The Journal of Cell Biology
JCB
T Published August4,2003 Owen Pornillos, Introduction of thehumanHrsprotein HIV Gagmimicsthe Tsg101-recruiting activity (the “latedomain”)bindsdirectlytotheNH addition totheP(S/T)APsequencefoundinHIV-1Gag, factors thatcancatalyzereleaseoftheenvelopedvirion.In to facilitateviralbuddingbyrecruitingadditionalcellular (Tsg101; forreviewseePornillosetal.,2002c).Tsg101appears site forthecellularprotein,tumorsusceptibilitygene101 canbeeither SerorThr;Fig.1),whichisadocking position domain tetrapeptidemotifP(S/T)AP(wherethesecond Demirov etal.,2002b).AllHIV-1strainscontainthelate Gag p6region(Göttlingeretal.,1991;Huang1995; the presenceofa“latedomain”locatedinCOOH-terminal of HIV-1virionsandGagVLPsfrommostcelltypesrequires (VLPs) thatresembleauthenticHIVvirions.Efficientrelease proteins, HIV-1Gagcanformextracellularvirus-likeparticles see Göttlinger,2001).Evenintheabsenceofanyotherviral and formsthestructuralshellofimmaturevirus(forreview The HIVGagproteinorchestratesviralassemblyandbudding, 2 1 that Tsg101 bindstheCOOH-terminalregionofendo- endosomal membrane hasnotbeenknown. Now, wereport which Tsg101 isrecruitedfromthecytoplasm ontothe vesicular bodies(MVBs).However, themechanism by the lumenoflateendosomalcompartmentscalledmulti- Tsg101 isrequiredforbiogenesisofvesicles thatbudinto ubiquitin E2variant (UEV)domainof Tsg101. Inthecell, Marielle Payne, http://www.jcb.org/cgi/doi/10.1083/jcb.200302138 The Journal ofCellBiology, Volume 162, Number 3,August 4,2003 425–434 multivesicular body proteinsorting; budding;virions;ubiquitin;vacuolar Key words: virus of MolecularBiology,10550NorthTorrey PinesRoad,LaJolla,CA92037. O. Pornillos’presentaddressisTheScripps ResearchInstitute,Department Tel.: (801)595-8203.Fax:581-7959. email:[email protected] University ofUtah,20N,1900E, Rm. 211,SaltLakeCity,UT84132. Address correspondencetoWesleyI.Sundquist,Dept.ofBiochemistry, this paper. O. Pornillos,D.S.Higginson,andK.M.Straycontributedequally to
Myriad Genetics, Salt LakeCity, UT84108 ofBiochemistry,Department University ofUtah SchoolofMedicine, Salt LakeCity, UT84132
TheRockefeller University Press, 0021-9525/2003/08/425/10$8.00 Article
A conserved P(S/T)APtetrapeptide motifwithinGag Tsg101 tofacilitatethefinal stagesofvirusbudding. he HIV-1 Gagproteinrecruitsthecellularfactor
1
1 Gong-Ping He,
Daniel S.Higginson,
2 Hubert E. Wang, 1 Kirsten M.Stray, Kirsten 2 -terminal 2 ScottG.Morham, 1 virus bindtotheNH al., 2002;Timminset2003). 2001; Stracketal.,2000;KikonyogoYasuda E3ligases(Xiangetal.,1996;Harty1999, ubiquitin any aminoacid),whichbindstheNedd4proteinfamilyof best characterizedoftheseisthePPXYmotif(whereX characterized inseveralotherenvelopedRNAviruses.The distinct latedomainsequenceshavealsobeenidentifiedand All fourP(S/T)APresiduesarerequiredforlatedomainactivity cally recognizesP(S/T)APsequences(Pornillosetal.,2002a). enzymes inthatitdisplaysahydrophobicgroovespecifi- al., 2002a).Tsg101UEValsodiffersfromthecanonicalE2 (Moraes etal.,2001;VanDemarkPornillos et active sitecysteineresiduerequiredforubiquitintransfer related toubiquitinE2–conjugatingenzymes,butlackthe 2003). UEVdomainsalsobindubiquitinandarestructurally and Allen,2002;Pornillosetal.,2002b;Timmins al., 2001;VerPlanketDemirov2002a;Myers domain ofTsg101(Garrusetal.,2001;Martin-Serrano et the Hrs mediated, inpart,by bindingofthe Tsg101 UEVdomainto kinase substrate (Hrs;residues222–777). This interaction is somal proteinhepatocyte growth factor–regulatedtyrosine gene 101; kinase substrate;MVB,multivesicular body;Tsg101,tumorsusceptibility fortransport;Hrs,hepatocytegrowthfactor–regulatedtyrosine required Abbreviations usedinthispaper:ESCRT, endosomalsortingcomplex VLP, virus-likeparticle;Vps,vacuolar proteinsorting. viral budding. ponents oftheMVBvesicle fissionmachinery tofacilitate this Hrsactivity, andthereby usurps Tsg101 andother com- the endosomalmembrane. HIV-1 Gagapparentlymimics indicate thatHrsnormallyfunctionstorecruit Tsg101 to that aremissingnative latedomains. These observations Tsg101 andrescuethebuddingofvirus-likeGagparticles Robert D.Fisher, The P(S/T)APlatedomainsofHIV-1,HIV-2,andEbola
348 UEV, ubiquitinE2variant;UIM,ubiquitin-interacting motif; PSAP 2 and Wesley I.Sundquist 351 motif.Importantly, Hrs 1 2 Jennifer E.Garrus, -terminal ubiquitinE2variant(UEV) 222–777 1 1
canrecruit
425
Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 426 Pelchen-Matthews etal.,2003).InHrs,the FYVEdomain some celllines (Göttlinger,2001;Raposo etal.,2002; tains signalsthattargetGag totheplasmamembranein N-myristoylated MAdomain bindsmembranes,andcon- proximal membrane-targeting domains.InHIV-1Gag,the similarities (Fig.1).First,both proteinscontainamino- the HrsandHIV-1Gagproteins exhibitseveralintriguing et al.,2002;Bache2003b). Tsg101; Bilodeauetal.,2002;BishopRaiborg dosome (andthereforeappearstofunctionupstreamof it interactswithubiquitylatedcargoproteinsontheearlyen- al., 2002;ShihetBache2003b)andbecause biogenesis (Piperetal.,1995;Odorizzi1998;Lloyd et cause itisrequiredforreceptordown-regulationandMVB factor thatrecruitsTsg101toendosomalmembranesbe- and Stenmark,2002).Hrsisanattractivecandidateforthe strate (Hrs;yeastVps27p;Raiborgetal.,2001a; (2) hepatocytegrowthfactor–regulatedtyrosinekinasesub- motifs (Garrusetal.,2001),including(1)Tsg101itself;and searches revealedseveralVpsproteinswithPTAPorPSAP of Tsg101totheendosomalmembrane.Proteindatabase interactions mightoccurandplayaroleintherecruitment T)AP motifshavenotbeendescribed,wereasonedthatsuch cruited tothemembranehasnotbeenelucidated. However, themechanismbywhichTsg101/ESCRT-Iisre- sents akeystepinthecommitmenttovesicleformation. Thus, recruitmentofTsg101tothemembranesurfacerepre- tein sortingandvesicleformation(Babstetal.,2002a,b). and otherproteinscomplexesrequiredtocompletepro- to recruitthedownstreamESCRT-II,ESCRT-III,Vps4, uitin modifications(Katzmannetal.,2001);and(2)ithelps interactions betweentheTsg101UEVdomainandubiq- functions: (1)itbindsubiquitylatedcargoproteinsviadirect Tsg101/ESCRT-I appearstoperformatleasttwoessential endosome duringMVBbiogenesis.Onceonthemembrane, that arerecruitedfromthecytoplasmontosurfaceof lysosome, wheretheyaredegradedbyhydrolasesandlipases. these vesiclesandtheirproteincargosintothelumenof (MVBs). MVBscanthenfusewithlysosomesandrelease late endosomalcompartmentstoformmultivesicularbodies into microdomainsthatultimatelybudassmallvesicles delivered toendosomalmembraneswheretheyaresorted al., 2002).Inthispathway,monoubiquitylatedproteinsare for destructioninthelysosome(forreviewseeKatzmannet membrane proteinssuchascellsurfacereceptorsaretargeted vacuolar proteinsorting(Vps)pathway,inwhichintegral 2001). Tsg101andESCRT-Iperformessentialrolesinthe transport-I (ESCRT-I)proteincomplex(Katzmannetal., subunit oftheendosomalsortingcomplexrequiredfor elements foundincellularproteins. speculate thattheTsg101UEVdomainmaybindP(S/T)AP ral P(S/T)APsequences,anditisthereforereasonableto Tsg101 UEVdomainpresumablydidnotevolvetobindvi- 2002a,b). However,despitethissequencespecificity,the within theTsg101UEVbindingsite(Pornillosetal., (Huang etal.,1995),andallfourmakeimportantcontacts The domainorganizationand biochemicalpropertiesof Although interactionsbetweenTsg101andcellularP(S/ ESCRT-I isoneofaseriessolubleproteincomplexes In thecell,Tsg101(yeastVps23p)normallyfunctionsasa The JournalofCellBiology
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Volume 162,Number3,2003 key aspectsofthesemodelsforHrsfunctionandviralmimicry. brane. Experimentsdescribedinthispaperweredesignedtotest cle formationtositesofviralbuddingontheplasmamem- tions ofHrs,andtherebyredirectthemachineryMVBvesi- Gag proteinhasevolvedtomimictheTsg101-recruitingfunc- brane throughadirectinteractionwithHrs;and(2)theHIV-1 which (1)Tsg101isnormallyrecruitedtotheendosomalmem- al., 2000;Freed,2002;Pornilloset2002c). Nedd4 substratesandforPPXYvirallatedomains(Rotinet This sequencematchestheconsensussitesforcellular ubiquitylates Hrs,theproteindoesharboraPPEYmotif. al., 2002),andalthoughitisnotyetknownwhetherNedd4 Rsp5p) familyofubiquitinligases(Katzetal.,2002;Polo UIM-containing proteinsaresubstratesfortheNedd4(yeast et al.,2002;RaiborgShih2002).Other Falquet, 2001;Bishopetal.,2002;LloydPolo motif(UIM;Youngetal.,1998;Hofmannand interacting requires acis-actingsequencemotifcalledtheubiquitin- receptor down-regulationthroughtheMVBpathway,and (Strack etal.,2000,2002).Hrsubiquitylationisessentialfor mains havebeenshowntorecruitubiquitinligaseactivities virus budding(forreviewseeVogt,2000),andvirallatedo- not beendemonstrated,ubiquitintransferisimportantfor role forHIVGagubiquitylationinretrovirusbuddinghas Gag proteinsaremonoubiquitylated.Althoughafunctional ently unstructured)regions.Third,bothHrsandHIV-1 contain P(S/T)APmotifswithinproline-rich(andappar- et al.,2001b;Katzmann2003).Second,bothproteins branes (Komadaetal.,1997;Odorizzi1998;Raiborg binds PI(3)PandtherebytargetsHrstoendosomalmem- P(S/T)AP andPPEYmotifsarealsoindicated. “steadiness box/Vps28bindingsite”(Fengetal.,2000).Locationsof variant; PRD,proline-richdomain;COIL,putativecoiled-coil;SBOX, interaction domainsbyaverticaldashedline.UEV,ubiquitinE2 binding domainsseparatedfromtheCOOH-terminalprotein–protein the sameaffinity toanine-aminoacidpeptide astothein- UEV binding epitope issmall,andthedomain bindswith from thehumanTsg101and Hrsproteins.TheTsg101 to emphasizetheirsimilarities,withtheNH Tsg101 andHrsproteins. Figure 1. bind toisolatedP(S/T)APmotifs fromHIV-1 First, wetestedwhethertheUEV domainofTsg101could Gag, Tsg101,andHrs Tsg101 UEVbindstheP(S/T)AP motifsfromHIV-1 Results The precedingobservationsareconsistentwithamodelin Domain organizationoftheHIV-1Gagandhuman TheHIVGagandHrsproteinsarealigned 2 -terminal membrane- NL4–3
Gagand
Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 personal communication). ited almostundetectableTsg101UEVbinding(Fisher,R., ined someisolatedP(S/T)APpeptidesequencesthatexhib- lute bindingaffinitiessignificantly.Indeed,wehaveexam- flanking thecentralP(S/T)APmotifcanmodulateabso- the P(S/T)APmotifsofallthreeproteins,butsequences PTAP peptide.Hence,theTsg101UEVdomaincanbind (Hrs) weakerthanTsg101UEVbindingtotheHIV-1 binding affinitieswerefourfold(Tsg101)andsevenfold tides fromTsg101andHrs(Fig.2).However,the the PTAPmotifwasmutatedtoLeu(Fig.2). bind toacontrolpeptideinwhichthesecondProresidue interaction wasspecific,astheTsg101UEVdomaindidnot BD orADalone didnotproducesignificant in whichoneof thetwoplasmidsencoded onlytheGal4p activity (Fig.3A,topleft).In contrast,controlexperiments strain J693,resultinginsignificant levelsof sion (Hrs-AD)werecotransfected intothereporteryeast sion (Tsg101-BD)andanHrs–Gal4p activationdomainfu- Plasmids expressingaTsg101–Gal4p bindingdomainfu- proteins wasexaminedindirected yeasttwo-hybridassays. The interactionoffull-lengthTsg101andwild-typeHrs Interaction offull-lengthTsg101andHrsproteins tact HIV-1p6protein( tetrapeptides depicted), butthatsequencesflankingthecentralP(S/T)AP motif doesnotsignificantlyaffecttheTsg101bindingaffinity(not Note thatsubstitutionofSforTatthesecondpositionP(S/T)AP fits tosimple1:1bindingmodelsusedobtain binding affinities. HIV-1 GagPTAPmotif(negativecontrol).Solidlinesshowtheoptimal motifs ofHIV-1Gag,Tsg101,andHrs,aswellamutantformthe UEV domaintoimmobilizedfusionpeptidesspanningtheP(S/T)AP the concentration-dependentbindingofpurifiedrecombinantTsg101 HIV-1 Gag,Tsg101,andHrs. Figure 2. dissociation constant(K dissociation librium responsesfitasimple1:1bindingmodelwith dependent bindingtotheGagPTAPpeptide,andequi- ments. Asexpected,Tsg101UEVexhibitedconcentration- Tsg101 UEVdomainbindinginBIAcorebiosensorexperi- T)AP motifsfromGag,Tsg101,andHrsweretestedfor Therefore, 10-aminoacidpeptidesspanningthecentralP(S/ The Tsg101UEVdomainalsoboundtoP(S/T)APpep- The Tsg101UEVdomainbindstheP(S/T)APmotifsfrom can modulateTsg101bindingaffinitysignificantly. d 20 Biosensorbindingisothermsshowing 25 C ) of21
M; Pornillosetal.,2002b).
1
M (Fig.2).This
-galactosidase -galactosidase
tivity ( Tsg101 UEVbindingtoseveraladditionalPSAP-likemotifs seen fortheTsg101M95Amutationmayreflectweak (approximately twofold).Thegreaterreductioninbinding reduced wild-typeTsg101binding,albeittoalesserextent coiled-coil Pro/Gln-rich region(e.g.,Hrs451–777; seeFig.3 spanned justthe HrsPSAPelement(e.g., 1–450)orthe weakly (ornotatall)toaseries ofsmallerHrsfragmentsthat 573 contributetoTsg101 binding. Tsg101boundonly 2003a), andwethereforeconclude thatHrsresidues560– into thePro/Gln-richregion boundwell(Bacheetal., rich regionofHrs(Hrs deletion thatremovedtheentireCOOH-terminalPro/Gln- did noteliminate)Hrsbinding,andafurthersix-aminoacid tion ofCOOH-terminalHrsresidues565–777reduced(but dues 222–777(Hrs equally welltoaCOOH-terminalfragmentspanningresi- able fortheTsg101/Hrsinteraction,asTsg101bound idues 1–221).Thesedomainswerealsocompletelydispens- tion). Asexpected,theHrs man HrstoyeastVps27p(Emr,S.,personalcommunica- substitution oftheHrs sixfold; Fig.3C).Incomplementaryexperiments,alanine (M95A) reducedHrsbindingsignificantly(approximately interactionwasnotmediatedthroughubiquitin. hybrid These experimentsindicatedthattheTsg101/Hrstwo- significant effectonTsg101/Hrsbinding(unpublisheddata). wise, alaninesubstitutionsintheHrsPPEYsequencehadno had noeffectontheHrs/Tsg101interaction(Fig.3B).Like- on theTsg101/Hrstwo-hybridinteraction(Fig.3,BandC). 2002a). Therefore,thesemutationsweretestedfortheireffects PTAP (M95A)orUb(N45A)binding(Pornillosetal., different sites,anddistinctTsg101pointmutationseliminate Hrs. Tsg101UEVbindsP(S/T)APpeptidesandubiquitinat the Tsg101UEVdomainandaubiquitinmodificationon and theHrsPSAPmotif;and/or(2)aninteractionbetween Hrs via(1)aninteractionbetweentheTsg101UEVdomain domain, wehypothesizedthatTsg101mightinteractwith Based ontheknownbindingpropertiesofTsg101UEV Requirements forTsg101/Hrsbinding length Tsg101-BDtypicallystimulated assays,thebindingoffull-lengthHrs-ADtofull- culture activity (Fig.3A).Similarly,insemi-quantitativeliquid construct (residues287–573) thatextended Tsg101 bindingtonearlybackground levels.AmurineHrs teins interactintheyeasttwo-hybridassay. fore, weconcludethatthefull-lengthTsg101andHrspro- bind theNH affinity binding.AssummarizedinFig.3D,Tsg101didnot were usedtodefinethecompleteHrsregionrequiredforfull sidual binding.Therefore,directedtwo-hybridexperiments PSAP interaction,HrsandTsg101exhibitedsignificantre- the sameprotein–proteininterface. with theideathatthesemutationsaffectedtwosidesof duce bindingoftheTsg101M95Amutation,consistent 583 In contrast,thePSAP-bindingmutationinTsg101 The ubiquitin-bindingmutationinTsg101UEV(N45A) Surprisingly, evenintheabsenceofTsg101UEV/Hrs PSGP 100-fold abovebackground(Fig.3,B–D).There- 586 and 2 -terminal VHSandFYVEdomainsofHrs(res- Hrs andHIVGagrecruitTsg101| 620 PSMP N ) astofull-lengthHrs.Incontrast,dele- N 348 623 C PSAP ; Hrsresidues222–559)reduced PSAP ) thatareconservedfromhu- mutationdidnotfurtherre- 351 sequence(Hrs -galactosidase ac- Pornillosetal. 15 residues PSAP ) also
427
Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 428 VLP releasewas dependentonthepresence ofafunctional Hermida-Matsumoto andResh,2000;Garrus etal.,2001). expressed wellandformedVLPs efficiently(Fig.4B,lane1; C; Cell).Asexpected,thecontrol Gag–GFPfusionprotein lyzed inWesternblotsofcytoplasmic extracts(Fig.4,Band VLP). IntracellularGagprotein expressionlevelswereana- pernatants aftersucrosecushion pelleting(Fig.4,BandC; VLP releasewasanalyzedbyWesternblottingofculturesu- tein expressionandforVLPrelease24–28hlater(Fig.4). with HIVGagexpressionconstructs,andanalyzedforpro- components oftheMVBpathway(Garrusetal.,2001). 2000), includingtherequirementforTsg101andother ral assemblyandbudding(Hermida-MatsumotoResh, proteins inhumancelllinesrecapitulatesmanyaspectsofvi- cultured humancells.ExpressionofHIVGag–GFPfusion Hrs weresufficienttosupportthebuddingofVLPsfrom Next, wetestedwhethertheprotein-recruitingfunctionsof Hrs latedomainactivity tative coiled-coilandatleastpartofthePro/Gln-richregion. (1) thePSAPmotif;and(2)asecondregionspanningpu- quires thepresenceoftwo(ormore)differentHrsregions: D). Thus,weconcludethatfullaffinityTsg101bindingre- Human embryonickidney293Tcellsweretransfected The JournalofCellBiology
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Volume 162,Number3,2003 (D) BindingofTsg101toHrsdeletionmutants. of wild-typeandmutant( absorbance (595nm)andSDsfromthreeindependentmeasurements.(C)Binding semi-quantitative CPRG yeast two-hybridinteractions(togetherwithappropriatecontrols)asmeasured in of Hrstowild-typeandmutant(N45A)Tsg101.BindingexperimentsinB–Dshow synthetic agarmedialackingtheappropriateaminoacidsandwereanalyzedfor (BD) andactivationdomain(AD)fusionconstructs.Cellsweregrownon formed withpairsofplasmidsexpressingtheindicatedGal4DNA-bindingdomain Figure 3. lift assayfor -galactosidase activity(blue)asdescribedinMaterialsandmethods.(B)Binding
Hrs/Tsg101 yeasttwo-hybridbindingassays.
replaced withtheHrs replaced PTAP motif(57%oftotalGag proteinreleased).TheHrs protein released)wasnearlyasstrongthenativeGag Hrs
protein thatwasmissingthe entire p6domain(Gag construct alsorescuedparticle releasewhenfusedtoaGag (Gag PTAP latedomain,asmutationofthemotiftoLIRL main activityoftheHrs released intotheculturesupernatant.Indeed,latedo- Hrs PTAP motif,theGFPpolypeptideofGag could replacethelatedomainactivityofcis-actingGag rus etal.,2001). cle release(Göttlingeretal.,1991;Huang1995;Gar- tains boththe normally targetHrstotheendosomalmembrane,butcon- lane 3),theGag and bindsTsg101(Fig.3D).AsshowninFig.4B(top, budding ofdefective Gagconstructsintrans (Fig.4C).This to testwhether Gag–Hrsfusionproteinscould rescuethe published dataandseebelow).
-galactosidase reportergeneactivity.Yeastcellswerecotrans-
To testwhethertheproteinrecruitmentactivitiesofHrs Analogous resultswereobtained inexperimentsdesigned
N N
PTAP fragmentismissingtheNH ; Fig.4A).Asdescribedintheprevioussection,
–GFP; Fig.4B,lane2)severelyattenuatedparti-
PSAP) Hrstowild-typeandmutant(M95A)Tsg101.
-galactosidase activityassays.Barsdepicttheaverage
348
PSAP
PTAP –Hrs 351 N motifandthePro/Gln-richregion, polypeptide(denotedGag N polypeptide(51%oftotalGag N fusionproteinwasefficiently 2 -terminal domainsthat (A)Filterpapercolony PTAP -GFP was p6 PTAP , un-
– N
Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 complementation ofdeficientGag (lane 4),Gag rano etal.,2001). Controlexperimentsdemonstrated that Gag proteinsthat arecompetentforbudding (Martin-Ser- proteins canbereleasedfrom cellsbyco-assemblingwith experiment tookadvantageof thefactthatdefectiveGag their VLP-buddingphenotypes.(B)TheHrs Gag p6. Figure 4. (lane 1),Gag–GFP2),Gag with 1.5 expression ofthedifferentGagconstructs.Cellswerecotransfected analyzing VLPrelease.Bottom,Westernblotshowingcytoplasmic Gag were transfectedwith0.5 expression oftheGag–GFPandGag–Hrsfusionproteins.293Tcells proteins. Bottom,Westernblotshowing cytoplasmic Gag–Hrs Top, WesternblotanalysisofVLPreleasebytheGag–GFPand the buddingarrestcausedbymutationofGagPTAPlatedomain. (lane 4),Gag PTAP (A)SummaryoftheGagandGag–Hrsfusionconstructs –GFP (lane2),Gag g plasmidDNAencodingGag Hrs cansubstituteforlatedomainfunctionsofHIV-1 PTAP PTAP –Hrs –Hrs NPSAP N C (lane5),oremptyvector6).(C)Trans g plasmidencodingGag–GFP(lane1), PTAP (lane5),orGag –Hrs PTAP p6 N –GFP (lane3),Gag budding.Top,Westernblot (lane3),Gag p6 and0.5 N PTAP polypeptiderescues –Hrs PTAP g emptyvector NC PTAP –Hrs (lane6). –Hrs NPSAP N
deed, the self (Poloetal.,2002),butdidnotdiminishVLPrelease.In- abolishes Hrsubiquitinbindingandubiquitylationofit- Gag Also, wetestedthereleaseofaGag and 3).Gag Gag–GFP PTAPsequence(Fig.4C,top,comparelane2 top, comparelane1and2),thatrescuerequiredthe the twoproteinswerecoexpressedinsamecell(Fig.4C, (Gag sequence within theHrs sequence reflected weakresiduallatedomainactivityofthePSAP lished data).Thelowlevelsofrelease,whenseen,presumably vs. 51%totalGagproteinreleased).Similarly, Gag in thePTAP-codingregionofGaggene.However, fectivity ofHIV-1proviralconstructsthatharbormutations type Gag–GFPproteincanrescueboththebuddingandin- defects (Martin-Serranoetal.,2001).Forexample,thewild- the infectivityofHIV-1proviralconstructswithbudding fusion proteinswithlatedomainactivitiescanalsorescue both incisandtrans. late domainactivityandcansupportHIV-1Gagbudding tif Hrs Hrs coexpression withGag assays. AsshowninFig.4B,Gag wild-type Gag–GFPcouldrescuethereleaseofGag Transmission EMwasusedto confirmthattheGag EM analysesofVLPs 348 this experiment,Gag protein (Fig.4C,lane6),althoughinsomerepetitionsof tivity, Hrs To testthesequencerequirementsforHrslatedomainac- Sequence requirementsforHrslatedomainactivity we concludethatfullHrslatedomainactivityrequiresthe tivity oftheR9 bled immature HIVvirionsinbothappearance andsize peared asenveloped, sphericalparticlesthat generally resem- bud efficiently.Incontrolexperiments, Gag–GFPVLPsap- amine thephenotypesof Gagconstructsthatfailedto late wellwiththerequirementsforTsg101binding. Gln-rich region,butnottheUIM.Theserequirementscorre- Hrs was notefficientlyrescuedintransbytheGag efficiency ascomparedwiththeGag corporation oftheHrs may reflectalterationsinparticlemorphologycausedbyin- cation assay(unpublisheddata),andwespeculatethatthis than Gag some degree(Fig.4C,lane5),butwasagainlessefficient a mutationintheUIM( Hence, weconcludethattheHrs released fromcells(Fig.4B,lane5),whereasGag In additiontorescuingVLPformationintrans,someGag PSAP N N N (Gag p6 PTAP PTAP proteinwasreleasedintheform ofVLPsandtoex- –Hrs PSAP PSAP 351 –Hrs 265 –Hrs PTAP elementandCOOH-terminalcoiled-coilPro/ proteinagainrescuedthereleaseofGag wasreleased(Fig.4B,lane4),butwithreduced PTAP LA N N p6 releaseslightly(unpublisheddata).Overall, –Hrs constructsmissingeitherthePSAPmo- 266 releasewasalsoefficientlyrescuedintransby –Hrs N Hrs andHIVGagrecruitTsg101| N PTAP constructdidnotefficientlyrescueinfec-
→ C ) werealsotestedinthetwoVLPrelease ALmutationactuallyappearedtoenhance proviralconstructinasinglecyclerepli- N N (Fig.4C,comparelaneand5). p6 222–777 PSAP wasreleasedatlowlevels(unpub- PTAP N ) orthePro/Gln-richregion 265
–Hrs polypeptide(seebelow).
C LA
polypeptide.TheGag
266
N
PTAP
N
(Fig.4C,top,lane4).
→ polypeptidehaspotent
PTAP PTAP
–Hrs AL).Thismutation
–Hrs
–Hrs
Pornillosetal.
N
constructwith
PTAP
N
N
control(27
C
–Hrs
wasnot
p6
p6
release
when
PTAP
PTAP PTAP p6
N to 429
– – –
C
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The Journal of Cell Biology Published August4,2003 430 (C–E) Transmissionelectronmicrographsofthin-sectioned293TcellstransfectedwithplasmidsexpressingGag another). Bars,100nm. examples ofclassic“latedomain”phenotypesinwhichtheassembledparticlesremainedtetheredtoplasmamembrane(or show immature HIV-1virionsandGagVLPs. density thatisnormallyobservedbeneaththemembranesof GFP layer,incontrasttotheevenlydistributedringofGag polypeptide appearedtocreatediscontinuitiesintheGag– (100–200 nmindiameter;Fig.5A).However,theGFP VLPs releasedfrom293TcellstransfectedwithplasmidsencodingGag–GFP(A)andGag Figure 5. quite pronouncedintheGag diameter). TheGag“discontinuity”phenotypewasalso were largerandmoreheterogeneousinsize(upto500nm were similarinappearancetotheGag–GFPparticles,but also releasedfromcellsintheformofenvelopedVLPsthat the Hrs (D), andGag and (2)VLPsformedbyGag membrane oftenappeareddiscontinuous,suggestingthattheCOOH-terminalGFPandHrspolypeptidesperturbedunderlyingGag lattice; formed enveloped,sphericalVLPsthatresembledauthenticimmaturevirionsexcept(1)theelectron-denseGaglayerbeneath theVLP Gag pressing Gag were notreleased efficiently(Fig.5,C–E).In cellsex- that to determinethephenotypes oftheGagfusionproteins some extent. As showninFig.5B,theGag EM imagesofthin-sectioned cellswerealsoexamined The JournalofCellBiology PTAP –Hrs N Electron microscopicanalysesofGag–GFPandGag–Hrsbudding. fusionproteinalteredparticle morphologyto PTAP PTAP N –Hrs formedenvelopedVLPsas expected, but –GFP, Gag N PSAP PTAP | (E).Thetopimagesshowclustersofparticlesassociatedwiththecellularsurface,andbottom
Volume 162,Number3,2003 –Hrs PTAP PTAP N exhibitedgreatersizevariationandwere,onaverage,somewhatlargerthanauthenticimmaturevirions. –Hrs –Hrs PTAP –Hrs N N C particles.Thus, , andGag N proteinwas PTAP – associate withmembranesand initiatesphericalparticleas- associate proteins were not releasedfromcellsefficiently, theydid Gag (Raposo etal.,2002;Pelchen Matthewsetal.,2003). HIV-1 canbudintoMVBcompartments inmacrophages in goodagreementwithrecent experimentsshowingthat gested thatatleastsomeoftheVLPsbuddedintracellularly, plasma membrane(unpublisheddata).Theseimagessug- were intheprocessof(orhadrecently)fusedwith peared thatvacuolarstructuresfilledwithassembledVLPs bottom images).Also,weobservedimagesinwhichitap- membrane viathinstalks(Fig.5,C–E,arrowsin the assemblingVLPsclearlyremainedtetheredtoplasma membrane connectivitiesinthin-sectionedimages,manyof C–E; topimages).Althoughitcanbedifficulttoestablish ated withthecellsurfacewerefrequentlyobserved(Fig.5, Hrs Overall, ourEManalysesestablished thatalthoughthe (AandB)Transmissionelectronmicrographsofthin-sectioned N PTAP PSAP –GFP, Gag , clustersofenvelopedsphericalparticlesassoci- PTAP –Hrs PTAP N (B).Notethatinbothcases,theproteins –Hrs N PTAP C , andGag –GFP (C),Gag PTAP –Hrs PTAP –Hrs toone N N PSAP
C
Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 Hrs derstanding MVB biogenesis,thecomplementary Gagpro- analyses ofthe cellularpathwayareclearly essential forun- quired forproteinsortingand MVBformation.Although with proteincargosandrecruits downstreamfactorsre- plasm ontothesurfaceof endosome,whereitinteracts indicate thatHrsrecruitsTsg101/ESCRT-I fromthecyto- labor plementary experimentsfromtheEmrandStenmark gous VLPsystem.Theseexperiments,togetherwithcom- dependent plasmamembranevesiclefissioninaheterolo- that COOH-terminalHrsfragmentscansupportTsg101- Tsg101 bindsHrs(andCOOH-terminalfragments),and (Murk etal.,2002).Wehaveshownthatfull-length Sorkin andVonZastrow,2002),MHC-IIpresentation 2002), receptordown-regulation(Katzmannetal.,2002; tant biologicalprocessesincludingdevelopment(Kramer, becausethispathwaymediatesanumberofimpor- interest The moleculareventsunderlyingMVBbiogenesisareof Implications forcellularHrsfunction Discussion port virusbuddingbecausetheNH Tsg101 tothesiteofbuddingmaynotbesufficientsup- Tsg101* levelsarelimiting.Alternatively,simplyrecruiting cient toblockVLPbuddingunderconditionswhere Hrs bindingcausedbytheTsg101M95Amutationissuffi- mutation, anditisthereforepossiblethatthereductionin Hrs Next, weexaminedwhetherthelatedomainactivityof The latedomainactivityofHrsisdependentonTsg101 therefore reflectedthedefect(s)inlatedomainactivity. structs occurredatalatestageinthebuddingprocess,and sembly. Thus,theblocktoparticlereleaseinthesecon- Gag–Hrs the Tsg101*M95Amutationalsoblockedreleaseof of theUEVdomain(Pornillosetal.,2002a).Interestingly, residue Met95toAlablockstheP(S/T)AP-bindingactivity lane 2).AsshowninFig.6,releaseofGag (Garrus etal.,2001;Fig.6,bottom,comparelane1and Tsg101 canbeefficientlydepletedusingRNAinterference M95A mutation,andVLPreleasewasreducedbytheHrs However, HrsbindingwasdiminishedbytheTsg101 could stillbindHrs(Fig.3C)andbecausetheGag somewhat unexpectedbecausetheTsg101M95Aprotein of Hrs fore, thisexperimentconfirmedthatthelatedomainactivity was expressed(Fig.6,comparelane2and3).There- ference–resistant Tsg101–FLAGprotein(denotedTsg101*) 2). VLPreleasewasrestoredwhenanexogenousRNAinter- from theproducercells(Fig.6,top,comparelane1and from 293TcellswasblockedwhenTsg101depleted binding bytheUEVdomainofTsg101. again emphasizesthefunctionalsignificanceofP(S/T)AP hibited bytheM95Amutation.Ineithercase,result Tsg101 UEVmayperformadditionalfunctionsthatarein- As mentionedearlierinthispaper,mutationofTsg101 N N atories (Bacheetal.,2003a; Katzmann etal.,2003), polypeptiderequiredthepresenceofTsg101.Cellular PSAP N isdependentonTsg101. N mutantproteinwasreleasedfromcells(Fig.4B). protein(Fig.6,lane4).Thisabsoluteblockwas 2 -terminal domainof p6 –Hrs N VLPs PTAP PSAP – for bothMVBbiogenesisandvirusbudding. mammalian cells,forthevesiclefissionactivitiesrequired tein releaseassayprovidesaneffectivefunctionaltest,in of Gag–Hrs binding activityofTsg101. Figure 6. lated proteincargos); and/or(3)membrane-dependent Hrs and otherendosomal membraneprotein(s) (e.g.,ubiquity- rano etal.,2003);(2)cooperative bindingofTsg101toHrs membrane, andTsg101can alsooligomerize;Martin-Ser- (1) avidityeffects(asmultiple copiesofHrsassembleatthe membrane isstillunclear,but plausiblepossibilitiesinclude allows Tsg101recruitmentonly afterHrsisboundtothe tween HrsandTsg101.Thenatureofthe“switch”that curs, atleastinpart,throughadirectbindinginteractionbe- CRT-I complex,andourexperimentsindicatethatthisoc- 2003b). ThiscomplexnextrecruitsthesolubleTsg101/ES- 1997; Raiborgetal.,2002;YamadaBache cargos, STAM1,STAM2,Eps15,andclathrin(Asaoetal., protein complexthatincludesHrs,ubiquitylated et al.,2000;Raiborg2001b;Bache2002). teins mayalsoplayimportantrolesinHrslocalization(Urbe al., 2002).Finally,interactionswithotherendosomalpro- the ubiquitinmodificationsoncargoproteins;Raiborg et (e.g., throughinteractionsbetweentheHrsUIMmotifand al., 2003).Hrslocalizationmayalsobecargo-dependent 1998; Urbeetal.,2000;Raiborg2001b;Katzmann displayed ontheendosomalmembrane(Odorizzietal., least inpart,byFYVEdomainbindingtoPI(3)Pmolecules MVB factorstobind,anditsrecruitmentismediated,at tually sortedintoMVBvesicles.Hrsisthefirstofsoluble where ubiquitylatedproteincargosaccumulateandareeven- cytoplasm ontosubdomainsoftheendosomalmembrane soluble complexesmustbesequentiallyrecruitedfromthe An intriguingaspectofMVBbiogenesisisthataseries onto theendosomalmembrane Sequential recruitmentofproteins RNA; and(2)lane5wasamocktransfection. and methodswiththefollowingexceptions:(1)lane1receivedno (Tsg101*). 293TcellswerecotransfectedasdescribedintheMaterials reexpression ofexogenous,siRNA-resistantTsg101-Flagproteins showing depletionofendogenousTsg101proteinwithsiRNA,and expression levelsofGag–Hrs On themembrane,Hrsisanessentialsubunitofamulti- Budding ofGag N VLPrelease.Middle,Westernblotshowingcytoplasmic Hrs andHIVGagrecruitTsg101| Top,Westernblotshowingrelativelevels p6 N –Hrs . Bottom,Anti-Tsg101Westernblot N isdependentontheP(S/T)AP- Pornillosetal.
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Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 432 presumably evolved toexitcellsasefficiently aspossible, also interactdifferently withTsg101because viruseshave the loweraffinityHrsPSAPsequence. Virusesandcellsmay the virustocompeteeffectively forTsg101bindingagainst affinity P(S/T)APelementof HIV-1Gagapparentlyallows MVB vesicleformationandbudding (Fig.7B).Thehigher in ordertohijackthemachinery thatnormallycatalyzes mal protein-recruitingfunctionsofthecellularHrsprotein HIV-1, HIV-2,andEbolahaveevolvedtomimicthenor- Our analysesalsoindicatethatthestructuralproteinsof Viral recruitmentofTsg101 al., 2002;PellicenaandMiller,2002). to createdirectionalityinothercellularpathways(Francis et are usedbyotherclassesofproteins,suchasproteinkinases, gous autoinhibition/conformationalswitchingmechanisms model remainstobetestedrigorously,wenotethatanalo- of bothHrsandubiquitylatedproteincargos.Althoughthis nism forinsuringthatactivationoccursonlyinthepresence cargos, whichwouldprovideacooperativebindingmecha- domain interactingwithubiquitinmodificationsonprotein might alsoinvolvetheubiquitin-bindingsiteonUEV PSAP elementofHrs(i.e.,intrans).Thisactivationevent in whichtheTsg101UEVdomainswitchestobind 7 A).Hrsbindingcouldthendriveaconformationalchange UEV domainbindsitsownPTAPelement(i.e.,incis;Fig. ily inanautoinhibitedconformationwhichtheTsg101 the solublecytoplasmicESCRT-Iproteinmayexistprimar- events inMVBbiogenesis).Inthismodel,weenvisionthat CRT-II and/orMVBcargobinding(orforotheressential serves primarilyastheswitchthat“activates”Tsg101forES- sites ofvesiclebudding,whereastheHrsPSAPelement downstream HrssiteservesprimarilytorecruitTsg101 tion. However,anattractivealternativemodelisthatthe crease theaffinityandspecificityofHrs/Tsg101interac- may playthedominantroleinTsg101recruitment. in allcontexts,indicatingthatthedownstreamHrselements completely eliminatecomplexformationandVLPbudding that disruptedtheTsg101UEV/PSAPinteractiondidnot actions werefunctionallyimportant,althoughmutations gion andatleastpartofthePro/Gln-richregion.Bothinter- stream Hrselementsthatspanstheputativecoiled-coilre- Tsg101 UEVdomain,andasecondthatincludesdown- first whichinvolvestheHrsPSAPsequencebindingto affinity Tsg101bindingrequiresatleasttwoHrselements,a Unexpectedly, ourbindingexperimentssuggestedthatfull and activation Possible mechanismsofTsg101recruitment membrane. downstream factorsonlyafterESCRT-Iisboundtothe vated” insomefashiontoallowtherecruitmentofthese likely thatthemembrane-boundESCRT-Imustbe“acti- onto themembrane(Babstetal.,2002b).Again,itseems CRT-II and-IIIproteinsarerecruitedfromthecytoplasm al., 2000;Bacheet2002). conformational changesorphosphorylationevents(Urbeet The useofmultiplecontactsitescouldsimplyservetoin- In thenextstagesofMVBbiogenesis,solubleES- The JournalofCellBiology |
Volume 162,Number3,2003 membrane fission. perform subsequentstepsnecessaryforvesicleformationand fect aconformationalchangethatactivatestheproteinto main, thevirusmaysimultaneouslyrecruitTsg101andef- binding tightlytothePTAP-bindingsiteinUEVdo- MVB vesicles.Thisraisestheintriguingpossibilitythatby whereas cellsmustcarefullyregulatethefluxofcargointo render pIRES-TsgFlagresistanttosiRNA(denotedpIRES-Tsg*Flag)werein- Inc.) togivepIRES-TsgFlag(Garrusetal.,2001).Sevensilentmutationsthat plified productwasclonedintopIRES2-EGFP(CLONTECHLaboratories, fication withadownstreamprimerencodingtheFlagsequence.Theam- Flag epitopetothe3 A Tsg101mammalianexpressionvectorwasconstructedbyfusingthe Plasmid constructs Materials andmethods Figure 7. to facilitateviral budding. redirects Tsg101 and theESCRT-Icomplextoplasma membrane how HIVGagmimicstheTsg101-recruiting functionofHrsand COOH-terminal regionsofTsg101 andHrs.(B)Modelillustrating proteins mightbridgeorcontribute totheinteractionbetween text fordetails).Notethatourdata allowthepossibilitythatother interaction andapossibleactivation mechanismforTsg101(see MVB andHIVbudding. Models forTsg101recruitmentand activationduring endofthefull-lengthTsg101geneusingPCRampli-
(A)ModelillustratingsitesofTsg101/Hrs
Downloaded from from Downloaded on June 22, 2016 22, June on jcb.rupress.org The Journal of Cell Biology Published August4,2003 TC tains theRev-independentHIV-1 introduced intopIRES-Tsg*FlagusingKunkelmutagenesis. mutagenesis (Garrusetal.,2001).N45AandM95Amutationswerealso al., 2001).Theseexperimentswererepeatedatotalofsixtimes. region withtheappropriateHrssequences.Gag struct byreplacingthelastthreeaminoacidsofGagandGFP-coding (o-nitrophenyl- DNA sequencing.Fullcloningdetailsareavailablefromtheauthors. Hrs withtheGal4pactivationdomain(AD).Allconstructswereverifiedby plasmids encodedin-framefusionsofTsg101withtheGal4pBDand were subclonedintotheplasmidMP29(MyriadGenetics).Theresulting ics), whichencodestheGal4pDNA-bindingdomain(BD).Hrsconstructs from pIRES-TsgFlagandsubclonedintotheplasmidMP30(MyriadGenet- BE276844; fromAmericanTypeCultureCollection,Manassas,VA). PCR amplifiedfromanEST(GenBank/EMBL/DDBJaccessionno. were either astopcodonortheappropriateHrssequences.constructs structs werecreatedbyreplacingthep6-andGFP-codingregionswith rpm (4 through a20%sucrosecushionin microcentrifugefor90minat13,000 VLPs from1.2mlofsupernatants from transfectedcellswerepelleted Western blots siRNA duplexesand1 (Tsg101*). Thesecondtransfectionwasperformedwiththesame50-nM wild-type ormutantRNAi-resistantpIRES-Tsg*Flagexpressionvector was with50nMsiRNAduplexesandeither2 293T cellswerecotransfectedtwiceat24-hintervals.Thefirsttransfection UCU CGUCdTdT;antisense,5 nucleotides 413–433.siRNAsequences:sense,CCUCCAGUCUUC Synthetic 21-ntsiRNAduplexesweredesignedtotargetTsg101atcoding formed asdescribedpreviously(Elbashiretal.,2001;Garrus2001). 28 hlaterandanalyzedbyWesternblotting(seebelow). into 293Tcells.Supernatantsandcytoplasmiclysateswereharvested24– DNA encodingGag–GFPandGag–Hrsfusionproteinswastransfected essentially asdescribedpreviously(Garrusetal.,2001).Inbrief,plasmid 2000 (Invitrogen;Garrusetal.,2001).VLP-buddingassayswereperformed sion plasmidsasindicatedinthefigurelegendsusingLipofectAMINE™ imum essentialmedium(6-wellplates)andweretransfectedwithexpres- Human embryonickidney293Tcellsweregrownin2mlDulbecco’smin- Tissue cultureandVLP-buddingassays Yeast strainJ693( Yeast two-hybridbindingassays (Jenkins etal.,2001;Pornillos2002a). GST–peptide fusionconstructswerealsoobtainedasdescribedpreviously (Garrus etal.,2001),andpurifiedrecombinantTsg101UEVdomain GST–peptide fusionconstructswereperformedasdescribedpreviously BIAcore biosensormeasurementsofTsg101UEVbindingtoimmobilized Biosensor bindingassays troduced intothewild-typeTsg101-codingregion 24–30 handanalyzedbyWesternblotting(seebelow). supernatants andcytoplasmiclysateswereharvestedafteranadditional ing Institute,NewYork,NY).Gag mida-Matsumoto andResh,2000;agiftfromMarilynSloan-Ketter- titation of cate withCPRG(chlorophenolred at random,pooled,grownonsyntheticliquidmedia,andassayedintripli- Gag Gag PTAPmotiftoLIRLasdescribedpreviously(Garrusetal.,2001). ids. Toidentifycoloniesproducing controls), andplatedonsyntheticmedialackingtheappropriateaminoac- various pairsofBDandADconstructs(usingemptyvectorsas gal80 lys2 1% NP-40,and0.1% SDS)andincubatedonicefor4 min.Sampleswere directly into30–35 Co.). Transfected293Tcells(onewell froma6-wellplate)wereharvested with antisera,andproteinbandswere detectedbyECL(PierceChemical l sampleswereseparatedbySDS-PAGE, transferred,blocked,blotted with X-gal(5-bromo-4-chloro-3-indoyl- filter paperdisks,lysedbyfreeze-thawinginliquidnitrogen,andassayed All GagandGag–HrsconstructswerebasedonpGag–GFP,whichcon- For yeasttwo-hybridassays,Tsg101constructswerePCRamplified Functional siRNAknockoutandrescueofTsg101expressionwereper- TTC PTAP C) andresuspendedin25–30 TCT –Hrs fusionconstructswerecreatedfromtheGag -galactosidase activity,colonies( :: CGT GAL1p-HIS3 C - D 433 MAT -galactopyranoside) asdescribedpreviously(Garruset (mutatednucleotidesareunderlined)usingKunkel l RIPAbuffer(10mM TrisCl,pH7.0,150mMNaCl, g Gag ade2his3leu2trp1cyh2ura3 ; Bendixenetal.,1994)wascotransformedwith p6 GACGAGAGACUGGdTdT. –Hrs HBX2 PTAP l of1 GagsequencefusedtoEGFP(Her- -galactosidase, cellswereliftedonto N –GFP wascreatedbymutatingthe expressionvector.VLP-containing - D - D -galactopyranoside) orONPG -galactopyranoside). Forquan- SDSgelloadingbuffer.5–8- 20 perplate)wereselected g pIRES2-EGFPor2 p6 andGag :: GAL1p-LacZ gal4 413 PTAP AA p6 CCT –GFP con- –Hrs con- CCA g G- Garrus, J.E.,U.K.von Schwedler,O.W.Pornillos,S.G.Morham, K.H.Zavitz, Feng, G.H.,C.J.Lih,andS.N.Cohen. 2000. TSG101proteinsteady-statelevelis Elbashir, S.M.,J.Harborth,W.Lendeckel,A.Yalcin,K.Weber,andT.Tuschl. Demirov, D.G.,J.M.Orenstein,andE.O.Freed.2002b.Thelatedomainofhu- Demirov, D.G.,A.Ono,J.M.Orenstein,andE.O.Freed.2002a.Overexpression Freed, E.O.2002.Viral latedomains. Francis, S.H.,C.Poteet-Smith,J.L.Busch, R.Richie-Jannetta,andJ.D.Corbin. Bendixen, C.,S.Gangloff,andR.Rothstein.1994.Ayeastmating-selectionscheme Bishop, N.,A.Horman,andP.Woodman.2002.MammalianclassEvpsproteins Bilodeau, P.S.,J.L.Urbanowski,S.C.Winistorfer,andR.C.Piper.2002.The Bache, K.G.,C.Raiborg,A.Mehlum,andH.Stenmark.2003b.STAMHrs Bache, K.G.,A.Brech,Mehlum,andH.Stenmark.2003a.Hrsregulatesmulti- Bache, K.G.,C.Raiborg,A.Mehlum,I.H.Madshus,andH.Stenmark.2002. Babst, M.,D.Katzmann,W.Snyder,B.Wendland,andS.Emr.2002b.Endo- Babst, M.,D.Katzmann,E.Estepa-Sabal,T.Meerloo,andS.Emr.2002a.Escrt- Asao, H.,Y.Sasaki,T.Arita,N.Tanaka,K.Endo,H.Kasai,Takeshita, References Accepted: 4June2003 Submitted: 24February2003 W.I. Sundquist. ing theirresultsbeforepublication. advice. WealsothanktheStenmarkandEmrlaboratoriesforcommunicat- croscopy; andmembersoftheSundquistlabfortechnicalassistance periments; NancyChandlerandBarbieGanserforhelpwithelectronmi- We thankRebeccaRichandDavidMyszkaforhelpwiththeBIAcoreex- transmission electronmicroscope(Tecnai-12;Philips). described previously(Garrusetal.,2001).Sampleswereimagedona (von Schwedleretal.,1998).Cellswerepreparedforthin-sectionEMas sucrose cushionsandpreparedforthin-sectionEMasdescribedpreviously VLPs from80mlpooledculturesupernatantwerepelletedthrough20% Electron microscopy 4A10 (1:1,000;GeneTex,Inc.)wasusedtodetectTsg101. 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