Proc.Nati.Acad.Sci.USAVol.77,No.7,pp.3870-3874,July1980BiochemistryTransportofvesicularstomatitisvirusglycoproteininacell-freeextract(endoplasmicreticulum/Golgicomplex/oligosaccharideprocessing/membraneassembly)ERIKFRIESANDJAMESE.ROTHMANDepartmentofBiochemistry,StanfordUniversity,Stanford,California94305CommunicatedbyI.RobertLehman,April7,1980ABSTRACTWedescribeacell-freesysteminwhichthemembraneglycoproteinofvesicularstomatitisvirusisrapidlyandefficientlytransportedtomembranesoftheGolgicomplexbyaprocessresemblingintracellularproteintransport.Trans-portinvitroisenergydependentandisaccompaniedbyter-minalglycosylationofthemembraneglycoprotein(dependentuponUDP-lcNAcandresultinginresistancetoendo-f-N-acetylglucosaminidaseH).Theelucidationofthemechanismsofassemblyofcellularmembranesandoftheorganellesthatthesemembranesdefineposesamajorchallengetocellbiologists.Inparticular,itwillbeimportanttounderstandhowdistinctsetsofproteinsaredeliveredtothedifferentsubcellularorganellessoastoconferuponthemtheirdistinctfunctions.Howisthishighlyspecificintracellulartransportandsortingofproteinsaccomplished?Anessentialsteptowardsobtainingamoleculardescriptionofthesecomplexcellulareventswillbetoachieveconditionsunderwhichthesametransportofnascentproteinscanproceedincell-freeextracts,foronlythencanthetoolsofbiochemistrybefruitfullyapplied.Wedescribehereexperimentsdemon-stratingthatitispossibletoobtaintransferofproteinbetweenspecificorganellesinacell-freeextractbyareactionresemblingthatofintracellularproteintransport.ForthesestudieswehaveutilizedChinesehamsterovary(CHO)cellsinfectedwithve-sicularstomatitisvirus(VSV)asawell-definedsysteminwhichtoinvestigatethetransportofamembraneproteindestinedfortheplasmamembrane(1).Theviralglycoprotein(Gprotein)thatwillresideinthemembraneofthematurevirionistheonlyglycoproteinsyn-thesizedinVSV-infectedcells.Likecellularsurfaceglycopro-teins,Gissynthesizedintheendoplasmicreticulum(ER),istransportedtotheGolgicomplex,*andisthentransportedtotheplasmamembrane(1).Clathrin-coatedvesiclesappeartomediatebothofthesetransportsteps(6).ThesmallgenomeofVSV(encodingonlyfiveproteins,allfoundinvirions)providesassurancethatthematurationofGfollowshost-specificpath-ways.DuringitssynthesisintheER,Gacquirestwomannose-richoligosaccharidesthataresubsequentlyprocessedasGpassesthroughtheGolgicomplex(2,3,7).Thischangeinoligosac-charidestructureprovidesanindirectmeansforassayingthearrivalofGattheGolgicomplex.Endo-Hhasprovedusefulinthisregard,becausethisenzymewillcleavethemannose-richprecursoroligosaccharidesofG,causingamarkedreductionofapparentmolecularweight,butwillnotattacktheGolgi-processedoligosaccharides(7).ThegeneralapproachwehavetakentoobtaintransportofGinvitroistoprepareextracts(postnuclearsupernatants)ofVSV-infectedCHOcellsthathadbeenbrieflyincubatedwith[35S]methionineinvivosoastolabelGintheER.ThisresultsinanextractinwhichallofthelabeledGproteinstillbearstheprecursoroligosaccharidesthatcanbecleavedbyEndo-H.TheproductionofanEndo-H-resistantformofGafterincubationofthecell-freeextractwouldbetakenasinitialevidenceoftransporttotheGolgicomplexinvitro.ToensurethatonlyeventsresultinginthetransferofGbe-tweenorganelleswouldbedetectedbythisindirectassay,wehaveutilizedextractspreparedfromaVSV-infectedmutantCHOcellline(clone15B)andaninvitrocomplementationscheme.Clone15BcellslackUDP-GlcNAcglycosyltransferaseI,anenzymefoundintheGolgicomplex(3)thatisneededtoinitiateprocessingoftheoligosaccharidesfrom"highMan"toEndo-H-resistant"complex"structures(2,8,9).Gistransportednormallywithininfected15Bcells(10)butalwaysremainssensitivetoEndo-H(11).Whenextractsof[asS]methionine-labeled,VSV-infected,clone15Bcellsareincubatedwithex-tractsofuninfectedwild-typeCHOcells,theappearanceofEndo-H-resistantformsofGprovidesstrongevidenceforthetransferofGfrom15BcellmembranestotheGolgicomplexderivedfromthewild-typecell,thesiteofoligosaccharideprocessing.MATERIALSANDMETHODSCellsandViruses.CHOcells(referredtoas"wildtype,"althoughnottheimmediateparentofclone15B)weremain-tainedinsuspension(12).TheCHOcellmutant(clone15B,obtainedfromStuartKornfeld,WashingtonUniversity)wasgrownasamonolayer(8).StockofVSV(Indianastrain)waspreparedfromCHOcells(12).PreparationofExtracts.Infectionof15Bcells.Sixplates(10-cmdiameter)atnearconfluency(about107cellsperplate)wereinfectedwith5-10plaque-formingunitsofVSVpercellinserum-freegrowthmedium(2mlperplate)containingac-tinomycinDat5jig/ml.At1hrafterinfection,8mlofcom-pletegrowthmediumwasaddedtoeachplate.At4hrafterinfection,cellswereremovedbytrypsinizationasfollows:Eachplatewasrinsedwith5mlofphosphate-bufferedsaline(Pi/NaCl;perliter:0.20gofKCI,0.20gofKH2PO4,8.0gofNaCl,and1.15gofNa2HPO4,pH7.4)andthenrinsedquicklywithAbbreviations:VSV,vesicularstomatitisvirus;G,viralglycoprotein;ER,endoplasmicreticulum;CHO,Chinesehamsterovary;Endo-H,endo-w-N-acetylglucosaminidaseH;Pi/NaCl,phosphate-bufferedsaline;CCCP,carbonylcyanidem-chlorophenylhydrazone;EGTA,ethyleneglycolbis(f3-aminoethylether)-N,N,N',N'-tetraaceticacid.*Weusetheterm"Golgicomplex"torefertotheintracellularsitesinCHOcellsinwhichtheoligosaccharidesofGareprocessedbytrimmingofManresiduesandadditionofterminalsugars(GlcNAc,Gal,sialicacid),makingthemresistanttocleavagebyendo-f3-N-acetylglucosaminidaseH(Endo-H).Forothercelltypesthesemodificationshavebeenshownbysubcellularfractionationandelectron-microscopicautoradiographytooccurintheGolgicomplex(2-5)asitisdefinedmorphologically.3870Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked"ad-vertisement"inaccordancewith18U.S.C.§1734solelytoindicatethisfact.Proc.NatI.Acad.Sci.USA77(1980)38713mlofPi/NaClcontaining0.5goftrypsinand0.2gofEDTAperliter.After5-10min,cellsweresuspended,washedinice-coldPi/NaCl,andthensuspendedin15mlofmethio-nine-freeJoklik'sminimalessentialmedium(GIBCO)con-taining10%dialyzedfetalcalfserum,1%nonessentialaminoacids(GIBCO),and20mMHepes-NaOH(pH7.3).Labelingofcells.Aftera5-to10-minequilibrationat370C,[35S]methionine(1-5mCi,>1000Ci/mmol,Amersham;1Ci=3.7X1010becquerels)wasadded.Aftera5-minpulse-la-beling,30mlofice-coldPi/NaClcontaining0.10gofCaCl2and0.059gofMgSO4perliterwasadded.Cellswerecentri-fugedat4VC,quicklywashedoncewiththesameice-coldbuffer,andresuspendedin6mlofice-coldPi/NaCl(withMg2+andCa2+)containing5MMcarbonylcyanidem-chlorophen-ylhydrazone(CCCP),andkeptonicefor5minbeforetheadditionofanother24mlofwarmPi/NaCl(withMg2+andCa2+)containing5,uMCCCP.Thecellswerethenincubatedfor20minat37C.Lysisofcells.Allsubsequentmanipulationswereat0-4°C.Cellswer
View Full Document