacidresidues212to425wasamplifiedbyPCRusingprimers59-GGA-TCCAAGTGCTAAGAAGCC-39and59-TTAGAGCTCGTCATGGGCGGC-ATC-39andligatedin-frametopRSET-B(digestedwithBamHIandHindIII).Theresultingconstructs,PREST:EPSIN-MandpRSET-Cal,wereintroducedintoEscherichiacoli.Expressionoftherecombinantproteinswasinducedwithisopropylthio-b-galactosideandtheproteinswerepuri-fiedusinganickel-nitrilotriaceticacidagarosecolumn(Qiagen)accordingtothemanufacturer’sinstructions.Affinity-purifiedrecombinantproteinswereusedtoimmunizerabbits.Theantibodywasaffinity-purifiedusingrecombinantproteinasdescribedpreviously(HarlowandLane,1988).

EPSIN1inVacuolarTrafficking2271

Immunohistochemistry

Immunohistochemistrywasperformedasdescribedpreviously(Frigerioetal.,1998;Parketal.,2004).Thefixedprotoplastswereincubatedwithappropriateprimaryantibodies,suchasratmonoclonalanti-HA(RocheDiagnostics)orrabbitanti-clathrinantibodies,inTSWbuffer(10mMTris-HCl,pH7.4,0.9%NaCl,0.25%gelatin,0.02%SDS,and0.1%TritonX-100)at48CovernightandthenwashedwithTSWbufferthreetimes.Theprotoplastswereincubatedwithtetramethylrhodamineisothiocyanate–orfluoresceinisothiocyanate–conjugatedgoatanti-rabbitIgG(Sigma-Aldrich)oranti-ratIgG(Zymed)antibodiesinTSWbufferandwashedthreetimeswithTSWbuffer.

ProteinPreparation,ProteinGelBlotAnalysis,andImmunoprecipitation

Cellextractsfromprotoplastswerepreparedasdescribedpreviously(Jinetal.,2001).Toobtaintheproteinsfromtheculturemedium,coldtrichloroaceticacid(100mL)wasaddedtothemedium(1mL)andproteinaggregateswereprecipitatedbycentrifugationat10,000gat48Cfor5min.Theproteinaggregatesweredissolvedinthelysisbuffer.Thesupernatantwasusedforproteingelblotanalysiswithanti-GFP,anti-EPSIN1,anti-VSR,andanti-HAantibodies(RocheDiagnostics),asde-scribedpreviously(Jinetal.,2001).TheproteinblotsweredevelopedwithanECLdetectionkit(AmershamPharmaciaBiotech),andimageswereobtainedusinganLAS3000image-capturesystem(Fujifilm).

Immunoprecipitationwasperformedasdescribedpreviously(Parketal.,2005).Briefly,proteinextracts(100mgoftotalprotein)inimmuno-precipitationbuffer(10mMTris-HCl,pH7.5,150mMNaCl,2.0mMEDTA,0.2mMphenylmethylsulfonylfluoride,and1%[v/v]TritonX-100)supple-mentedwithanEDTA-freeproteaseinhibitorcocktailwerefirstincubatedwithProteinA–Sepharose(CL-4B;Amersham)for30minandcentrifugedat10,000gfor5minat48Cforpreclearing.Subsequently,4mgofanti-HAantibody(12CA5;RocheDiagnostics)wasaddedtothesupernatantandincubatedfor3hat48C.TheimmunocomplexeswereprecipitatedbyincubatingthemwithProteinA–agarosefor1hat48C.Thepelletwasthenwashedthreetimeswithimmunoprecipitationbuffer,suspendedinho-mogenizationbuffer,andsubjectedtoimmunoblotanalyses.ProteinPull-DownAssay

GSTandMBPfusionconstructswereintroducedintotheE.coliBL21(DE3)LysSstrain,andexpressionofthefusionproteinswasinducedbyisopropylthio-b-galactoside(1mM)at378C.GSTandMBPfusionproteinswerepurifiedusingglutathione–agarose4B(Peptron)beadsandamyloseresin(NewEnglandBiolabs),respectively,accordingtothemanufacturers’instructions.

Forproteinpull-downassays,partiallypurifiedrecombinantGSTorMBPfusionproteinsboundtoglutathione–agarosebeadsoramyloseresin,respectively,weremixedwithtotalproteinextractsin100mLofproteinpull-downbuffer(40mMHEPES-KOH,pH7.5,10mMKCl,3mMMgCl2,0.4Msucrose,1mMEDTA,1mMDTT,and0.2%TritonX-100)andincubatedat48Cfor1hwithagitation.Thebeadswerethenpelletedbycentrifugationat2000gfor1minat48Candwashedfourtimeswithproteinpull-downbuffer.Boundproteinswereeluted,fractionatedby10%SDS-PAGE,andsubjectedtoproteingelblotanalysisusingappro-priateantibodies.

GenerationofTransgenicPlants

Thecauliflowermosaicvirus35SpromoterandHA:EPSIN1wereinsertedintothepolylinkerregionofpBIB-HYG(Becker,1990),andtheresultingconstructwasintroducedintoplantsbyAgrobacteriumtumefaciens–mediatedtransformation(CloughandBent,1998).

2272ThePlantCell

AccessionNumbers

ArabidopsisGenomeInitiativelocusidentifiersforthegenesmentionedinthisarticleareasfollows:EPSIN1,At5g11710;EPSIN2,At2g43170;EPSIN3,At3g59290;AALP,At5g60360;VSR1,At3g52850;PEP12p,At5g16830;a-ADR,At5g22770;g-ADR,At1g23900;d-ADR,At1g48760;VTI11,At5g39510;andVTI12,At1g26670.SupplementalData

Thefollowingmaterialisavailableintheonlineversionofthisarticle.SupplementalFigure1.EPSIN1IsNotPermanentlyAssociatedwithClathrin-CoatedVesicles.

ACKNOWLEDGMENTS

TheT-DNAinsertionmutantwasobtainedfromtheABRC(OhioStateUniversity).ThisworkwassupportedbyagrantfromtheCreativeResearchInitiativesProgramoftheMinistryofScienceandTechnology(Korea).

ReceivedOctober29,2005;revisedMay26,2006;acceptedJuly5,2006;publishedAugust11,2006.

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Arabidopsis EPSIN1 Plays an Important Role in Vacuolar Trafficking of Soluble Cargo Proteins in

Plant Cells via Interactions with Clathrin, AP-1, VTI11, and VSR1Jinhee Song, Myoung Hui Lee, Gil-Je Lee, Cheol Min Yoo and Inhwan HwangPlant Cell 2006;18;2258-2274; originally published online August 11, 2006;

DOI 10.1105/tpc.105.039123

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