MOLIECULARANDCELLULARBioLociN,June1989.p.2706-2714Vol.9,No.60270-7306/ - PDF document

MOLIECULARANDCELLULARBioLociN,June1989.p
MOLIECULARANDCELLULARBioLociN,June1989.p.2706-2714Vol.9,No.60270-7306/89/062706-09$0()'.00/0CopyrightV1989.AmericanSocietyforMicrobiologyCharacterizationoftheYeastKEXIGeneProduct:aCarboxypeptidaseInvolvedinProcessingSecretedPrecursorProteinsANTONYCOOPERANDHOWARDBUSSEY*Dep(artmiienitofBiology,McGillUniversity,1205AvenueDr.Pentfield,Montreal,Quehec,CalnadaH3AIBIReceived23December1988/Accepted23March1989WehaveidentifiedandpartiallycharacterizedtheSaccharomycescerevisiaeKEXIgeneproduct,Kexlp,toassessitsroleinprocessingsecretedproteinprecursors.Anti-Kexlpantibodiesidentifieda113-kilodaltonproteinthatwasabsentincellsinwhichtheKEXIgenehasbeendisruptedandthatwasmoreabundantincellsoverexpressingtheKEXIgene.Kexlpwasfoundtobeamembrane-associatedglycoproteinwithN-linkedcarbohydrate.TheN-linkedoligosaccharide(s)wasmodifiedinaprogressivemanneraftersynthesis,causingtheglycoproteintoslowlyincreaseinmassto115kilodaltons.AfteraKex2p-mediatedcleavageeventatspecificpairsofbasicaminoacids,cL-factorandKikillertoxinprecursorshaveCOOH-terminaldibasicresidueextensionsandrequireacarboxypeptidaseB-likeenzymetoprocesstheprecursorstomaturity.Acarbox-ypeptidaseactivity,withapparentspecificityforbasicaminoacids,wasdetectedinKEXIcells.DisruptionoftheKEX1geneabolishedthisactivity,whileoverexpressionofKEX1increasedit.Ourresultsprovidebiochemicalevidenceconsistentwithearliergeneticwork,thatKEXIencodesaserinecarboxypeptidaseinvolvedintheprocessingofprecursorstosecretedmatureproteins.Hormonesandneurotransmitterssecretedbyeucaryoticcellsareknowntobeproducedasprecursorswhicharethenproteolyticallyprocessedtoyieldmaturepolypeptides(8.24,35).Withinmanyprecursors.pairsofbasicresiduesflankmaturepolypeptidesandactassitesforendoproteolyticcleavage.FurtherprocessingoftenrequiresremovalofthebasicaminoacidsbyacarboxypeptidaseB-likeenzyme.SuchaprocessingsystemoccursintheyeastSaucharo-mvcescerevisiae(3,13).Thex-factorpheromoneandKlkillertoxinarethebest-characterizedexamplesofsecretedproteinsderivedfromsuchprocessedprecursors.a-Factorisa13-amino-acidpeptidepheromoneinvolvedinthematingofhaploidyeastcells.Theot-factorpeptideisencodedbytwoseparategenesMFoQIandMFox2(22.34)whichencodea165-or120-amino-acidprecursor,respectively.Thelargerprecursorcontainsfourrepeatsofthepheromone.whilethesmallerencodesatandemrepeat.SeparatingeachrepeatisthepeptidesequenceLys-Arg-Glu-Ala-(Glu/Asp)-Ala-Glu-Ala.ProcessingisinitiatedbytheKEX2geneproduct(Kex2p)whichendoproteolyticallycleavesafterthepairofbasicresidues(19).TheSTE13geneproduct(20)removestheGlu-AlarepeatsfromtheN-terminusofthepheromone,resultinginana-factorpeptidewithaLys-Ai-gCOOH-terminalextension(aF-KR).KillertoxinisasecretedheterodimericproteinproducedbystrainsofS.cereisit(iwhichcontainacytoplatsmicallyinheriteddouble-strandedRNAgenome(36).TheKltoxinisproducedasaprecursorwiththetwosubunitsofthematuretoxin.(xandP,separatedbytheyregion.FlankingtheyregionarepairsofbasicresiduesthatKex2pcleavestoreleasethecx-nprotein.The(xsubunitofthisprocessingintermediatepossessesaCOOH-terminalextensionoftwobasicresidues.Onthebasisofpreliminarystudies,amembrane-associ-atedactivity,termedysca,hasbeenproposedtobethecarboxypeptidatseinvolvedinprocessingtheCOOHtermi-*Correspondingauthor.nusofx-factor(1).ThisactivityhasbeenattributedtotheKEXIgene(38).Theprecursorprocessingofboth(x-factorandKikillertoxinrequiresacarboxypeptidaseB-likeenzymetoremovepairsofbasicresiduesattheirrespectiveCOOHtermini.Earliergeneticandmolecularstudies(7)providedstrongindicationsthattheKEXIgeneproduct(Kexlp)fulfillsthisfunction.HerewecharacterizeKexlpanddemonstratethatitisindeedacarboxypeptidase,withapparentspecificityforbasicresidues,involvedinprocessingsecretedproteinsinS.cere'isi(ae.MATERIALSANDMETHODSMaterials.Restrictionendonucleases,T4DNApolymer-ase,T4DNAligase.andK.enowfragmentwerepurchasedfromeitherNewEnglandBioLabs,Inc.(Beverly,Mass.),BethesdaResearchLaboratories,Inc.(Gaithersburg,Md.),orPharmacia(Uppsala,Sweden)andwereusedasrecom-mendedbythesuppliers.Tunicamycin,cycloheximide,en-do-p-N-acetylglucosaminidaseH(EndoH),phenylmethyl-sulfonylfluoride(PMSF),andTritonX-100werepurchasedfromBoehringerMannheimBiochemicals(Indianapolis.Ind.).SequencingwasperformedbyusingtheSequenasekit(U.S.Biochemicals,Cleveland,Ohio).EconofluorandEn3HancewereobtainedfromDupont,NENResearchProducts(Boston.Mass.).Isopropyl-3-D-thiogalactopyrano-side(IPTG).N-tosyl-L-phenylalaninechloromethylketone(TPCK).Nx-p-tosyl-L-lysinechloromethylketone(TLCK).Nx-p-tosyl-L-argininemethylester(TAME),andNx-p-tosyl-L-lysinemethylester(TLME)wereobtainedfromSigmaChemicalCo..St.Louis.Mo.[_y32P]dATPand[35S]methioninewerepurchasedfromAmershamCorp.(ArlingtonHeights.Ill.).Trans35SlabelwasfromICNBiochemicals(Irvine.Calif.).Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)supplieswerefromBio-RadLaboratories(Richmond.Calif.).Bacterialstrainsandprocedures.EshIericlii(coliMC10612706CHARACTERIZATIONOFTHEYEASTKEXIGENEPRODUCT2707(5)wasusedforroutinegrowthandmaintenanceofplas-mids,whileE.coliDH1(25)wasusedforproductionofthefusionprotein.E.coliUT580wasthestrainusedforsingle-strandedDNA(ssDNA)production.Allmediaforroutinegrowthandmaintenanceofstrainswerestandard(25).Ampicillin,whenrequired,wasusedat100p.gml-'.Yeaststrainsandprocedures.S.ceirevisiaestrainsusedwerecl3ABYS86(hereaftercalledS86)(MATotAiira3-2le2Ihisprl1prb2prcIcpsl),Sc25k(MATo(ldell/eii2-3.112ira

i3-52[KIL-k1])(7),Sc25k-13(7),andM190.7-
i3-52[KIL-k1])(7),Sc25k-13(7),andM190.7-6C(MATasstlsst2).HAB211(secI8le,,2-3,112ideld)andHAB213(sec1leu2-3,112adtlel)havebeendescribedpreviously(23).HMSF6(MATase(7)wascrossedwithC627-4B(MATotaidelleiu2-3,112his3-11,15).Thediploidsweresporulated.andasciweredissected.OnesporewasselectedandcalledHAB212(sec7leii2-3,112(adelhis3-11,15).TheyeastgrowthmediausedwereYEPDcompleteme-dium(0.5%yeastextract[DifcoLaboratories,Detroit,Mich.],1%Bacto-Peptone[Difco],0.1%NaCl,0.1%KH2PO4,0.05%MgSO4,21%glucose)andminimalmedium(0.67%yeastnitrogenbase[YNB][Difco],2%glucose,supplements[2.5,ugml-']requiredbyauxotrophicstrains).S.cerev'isiaewastransformedbythemodifiedlithiumacetateprocedure(37).One-stepgenedisruptionwasper-formedasdescribedpreviously(31).StrainsS86andSc25kweredisruptedattheKEXIlocuswiththeURA3gene(7)togeneratestrainsS86-16andSc25k-16,respectively.TheDNAconstructusedforthesedisruptionshasbeende-scribedpreviously(7).Site-directedmutagenesis.A1.5-kilobase(kb)XbaI-SallfragmentcontainingtheinitiationcodonofKEXIwasin-sertedintoBluescriptKSM13+(Stratagene,SanDiego,Calif.)andtransformedintoE.coliUT580,andssDNAwasisolatedasdescribedpreviously(37),usingM13K07helperphage(Pharmacia).Oligonucleotide-directedmutagenesiswasperformedonthisssDNA,usingthemutagenicoli-gonucleotide5'-AGATAAAGATCTGAATAGA-3'asde-scribedbytheMuta-geneinvitromutagenesissystem(Bio-Rad).Thedesiredmutantwasinitiallydetectedbycolonyhybridization,usingthe32P-end-labeledoligonucleotideasaprobe(25).ThepositiveclonewasconfirmedasthecorrectmutantbythepresenceofanewlycreatedBgIIIsite.DeletionoftheKexlpmembrane-spanningdomainwasaccomplishedbyusingloop-outmutagenesis.A1.4-kbXl/ol-HinidIIIfragmentcontainingtheCOOH-terminalthirdoftheKEXIgenewasinsertedintoBluescriptKSM13-,andssDNAwasisolated.MutagenesiswasperformeduponthisssDNAasdescribedaboveusingthemutagenicoligonucle-otide5'-GAGATCTCAATGTATAAACACAAAGATAGAAGAGTGAGGAGAAAA-3'.ThemutationwasidentifiedinitiallybytheintroductionofaBgIIIsiteandconfirmedbysequencing.Invitrotranscriptionandtranslation.The3.1-kbHinidIIIfragmentcontainingtheentirecodingregionoftheKEXIgenewasinsertedintotheSP6polymerasetranscriptionvector,pSP64,generatingpSP64-H5.KEXImRNAwasproducedbySP6RNApolymeraserunofftranscriptionfromlinearizedpSP64-H5asdescribedbythesupplier(PromegaBiotec,Madison,Wis.).TheresultinguncappedmRNAtranscriptsweretranslatedinarabbitreticulocytelysate(Amersham),asrecommendedbythesuppliers,inthepresenceof30to40,uCiof[35S]methionine.SDS(finalconcentration,1%)wasaddedtothetranslationproductswhichwerethenincubatedatroomtemperature,dilutedwithimmunoprecipitation(TNET)buffer(1%TritonX-100,150mMNaCl,1mMEDTA,150mMTrishydrochlorideA.B.KEXIEcoR1729619.637DGaBmHIFIG.1.(A)PredictedstructureofKexlp.Theconservedserineregionattheactivesite(residuesGESYA)isindicated.©.Aspar-agineresidueswiththepotentialtobeglycosylatedandthatwereaminoterminaltothemembranespanningdomainareshown.(B)ThelacZ-KEXlgenefusionconstruct.pWR590-KX1,usedforfusionproteinexpression.VectorpWR590containsthelhipro-moter(P)andoperator(0)aindthecodingsequenceforapproxi-matelv590aminoacidsoflacZ.[pH8.0]),andimmunoprecipitatedwithanti-Kexlpantise-rumatsdescribedbelow.Antiseraproduction.AntibodieswereraisedagainstKEXlpbyconstructionandoverexpressionofalacZ-KEXIgenefusioninE.coliessentiallyasdescribedpreviously(15).Thegenefusioncontainedthepromoterandcodingregionsfortheamino-terminalhalfoftheE.colilacZgenefusedinframetoa1-kbopenreadingframesegment(E(oRI-BamHI)fromthecenteroftheKEXIgene(Fig.1).AlthoughthisportionoftheKEXIgenedoesnotcontainastopcodoninthecorrectreadingframe,astopcodonispresentinthevectortwocodonsdownstreamfromtheinsertedKEXIsequence.Transformantsweregrowninliquidculture,inducedwithIPTG,andthenlysedbyseveralfreeze-thawcycles.Theresultingwhole-cellextractswererunonSDS-polyacrylamidegelsandthenstainedwithCoomassiebluetodeterminethefusionproteinsizeandtoestimatetheextentofoverproduction.E.coliDH1trans-formedwiththegenefusion,pWR590-KX1,producedanovelproteinofapproximately105kilodaltons(kDa)afterinductionwithIPTG.Thisproteinwas30kDalargerthantheinduced,unfusedLacZprotein,aswasexpectedforaninframefusionproductwiththeKEXIfragment.Thefusionproteinwaspurifiedbygrowing200mlofE.VOL.9.19892708COOPERANDBUSSEYcoliharboringthefusionconstructpWR590-KX1tonear-stationaryphase.Cellswerepelletedandwashedtwiceinphosphatebuffer(50mMpotassiumphosphatebuffer[pH8.0],150mMKCI).Cellswereresuspendedin20mlofphosphatebuffer,sonicated,andthencentrifugedat12,000xgfor10minat4°CinaSS-34rotor.TheresultingpelletwassuspendedinSDS-PAGEsamplebuffercontaining3%SDSandrunonaSDS-polyacrylamide(7%)preparativegel.ThefusionproteinwasidentifiedbystainingwithCoomassieblue.Thegelslicecontainingthefusionproteinwasgroundupwith10mMTrishydrochloride(pH7.3)-0.9%NaCIandpassedrepeatedlythrougha20-gaugeneedle.Freundcom-pleteadjuvant(Sigma)andSDS(finalconcentration.3%)wasaddedtotheemulsion.TheemulsionwasinjectedintoNewZealandWhitefemalerabbits.Twosubsequentinjec-tions2and5weekslaterdidnotincludeFreundadjuvant;approximately50,ugoffusionproteinwasusedperinjec-tion.Thepolyclonalantiserumwascollected7weeksaftertheinitialinjection.Thesamplewaspartiallypurifiedandconcentratedbytheadditionofammoniumsulfatetoafinalconcentrationof45%whichwasthencentrifugedat10,000xgfor15min.Theresultingpelletwasdissolvedin25mMTrishydrochloride(pH7.5)anddialyzedextensivelyagainstthisbuffer.Antiserumwa

stestedforabilitytoimmunoprecipitateradi
stestedforabilitytoimmunoprecipitateradiolabeledinvitro-translatedKEXIgeneproduct.Theinvitroproductwasadjustedto1%SDS,incubatedat37°Cfor5min,dilutedwithimmunoprecipitation(TNET)buffer,andimmunoprecipitatedasdescribedbelow.Celllabelingandimmunoprecipitation.Thecelllabelingandimmunoprecipitationprocedureisbasedonthatalreadydescribed(23).Yeastcells(10ml)weregrowninminimalmediumtoadensityof107cellsml-',concentratedfourfold,andthenlabeledwith100p.CiofTrans35Slabelfor10minat30°C.Chaseconditionswereproduced,afterthepulse,bytheadditionofmethionineandcysteinetoafinalconcentra-tionof1mM.Cellstreatedwiththedrugtunicamycin(10,ugml-')werepreincubatedfor30minat30°Cbeforelabeling.Cycloheximide,whenused,wasaddedtoafinalconcentra-tionof100p.gml-'.Afterlabeling,thecellswerepelletedbyrapidcentrifugationinamicrofugefor1min,washedin1mlofice-coldbreakagebuffer(BB)(150mMNaCl,10mMNa,B407[pH8.0]),andrepelleted.Thecellswereresus-pendedin80,u1ofBBandthenlysedwithglassbeads.SDSwasaddedtoafinalconcentrationof1%,andthelysatewasboiledfor5minandmicrofugedfor1min.Thesupernatantwasextractedanddilutedwith1mlofTNETbuffer.Kexlpantiserum(10,u1)wasadded,andthiswasshakenovernightat4°C.Samples(100,ul)of20%(vol/vol)proteinA-Sepha-roseCL-4B(PAS)(Pharmacia)wereaddedandshakenat25°Cfor3h.Aftertheprecipitatewaswashedthreetimeswithwash-TNET(TNETwith0.1%TritonX-100),60p.lofSDSgelsamplebufferwasaddedforsolubilization.Themixturewasheatedfor5minat100°C.Afterthemixturewascentrifugedfor10s,thesupernatantwastransferredtonewtubes.AllsampleswerethenanalyzedbySDS-PAGE(7to10%polyacrylamidegradientgel).GelswerestainedforproteinwithCoomassieblue.Forfluorography,destainedgelsweretreatedwithEn3HancebeforeexposuretoX-Omatfilm(EastmanKodakCo.,Rochester,N.Y.).EndoHdigestion.ImmunoprecipitatedKexlpboundtoPASwaswashedthreetimeswithwash-TNETandthenoncewith1mlof100mMsodiumcitrate(pH5.5).ThepelletedPASwasthenresuspendedin200p.1of100mMsodiumcitrate(pH5.5)and10mUofEndoHwasadded.Thiswasshakenat37°Cfor18h.ThePASwaswashedwithwash-TNET,pelleted,andboiledinSDSgelsamplebuffer.andthesolubilizedextractwasanalyzedbySDS-PAGE.Sodiumcarbonateextractions.Labeledcellswerelysedwithglassbeadsasdescribedabove.Theresultingcrudehomogenatewasmixedwithanequalvolumeofice-cold0.2MNa,CO(pH11.5),vortexedextensively,andleftfor30minat4°C.Thislysatewasthencentrifugedat178,000xginanAirfuge(300rotor;BeckmanInstruments,Inc.,Fuller-ton,Calif.)for15minatroomtemperature.Thesupernatant(S1)wasneutralizedwithHCI,thepelletwasreextractedwithNa,C03givingasecondsupernatant(S2)andthepellet(P1).Thepelletwassuspendedinbreakagebuffercontaining3%SDS,heatedat60°Cfor15min,andthenboiledfor5min.AllsampleswerethendilutedwithTNETbuffer,immunoprecipitatedwithKEXIantiserum,andtreatedbySDS-PAGE,followedbyfluorography.Preparationofpelletbyusinghigh-speedcentrifugation.Thispelletpreparationprocedureisbasedupononeprevi-ouslydescribed(1).CellsweregrowntostationaryphaseinYNBat30°C,washedtwiceinice-coldwater,andweighed.Succinicacid(50mM;pH6.0)andglassbeadswereaddedtothecellsinaratioof1.5mlofbuffer/2gofbeads/1gofcells.Thecellswerelysedinaliquid-CO-cooledBraunhomoge-nizerwithsix45-spulses.Thecrudehomogenatewasremoved,thebeadsandflaskwerewashedtwicewithbuffer,andthesewasheswerecombinedwiththehomogenate.Thehomogenatewascentrifugedat2,500xgfor10minat4°CinaSA-600rotor(IvanSorvallInc.,Norwalk,Conn.).ThepelletwassuspendedbyhomogenizationwithanElvehjemhomogenizerin50mMsuccinicacid(pH6.0)-200mMKClandthencentrifugedat150,000xgfor40minat4°CinaTi7O.1rotor(Beckman).Thepelletwassuspendedbyho-mogenizationin50mMsuccinicacid(pH6.0)andstoredinaliquotsat-70°C.Solubilizationofthemembraneswasaccomplishedbydilutingthesamplesin50mMsuccinicacid(pH6.0)andaddingTritonX-100toafinalconcentrationof1%.Thiswasstirredat4°Cfor60minandthencentrifugedat175,000xgat4°CinaTLS-55rotor(Beckman).Thesupernatantwasusedimmediatelyorstoredforshortperi-odsoftimeat-70°C.Carboxypeptidaseassay.Thecarboxypeptidaseassayem-ployed(29a)isperformedina100-,ulvolumeat30°Cfor30min.Thereactionmixcontained50mMsuccinicacid(pH6.0),[3H]benzoyl-phenylalanine-alanine-arginine(BPAA)(Dupont,NEN)andunlabeledBPAA(PeninsulaLaborato-ries,Belmont,Calif.).SyntheticaF-KRprocessing.FivenanogramsofsyntheticoxF-KR(7)wasincubatedovernightat30°Cina10-plIvolumeof50mMsuccinicacid(pH6.0-0.1%TritonX-100withcarboxypeptidaseB(BoehringerMannheim)orwithavari-etyofyeastpelletfractionsobtainedbyhigh-speedcentrif-ugation.Theincubatedreactionswerespottedontoa1%YEPDmedium(pH3.5)plate(6)seededwithtesterstrainM190.7-6C,andtheplateswereincubatedovernightat30°C.RESULTSIdentificationoftheKEXIgeneproduct,Kexlp.KEXImRNAwasproducedinvitrofromanSP6promoter,trans-latedinvitroinarabbitreticulocytelysatesystem,andanalyzedbySDS-PAGEandfluorography.AdditionofKEXImRNA'tothelysateproducedonenovelproteinproductmigratingatanapparentmolecularmassof107kDa(Fig.2,lane2),largerthan82.2kDa,whichisthesizepredictedfromtheKEXInucleotidesequence.WebelievethataverynegativelychargedportionoftheproteinisMOL.CELL.BIOI-CHARACTERIZATIONOFTHEYEASTKEXIGENEPRODUCT2709mRNA94kD-Immunoppt.PtAbpiAbKEXIKEXIkexlotKEXI4:4*.4_-116kDi23468kD-1234FIG.2.Invitrotranslationandimmunoprecipitation(Immu-noppt.)ofKexlp.Lane1,ProteinsproducedfromendogenouslysatemRNA;lane2,productofinvitrotranslationofSP6RNApolymerasetranscriptsoftheKEXIopenreadingframe.Theinvitro-translatedproductwassolubilizedwithSDSbeforeimmuno-precipitationusingp

reimmuneserum(Pi;lane3)oranti-Kexlpantis
reimmuneserum(Pi;lane3)oranti-Kexlpantiserum(Ab;lane4).AllsampleswerethenelectrophoresedonSDS-PAGEandvisualizedbyfluorography.responsibleforconferringtheanomalousapparentmolecularmass.Inapredictedtractof105residues(residues506to611)ofKexlp,thereare55asparticacidorglutamicacidresidues(7);insomeplaces,thesenegativelychargedaminoacidsformstretchesofupto11consecutiveresidues.ThisabundanceoflocalizednegativechargemayinhibitSDSbindinganddiminishthecharge/massratiooftheprotein.ThiswouldresultinaslowermigrationrateinSDS-PAGEandleadtoanapparenthighermolecularmass.ThemouseosteopontinalsohasaregionofnegativelychargedresiduesandexhibitssimilaranomalousmigrationonSDS-polyacryl-amidegels(D.T.Denhardtpersonalcommunication).ToidentifyKexlpinvivo,anantibodywaspreparedagainstalacZ-KEX1genefusionproduct.Theresultingpolyclonalantiserumwastestedforitsabilitytoimmuno-precipitateinvitro-translatedKexlp(Kexlp-IVT).Treatingtheproductoftheinvitro-translatedKEXImRNAwiththeanti-KexlpantiserumandPASimmunoprecipitatedasinglespeciesof107kDa(Fig.2,lane4).Nosuchproteinwasimmunoprecipitatedwiththepreimmuneserum(Fig.2,lane3).ToaidinvivoidentificationandcharacterizationofKexlp,theKEXIgenewasoverexpressed.Byinvitromutagenesis,aBglIIsitewasintroducedseveralnucleotidesupstreamfromtheKEXIinitiationcodon.Theresulting3.0-kbBglII-HindIIIKEXIfragmentwasinserteddown-streamfromtheyeastADHIpromoterintheplasmidpVT103-L(37),creatingpKX1-8.TheplasmidpVT103-Lcontainstheleui2dalleleasaselectablemarkerwhich,duetoitstruncatedpromoter,resultsinahighplasmidcopynumber.YeaststrainsS86andS86-16(KEXIdisruption;kexl::URA3[kexl°]),transformedwitheitherpVT103-LorpKX1-8,werelabeledfor10min,lysed,andthenimmunoprecipi-tatedwithanti-Kexlpantiserum.Aproteinof113kDa(Fig.3,lane2)wasprecipitatedfromtheKEXIstrain(S86)butnotfromthekexlostrain(S86-16).Aproteinofthesamesize,but50-foldmoreabundant,waspresentintheKexlp-overproducingstrainS86-16(pKX1-8)(Fig.3,lane4).Thisproteinwasnotprecipitatedbythepreimmuneserum(Fig.-97kDFIG3.IdentificationofKexlpinS.cerevisiae.Kexlpwasimmunoprecipitatedfrom35S-labeledyeastwhole-cellextractsusingpreimmuneserum(Pi)oranti-Kexlpantiserum(Ab)beforeanalysiswithSDS-PAGE.S86(KEXI)wasimmunoprecipitatedwithpreim-muneserum(lane1)oranti-Kexlpantiserum(lane2).S86-16(ke.x10)wastransformedwitheitherthevectorpVT103-L(lane3)ortheKexlp-overproducingplasmid,pKX1-8(lane4)beforeimmunopre-cipitationwithanti-Kexlpantiserum.3,lane1).Aproteinofthesamesizewasimmunoprecipi-tatedfromanotherKEXIstrain,Sc25k,andwasmissingfromtheisogenicstrainSc25k-16thatwasdisruptedatthekexl")locus(datanotshown).Kexlpproducedinvivowas6kDalargerthanKexlp-IVT(113versus107kDa).Itislikelythatglycosylationisresponsibleforatleastsomeofthissizedifference.ThepredictedaminoacidsequenceofKexlprevealsfourpoten-tialsitesforN-linkedglycosylation(7).Treatmentofcellswithtunicamycin,adrugwhichinhibitsN-linkedglycosyl-ation,resultedina109-kDaproteinbeingimmunoprecipi-tatedfromlabeledcells(Fig.4).However,thisproteinwasstill2kDalargerthantheKexlp-IVT.Invivo-translatedKexlplikelyhasitssignalpeptide(7)removed.Thus,thesizedifferencebetweentheKexlp-IVT(whichretainsthesignalpeptide)andtunicamycin-treatedKexlpmaybegreaterthanthe2kDaindicated.ThisincreasemaybeduetoaposttranslationalmodificationorthesignallessKexlpmaymigrateanomalously.ProgressivemodificationofKexlp.S86-16(pKX1-8)cellswerelabeledfor10minandchasedfortheindicatedtimesinthepresenceorabsenceoftunicamycin.Intheabsenceoftunicamycin,newlysynthesizedKexlpshowedagradualincreaseinapparentmolecularmassfrom113to115kDaduringthe90-minchase(Fig.4).Withtheadditionoftunicamycin,noincreaseinapparentmolecularmassoc-curredduringthechase.Thisresultoccurredinboththepresenceandabsenceofcycloheximideduringthechase(Fig.5,lanes4and5).ThisprogressiveglycosylationwasnotlimitedtotheS86backgroundbutwasalsoseeninSc25kaswellasinthevarioussecmutants(Fig.6).chosemin-tunicamycrn+tunicomycin015306090015306090owo_ow"-116kDIVTIVTFIG.4.ProgressivemodificationofKexlpfromS86-16(pKX1-8)intheabsenceandpresenceoftunicamycin.Cellswerelabeledfor10minandthenchasedfortheindicatedtimesbeforeextractionandimmunoprecipitation.Kexlp-IVT(IVT)wasalsoimmunoprecipi-tated.VOL.9,1989ii".I4--*.,:,.J..A-.."-.k'.2710COOPERANDBUSSEYAOLSSNa2CO3vsPSis2PlSSNaPCOBEvT--s_3-VI0X234567FIG.5.Pulse-chaselabelingofKexlpfrorThecontentsofeachlanewereimmunoprecipitatantiserum.Lanes1and7,Kexlp-IVT;lane2.camycin-treatedcellsthatwerelabeledfor10nfromcellslabeledfor10min;lane4.Kexlpfromminandthenchasedfor90min:lane5.Kexlpclthepresenceofcycloheximide;lane6.KexlpchothentreatedwithEndoH.ThetunicamycinresultimpliesthatN-linkisinvolvedinthegradualincreaseintheaplmassofKexlp.TofurthertestthisimplicadigestionwasperformedonKexlpfromcemin(Fig.5).SuchtreatmentreducedtheapmassofthechasedKexlptothatofthetunKexlp(Fig.5,lanes2and6)asdidEndoH10-min-pulsedKexlp(datanotshown).Anattemptwasmadetodeterminewherepathwayprogressivemodificationtakesplaitheuseofseveraltemperature-conditionaltory)mutantswhichdefineasetoffunctisecretoryproteintransport(27).Attherestureof37°C,thefollowingmutantsblockpositions:secl8(endoplasmicreticulum)apparatus,sec7(Golgiapparatus-*secret(secil(secretoryvesiclefusionwithplasThreeseparatestrains(23),eachpossessingsecmutations,weretransformedwithpKXatthepermissivetemperatureof24°Ceithertemperatureorwereswitchedto37°C.Afterwerelabeledfor10minandthenchasedfcwasthenimmunoprecipi

tatedfromtheccabove.Inthesec18strain(Fig
tatedfromtheccabove.Inthesec18strain(Fig.6),progres:ofKexlpwasblockedattherestrictivetesec18mutantbutnotinthesedlmutant.Intheprogressivemodificationproceededpermissivetemperaturebutoccurredonlyrestrictivetemperature.Progressivemodifisec1824C37CPCPCsec724C37CPCPCFIG.6.ModificationofKexlpinsecretionThreedifferentsecstrains(HAB211.HAB212.transformedwithpKX1-8.Thestrainswerepultheneitherharvested(pulse[P])orchasedfo(chase[C])beforeimmunoprecipitationandSDSorpulse-chaseswereperformedateithertheptrestrictive(37°C)temperature.S86-16(pKX1-8).edwithanti-KexlpKexlpfromtuni-nin:lane3.Kexlpcellspulsedfor10hasedfor90mininisedfor90minandcedglycosylationparentmoleculartion,anEndoHlIschasedfor90parentmolecularicamycin-treatedtreatmentoftheZinthesecretoryce.Thisrequired1234567891011FIG.7.MembraneassociationofKexlp.S86-16transformedwithpKX1-8waslabeledandlysedasdescribed.Thevariousfractionswereimmunoprecipitatedwithanti-Kexlpantiserum.Thewhole-cellhomogenate(Homo,lane1)wascentrifugedat(12,000xg:,low-speedspin[LSS])togiveasupernatant(S;lane2)andpellet(P;lane3).Thewhole-cellhomogenatewastreatedwithNa,C03andthencentrifuged(178.000xg).resultinginasupernatant(Si;lane4)andpellet.ThepelletwasreextractedwithNa,C03togiveasecondsupernatant(S2;lane5)andpellet(lane6).Asimilarprocedurewasperformed(lanes7through11)uponS86-16trans-formedwithKexlpAMS(Kexlpwiththemembrane-spanningdo-maindeleted)fromplasmidpKX1-18.yeastses(secre-occursubsequenttothesec18blockandatleastpartiallyinionsrequiredfortheregionofthesec7block.trictivetempera-Kexlpismembraneassociated.TheKEXJgenesequenceattherespectivepredictsapolypeptidewithtwomarkedlyhydrophobicse-[ER]-*Golgiquences(7).Thefirst,situatedattheNterminusisundoubt-oryvesicle),andedlyasignalforERentry,asKexlpmustenterthesecretory,mamembrane).pathwaytobecomeglycosylated.Thesecondhydrophobiconeoftheabovesegment,situated100residuesfromtheCOOHterminusof1-8.CellsgrowntheKEX/-encodedprotein,contains18aminoacidsresiduesrremainedatthisandmayfunctionasamembrane-spanningdomain.r60min,thecellsWhole-cellextractsoflabeledcells,whenspunat12,000ir90min.Kexlpxgfor1min,gaveapelletthatcontainedessentiallyallofllsasdescribedtheKexlp(Fig.7,lane3).ThiscouldresultfromKexlpsivemodificationbeingeitheranextrinsicorintegralmembraneproteinoramperatureinthesolubleproteininamembrane-enclosedcompartment.Tothese(7mutant,distinguishbetweenthesepossibilities,thewhole-cellex-normallyatthetractwastreatedwithNa,C03(pH11.5)andcentrifugedatzpartiallyatthe178,000xgfor15min.Thisalkalitreatmentwillconvert[cationmustthusclosedvesiclestoopenmembranesheets;themembrane-enclosedsolubleproteinsandextrinsicmembraneproteinsarereleasedinsolubleform,whileintegralproteinsremainassociatedwiththemembranepellet(12).Kexlpwasnotsec1foundintheNa,C03supernatantbutwasfoundinthepellet24C37C(Fig.7,lanes4through6),suggestingafirmassociationPCPCbetweenKexlpandthelipidbilayer.IncompleterecoveryofKexlpisduetodifficultiesinsolubilizingtheNa,CO3-treatedpelletbeforeimmunoprecipitation.Thenucleotidesequenceencodingthepredictedmem----_brane-spanningdomainofKexlpwasspecificallyremovedbyinvivomutagenesis,creatingtheconstructpKX1-18zAMS(deletedmembranespanning).S86-16transformedwithpKX1-18AMSwaslabeledfor10minandimmunoprecipi-tatedwithKexlpantiserum(Fig.7B,lane7).ThewholecellandHABe1ciwerelysatewastreatedasdescribedtodeterminewhetherKexlp-lsedfor10minandAMSwasstillmembraneassociated.Eightypercentofrafurther90minKexlp-AMSwassolubleafterthelow-speedspin(Fig.7B,-PAGE.Thepulseslane8).TreatmentofthecrudelysatewithNa,CO3didnotermissive(24°C)orreleasetheremaining20%oftheproteinwhichremainedwiththepellet(Fig.7B.lane11).MOL.CELL.BIOL.woaliklaCHARACTERIZATIONOFTHEYEASTKEXIGENEPRODUCT2711DetectionofKexlpactivity.TominimizethepossibilitythatKexlpactivitymightbemaskedbyamoreabundantproteaseactivity,theKexlpactivitysearchwasdoneinstrainS86whichisdeficientinthetwocarboxypeptidases,carboxypeptidaseY(CPY)andS(CPS),andthetwoprote-ases,proteinaseAandproteinaseB.EarlierworkimplicatedKexlpasthecarboxypeptidaseresponsibleforprocessingsecretedproteinswhichhaveundergoneaKex2pcleavageevent(7).Kexlpshould,therefore,bespecificforbasicaminoacids.Toinvestigatethisassumption,anassaywasemployed(Rossieretal.,inpress)usingBPAAasasubstrate.ThebasisoftheassayreliesuponachangeinsolubilityofBPAAafterundergoingproteolyticcleavage.Thesubstrate,BPAA,isinsolubleintheliquidscintillantEconofluorand,therefore,itsdisinte-grationinEconofluorwillnotregisterinaliquidscintillationcounter.WhentheCOOH-terminalarginineresidueiscleavedfromBPAA,itresultsinaproduct,BPA,whichissolubleinEconofluorandcanthereforebedetectedbyscintillationspectroscopy.AsKexlpismembraneassociated,apelletfractionob-tainedbyhigh-speedcentrifugationwaspreparedfromthefollowingstrains;S86S86-16(pVT103)andS86-16(pKX1-8).ThesefractionsweresolubilizedwithTritonX-100andassayedforactivityagainstthesubstrateBPAA.Thespe-cificactivities(inpicomolesofBPAproducedperminutepermicrogramofprotein)wereforS86-16(pVT1031)(kexlo),29forS86(KEXI),and187forS86-16(pKX1-8)(overproducingKexlp).ActivitywasfoundintheS86fractionthatwasabsentfromtheS86-16-derivedfraction.ThisactivitywassixfoldmoreabundantintheKexlp-overproducingstrain,S86-16(pKX1-8.ThatthedegreeofenhancedactivitywasnotgreaterintheKexlpoverpro-ducermaybeduetothevariablelevelofoverproductionamongpKX1-8transformants,combinedwiththeobserva-tionthatthisleveldiminisheswithsub

culturing.Preliminaryresultssuggestthato
culturing.PreliminaryresultssuggestthatonlyasmallfractionoftotalKexlpactivityisdetectablewhenintactcellsareassayed,suggest-ingthatmostoftheactivityissequesteredintracellularly.AnumberofdifferentbuffersatdifferentpHsweretestedtomaximizeactivity,andthebestresultswereobtainedusing50mMsuccinicacidatpH6.0.Underthesecondi-tions,theK,,MforBPAAwasapproximately250,uM.TheresponseofKexlpactivitytoavarietyofagentsisshowninTable1.ChelatingagentshavelittleornoinhibitoryeffectontheKexlpactivity,whilecalciumandmagnesiumhavenoactivatingeffect,implyingthatKexlpisnotametallopro-teaserequiringeitherofthesedivalentcations.Thesensitiv-itytoPMSFimpliesthatKexlpisaserineprotease.Thebasisoftheheavymetalinhibitionisunknown.CarboxypeptidaseactivityofKexlpappearstobespecificforbasicresidues.CleavageofthesubstrateBPAAindicatesthatKexlpiscapableofcleavingaCOOH-terminalbasicresidue.TofurtherexaminetheproposedroleofKexlpintheprocessingofprecursors,weattemptedtodetermineiftheKEXIgeneproductwasspecificforbasicaminoacidresidues.UnlabeledBPAAwasincubatedwithCPY(BoehringerMannheim)orwithsolubilizedpelletextractsobtainedbyhigh-speedcentrifugation,fromakexlostrainorastrainoverexpressingKEXI.Thereactionproductswereanalyzedbythin-layerchromatographyandcomparedwiththefreeaminoacidsphenylalanine,alanine,andarginine(datanotshown).CPY,ayeastcarboxypeptidasecapableofcleavingmostaminoacidsfromtheCOOHterminusofpeptides(17),removedallthreeresiduesfromBPAA,whileKexlpTABLE1.InhibitorsofKexlpactivity"Inhibitr.ConcnKexlpactivitvInhibitor(mM)('4ofcontrol)None100TPCK199597TLCK167544TLME174551TAME156537MgCl22.5104CaCI22.5101EDTA199EGTA"177CoCIk0.550ZnCI,2.57HgCI.0.05)PMSF4"SolubilizedS86extractswereassayedwith250,uMBPAAinthepresenceoftheindicatedcompounds.TPCK.TLCK.TLME.andTAMEweresolu-bilizedindimethylsulfoxide.andtheireffectwascomparedwithacontrolcontainingdimethylsulfoxide."EGTA.Ethyleneglycol-bis(q-aminoethylether)-N.N.N'.N'-tetraaceticacid.cleavedonlythearginineresidue.NoargininewasreleasedfromBPAAafterincubationwiththeextractfromthekexl"strain.TheactivityofKexlpwasdeterminedbyusingBPAAasasubstrateinthepresenceofN-blockedpeptidestoobservewhethertheintroducedpeptidescouldactascompetitiveinhibitors.Ifso,itwouldindicatethatthepeptidewasalsoasubstrateforKexlp.Theresults(Table2)indicatethatonlythepeptideswithabasicresidueattheCOOHterminuswerecapableofinhibitingKexlpactivityagainstBPAA.ProductinhibitionwasusedasameanstofurthertestthespecificityofKexlpactivity.IfanaminoacidisasubstrateforKexlpwhenpresentattheCOOHterminusofapeptide,thentheadditionoffreeaminoacidtothereactionmixshouldinhibittheproteolyticcleavagereaction.However,iftheaminoacidisnotasubstrateforKexlp,thenadditionofthisfreeaminoacidshouldhavenoinhibitoryeffect.Oftheaminoacidstested(solubilityproblemspreventedusfromtestingtyrosineandcysteine),onlythebasicaminoacids,lysineandarginine,arecapableofinhibitingKexlpactivity(Table3).TABLE2.CompetitiveinhibitionofKexlpactivity"CompetitorKexlpactivity(%)Hippuricacid............................................100Hippuryl-His-Leu............................................105Hippuryl-Phe............................................99Hippuryl-Gly-Gly............................................97Hippuryl-Gly-Lys............................................68Hippuryl-Arg............................................48Hippuryl-Lys............................................35"SolubilizedS86extractswereassayedwith250,uMBPAAinthepresenceofthecompoundsshown.Thesecompoundsweredissolvedindimethylsulfoxide.addedtoafinalconcentrationof1mM.andcomparedwithacontrolcontainingdimethylsulfoxide.Hippuricacidalonecausedanactiva-tionofKexlpactivityto1309ofuninhibitedlevels.Toallowaninternallyconsistentcomparison,theactivitieswerenormalizedwithrespecttothehippuricacid-activatedlevel.VOL.9.19892712COOPERANDBUSSEYTABLE3.ProductinhibitionofKexlpactivity"KexlpactivityAmino)acid(esofcontrol)Asp.......................................108Gln.......................................108Glu.......................................107Phe.......................................106Gly.......................................103Thr.......................................102Leu.......................................100Pro.......................................100Ser.......................................100Asn.......................................98Ile98Met.......................................97Val.......................................97Ala96Trp.......................................91His.......................................84Lys.......................................62Arg.......................................43'SolubilizedS86extractswereassayedwith400±uMBPAAinthepresenceofvariousaminoacidspresentat20mM.TheaminoacidstocksolutionsweremadefreshandhadbeenadjustedtothesamepHbeforeaidditiontothereactionmix.MaturationofotF-KR.aF-KRisanintermediateofax-factorprocessingwhichhasundergoneKEX2-andSTE13-mediatedproteolyticcleavage.Itpossessesthematureax-factorNterminus,whiletheCOOHterminuscontainsanextensionofLys-Arg(KR).otF-KRwassynthesized(7)andutilizedasanaturalsubstrateforthecarboxypeptidaseresponsibleforprocessingOLF-KRtomatureat-factor.ax-factorwillcausezonesofgrowthinhibitionwhenspottedontoaplateseededwithastrainsupersensitivetoax-factor(MATasstlsst2).Thesizeofana-factor-inducedhaloisproportionaltothelogofthepheromoneconcentration.aF-KRexhibitedverylittlebio

logicalactivity(Fig.8A),yetwhentreatedin
logicalactivity(Fig.8A),yetwhentreatedinvitrowithcarboxypeptidaseB,theo-F-KRwasprocessedtoamorebiologicallyactiveform,mostlikelymatureax-factor(Fig.8B).Whenincubatedwithapelletextract,obtainedbyhigh-speedcentrifugation,fromastrainFIG.8.ActivationofaF-KRbyKexlp.SampleswerepreparedasdescribedinMaterialsandMethodsandanalyzedfortheirabilitytoactivateaF-KR.(A)aF-KR;(B)aF-KRincubatedwithcarbox-ypeptidaseB;(C)aF-KRincubatedwithapelletfraction,obtainedbyhigh-speedcentrifugation,fromS86-16(pVT103-L)(kexlt):(D)aF-KRincubatedwithapelletfraction,obtainedbyhigh-speedcentrifugation,fromS86-16(pKX1-18)(overexpressingKEXI).overproducingKexlp,theaoF-KRwasconvertedtoafarmorebiologicallyactiveform(Fig.8D),yetthesameextractfromakexl(straincausedonlyasmalldegreeofactivationofo-KR(Fig.8C).DISCUSSIONCertainproteinsdestinedforsecretionfromeucaryoticcellsareproducedasprecursorswhich,duringtheirpassageofthesecretorypathway,areprocessedtomaturepeptidesbyspecificproteases(9).Suchproteinsincludeneurotrans-mitters,peptidehormones,andtheyeastproteinsa(-factorandKlkillertoxin.Theprecursorsofa-factorandKikillertoxinareinitiallycleavedatpairsofbasicresiduesbytheKEX2-encodedendoprotease(3,13,19).TheKex2p-mediatedcleavageresultsinbotha-factorandthealphasubunitofKIkillertoxinhavingadibasicresidueextensionattheCOOHterminus.AcarboxypeptidaseB-likeenzymeisrequiredtoprocesstheseintermediatesintomaturepolypeptides.EarlierworkshowedthatkexlmutationsabolishtheproductionofactiveKlkillertoxin(4,39).Inaddition,suchmutantsproducelessactiveax-factorpheromonethanKEXIstrains(7).Whencloned,theKEXInucleotidesequenceshowedregionsofidentitywiththeyeastvacuolar,serinecarboxypeptidase,CPY(7).RegionsofidentityincludedresidueSer-146oftheCPYactivesitewhichisessentialforthepeptidebondhydrolysisreaction.DirectedmutationoftheconservedKEXIputativeactivesiteSer-198residueproducedaKex-phenotype(7).ThisraisedthepossibilitythattheKEXIproductwasthecarboxypeptidaseB-likeenzymerequiredforcompletingproteolyticprocessingofKlkillertoxinandox-factor.KEXIstrainscontainamembrane-associatedcarboxypep-tidaseactivityabsentfromkexl0strains.Thisactivitycor-relateswiththepresenceofKexlp;essentiallynoactivitycanbedetectedwiththeBPAAassayintheabsenceofKexlp,anduponKexlpoverproduction,thecarboxypepti-daseactivityincreases.Onthebasisofthesegenedisruptionandgenedosageresults,weconsiderthatKEXIisthestructuralgeneforthiscarboxypeptidase.TheKEXI-en-codedcarboxypeptidaserequiresaserineresiduetofunctionandisPMSFsensitive,stronglysuggestingthatKexlp,likeCPY,isaserineprotease.SpecificityofKexlp.ForKexlptofulfillthepredictedroleofthecarboxypeptidaseB-likeenzyme,itmustpossessgreatersubstratespecificitythanthebroadspecificityofCPY(17).OftheN-blockedpeptidesassayedwithKexlp,onlythosewithbasicresiduesattheCOOHterminuswerecapableofactingassubstratesfortheenzyme.AproductinhibitionstudydeterminedwhichaminoacidsinhibitedCOOH-terminalArgcleavagefromBPAA.Whenprovidedwithanexcessofproduct(freeaminoacid),thereactionbalanceisperturbedandtheforwardreactionisinhibited.Aselectionof18aminoacidsweretestedfortheirabilitytoinhibitKexlpactivityinthisway.Thetwobasicaminoacids,lysineandarginine,weretheonlyonesthatsignificantlyinhibitedKexlpactivity.Thefactthathistidine,whichispartiallypositivelychargedattheassaypH,inhib-itedKexlpactivitytoasmalldegreeisconsistentwiththisresult.a,F-KRisanaturalsubstrateforthecarboxypeptidaseinvolvedintheproteolyticprocessingofat-factor.Apelletfraction,obtainedbyhigh-speedcentrifugation,fromaKexlp-overproducingstrainwasabletoconvertaF-KRtoanactivebiologicalform,mostlikelymaturea.-factor.TheMOL.CELL.BIOL..CHARACTERIZATIONOFTHEYEASTKEXIGENEPRODUCT2713residualdegreeofactivationthatoccurredinakexl"fractionmaybeduetoaproteaseactivitythatcleavesaF-KRtoreleaseapeptidefragmentpossessingbiologicalactivity.Ofthetwochloromethylketoneinhibitors,TPCKandTLCK,onlyTLCKiscapableofinhibitingKexlpactivity.TLCKcontainsalysineresidueinitsstructure,andthispresumablyallowsittobindtotheKexlpactivesite.TPCK,withaphenylalanineresidueinplaceoflysine,isnotaninhibitorofKexlp.TPCKandTLCKareknowntomodifytheessentialhistidineresidueinthechargerelaysystemofvariousserineendoproteases(32,33).TPCKabolishesCPYactivitybymodifyingasinglehistidineresidue(16),andTLCKmayinhibitKexlpactivityinasimilarmanner.ThreeproteinsCPY,Kexlp,andarecentlydiscoveredhumanlysosomalprotectiveproteinHU54(14)formafamilybasedonaminoacidsequencehomology.IdentityextendstoincludetheessentialserineresidueofCPY.Onlyonehistidineresidueisconservedwithinthisfamilyandcorre-spondstoKexlpHis-470,whichmaybethehistidineresiduethatTLCKpotentiallymodifies.CPYisknowntoexhibitanesteraseactivity(17),anditappearsthatKexlpsharesthisproperty,asshownbytheabilityofTAMEandTLMEtoinhibitKexlpactivity.ThedegreeofspecificitydisplayedbyKexlpiscompati-blewiththatrequiredofacarboxypeptidaseB-likefunctioninprecursorprocessing.Mammalianfunctionalhomolog.SimilarprocessingtothatperformedbyKexlpisknowntooccurinothereucaryoticsystems.Theprecursorprocessingofmanyhormonesandneurotransmittersinvolvesendoproteolyticcleavageatmono-anddibasicresidues,thusnecessitatingtheneedforafunctionalhomologofKexlptoremoveCOOH-terminalbasicaminoacid(s).BovinecarboxypeptidaseE(CPE)isacandidateforsucharoleasaresultofitsspecificitytowardsbasicaminoacidsanditstissueandorganelledistribution,whichcoincideswithbioactivepeptideproduction(10).However,CPEandKexlpareno

tconsideredtoberelated;CPE,unlikeKexlp,i
tconsideredtoberelated;CPE,unlikeKexlp,isactivatedbyCoCl,andinhibitedbychelatingagents.Inaddition,CPEandKexlpsharenohomologyattheaminoacidlevel.ItisprobablethatCPE,whichisrelatedtocarboxypeptidaseAandB(11),belongstoadifferentfamilythanthatofKexlpandCPY.CharacteristicsofKexlp.Thehydrophobicdomain,situ-ated100residuesfromtheCOOHterminus,ispredictedtobemembranespanningandtoconfermembraneassociationonKexlp.Deletionofthisdomainresultedin80%ofKexlpbeingfoundinasolubleform.Itislikely,therefore,thattheamino-terminaldomainofKexlp,whichcontainsthepre-dictedactivesite,residesinthelumenofamembrane-enclosedcompartment.Inthisorientation,theproteasedomainofKexlp,locatedinthelumen,isexposedtosubstratestransversingthesecretorypathwaywhiletheCOOH-terminaltailofKexlpispositionedinthecytoplasm.AstheimmunoprecipitationandSDS-PAGEanalysiswasconductedonstrainsoverproducingKexlp,thereexiststhepossibilitythatsuchoverexpressioncouldleadtomislocal-izationofKexlp,resultinginheterogeneousmodifications.However,wesawnoindicationofsuchheterogeneityintheKexlpimmunoprecipitations.ThemodificationofN-linkedglycosylationofKexlpproceedsinanunusualmanner.TheN-linkedoligosaccha-rideisfirstattachedtoKexlpintheendoplasmicreticulum,asasecl8blockdoesnotpreventitsoccurrence.Then,inapost-secl8compartment(s),amodificationslowlyoccurstotheN-linkedoligosaccharidewherebyitssizeincreasesinanorderedmanner.Thesizeincreaseisnotlargeenoughtobeattributedtothepolymannoseouterchainadditionasseen,forexample,withinvertase(2).Thenatureofthisfurthermodificationisunknownbutlikelycandidatesarefurtherglycosylation,phosphorylation,orboth.KexlpcanactonlyafteraKex2pcleavageevent,henceKexlpmusteithershareasecretorycompartmentwithKex2porexistinapost-Kex2pcompartment.Workwitha-factorandKlkillertoxinindicatesthattheKex2pcleav-ageeventoccursinapost-sec18manner(4,21)andpoten-tiallyinapre-sec7compartment.ModificationtoKexlpN-linkedglycosylationcanbeusedtoindicatethelocaliza-tionoftheproteininthecell.Themodificationisabsentinasecl8mutant,occursfullyinasecimutant,andproceedspartiallyinasec7mutant.ThisimpliesthatKexlpexistsinacompartmentthatspansthesec7block.ItisnotknownwhereinthesecretorypathwaythattheSEC7geneproductacts,althoughthesec7mutationcanleadtoaccumulationofstackedcisternaeresemblingmammalianGolgivesicles(27).Kexlpislikelytobemaintainedwithinthesecretorysystem,asalmostnoKexlpactivitywasfoundatthecellsurface.Thisretentioncouldbeachievedinoneoftwomodes.ThefirstwouldinvolvethemaintenanceofKexlpwithinasinglecompartment.ThismodelwouldrequirethatthemodificationmachineryalsoresideinthiscompartmenttobeabletoactonKexlp.Ifthemodificationisglycosylinnature,itwouldimplythatthecompartmentinquestionwaslocalizedintheGolgiapparatus,asallthepost-ERglycosyl-ationenzymesthathavebeenlocalizedhavebeenfoundinthisorganelle(18).Analternativeandmore-speculativemodelwouldinvolveKexlptraversingthesecretorypathwayuntilreachingalateorpost-Golgipositionbeforeitwasrecycledtoacompart-mentearlierinthepathway.Witheachcycle,Kexlpcouldbeexposedtothemodificationmachinery,resultinginaprogressiveincreaseinthedegreeofKexlpmodification.Asec7blockcouldpotentiallystopsuchrecyclingandtherebylimittheextentofprogressivemodification.Aprecedentforsuchproteinrecyclinginthesecretorypathwayhasbeenfoundinbothanimalandyeastcells(30).SeveralanimalcellER-residingproteinsarethoughttoachievetheirlocalizationbycontinuallybeingretrievedfromtheGolgiapparatus.AshortCOOH-terminalsequenceisresponsibleforconferringtheERlocalizationontheseproteins(28).Asimilarreten-tionsignalexistsinS.cerevisiaewhich,whenattachedtothesecretedenzymeinvertase,preventsthesecretionofmostoftheenzyme(29).TheyeastSEC12protein(Secl2p)isconsideredtoparticipateinthetransportofsecretedproteinsfromtheERtotheGolgiapparatus(26).TheSecl2pisprogressivelymodifiedinasimilarmannertoKexlp.AlthoughnotattachedtoN-linkedglycosylation,themodi-ficationofSecl2poccursslowlyandispreventedbyasecl8block.TheauthorssuggestthepossibilitythatSecl2pmi-gratesrapidlyinacyclebetweentheERandGolgiappara-tus.ThischaracterizationofKexlpactivityidentifiesthere-mainingcomponentintheproteolyticprocessingofyeastpro-a.-factorandKlkillertoxinmaturation.ACKNOWLEDGMENTSWethankKathrynHillforhelpthroughoutthiswork,especiallywithmanuscriptpreparation.ThanksalsotoCharlieBooneforhelpanddiscussionsatvariousstagesofthiswork;toJeanRossierandKathyMettersforprovidingcarboxypeptidaseassayconditionsbeforepublication:toLloydFrickerfordiscussionsconcerningtheactivity,toMalcolmWhitewayforstrainM190.7-6C;toDieterWolfVOL.9.19892714COOPERANDBUSSEYforstraincl3ABYS86;toMargaretAhmadforassistancewiththeantiserum;andtoRobertLamarcheforMacintoshexpertise.WethanktheNationalSciencesandEngineeringResearchCoun-cilCanada,forstrategicandoperatinggralntsupport.A.C.isaCanadianCommonwealthScholar.LITERATURECITED1.Achstetter,T.,andD.H.Wolf.1985.Hormoneprocessingandmembrane-boundproteinasesinyeast.EMBOJ.4:173-177.2.Ballou,C.E.1982.Yeastcellwallandcellsuiface.p.335-360.ItiJ.Strathern.E.W.Jones.andJ.R.Broach(ed.).ThemolecularbiologyoftheyeastSacchiaromyces.ColdSpringHarborLaboratory,ColdSpringHarbor,N.Y.3.Bussey,H.1988.Proteasesandtheprocessingofpr-ecursorstosecretedproteinsinyeast.Yeast4:17-26.4.Bussey,H.,D.Saville,D.Greene,D.J.Tipper,andK.A.Bostian.1983.SecretionofSacchlaroclmcsucrevlisiaekillertoxin:processingoftheglycosylatedprecur-sor.Mol.Cell.Biol.3:1362-1370.5.Casadaban,M.,andS.N.

Cohen.1980.Anatlysisofgenecontrolsignals
Cohen.1980.AnatlysisofgenecontrolsignalsbyDNAfusionandcloninginE.coli.J.Mol.Biol.138:179-207.6.Chan,R.K.,andC.A.Otte.1982.Physiologicalcharacteriza-tionofSaccharoin'veescereivisiaemutantsSupersensitivetoGIarrestbyafactorandafactoipheromones.Mol.Cell.Biol.2:21-29.7.Dmochowska,A.,D.Dignard,D.Henning,D.Y.Thomas,andH.Bussey.1987.YeastKEX1geneencodesaputativeproteasewithacarboxypeptidaseB-likefunctioninvolvedinkillertoxinandalphafactorprecursorprocessing.Cell50:573-584.8.Douglas,J.,0.Civelli,andE.Herbert.1984.Polyproteingeneexpression:generationofdiversityofneuroendocrinepeptides.Annu.Rev.Biochem.53:663-715.9.Fisher,J.M.,andR.H.Scheller.1988.Prohormoneprocessingandthesecretorypathway.J.Biol.Chem.263:16515-16518.10.Fricker,L.D.1988.CarboxypeptidaseE.Annu.Rev.Physiol.50:309-321.11.Fricker,L.D.,C.J.Evans,F.S.Esch,andE.Herbert.1986.CloningandsequenceanalysisofcDNAforbovinecarboxypep-tidaseE.Nature(London)323:461-464.12.Fujiki,Y.,A.L.Hubbard,S.Fowler,andP.B.Lazarow.1982.Isolationofintracellularmembranesbymeansofsodiumcar-bonatetreatment:applicationtoendoplasmicreticulum.J.CellBiol.93:97-102.13.Fuller,R.S.,R.E.Stearne,andJ.Thorner.1988.Enzymesrequiredforyeastprohormoneprocessing.Annu.Rev.Physiol.50:345-362.14.Galjart,N.J.,N.Gillemans,A.Harris,G.vanderHorst,F.W.Verheijen,H.Galjaard,andA.d'Azzo.1988.ExpressionofcDNAencodingthe"protectiveprotein"associatedwiththelysosomalP-galactosidaseandneuromiinidase:homologytoyeastproteases.Cell54:755-764.15.Guo,L.,P.P.Stepien,J.Y.Tuo,R.Brousseau,S.Narang,D.Y.Thomas,andR.Wu.1984.Synthesisofthehumaninsulingene.VIII.ConstructionofexpressionvectorsforfusedproinsulinproductioninE.coli.Gene29:251-254.16.Hayashi,R.,S.Aibara,andT.Hata.1975.EvidenceforanessentialhistidineincarboxypeptidaseY.Reactionwiththechloromethylketonederivativeofbenzylocarbonyl-L-phenylal-anine.J.Biol.Chem.250:5221-5226.17.Hayashi,R.,Y.Bai,andT.Hata.1975.KineticstudiesofcarboxypeptidaseY.1.Kineticparametersforthehydrolysisofsyntheticsubstrates.J.Biochem.77:69-79.18.Hirschberg,C.B.,andM.D.Snider.1987.TopographyofglycosylationintheroughendoplasmicreticulumandGolgiapparatus.Annu.Rev.Biochem.56:63-87.19.Julius,D.,A.Brake,L.Blair,R.Kunisawa,andJ.Thorner.1984.Isolationoftheputativestructuralgeneforthelysine-argine-cleavingendopeptidaserequiredtheprocessingofyeastprepro-a-tactor.Cell36:309-318.20.Julius,D.,R.Schekman,andJ.Thorner.1983.Yeastaxfactorisprocessedfromalargeprecursorpolypeptide:theessentialroleofatmembrane-boUnddipeptidylaminopeptidase.Cell32:839-852.21.Julius,D.,R.Schekman,andJ.Thorner.1984.Glycosylation,andprocessingofprepro-a-factorthroughtheyeastsecretoi-ypathway.Cell36:309-318.22.Kurjan,J.,and1.Herskowitz.1982.StructureofaIyeastpheromonegene(MFalpha):aputativealpha-factorprecursorcontainsfouItandemcopiesofmaturealpha-factor.Cell30:933-943.23.Lolle,S.J.,andH.Bussey.1986.Invivoevidenceforposttrans-lationaltranslocationandsignalcleavageofthekillerprepro-toxinofSaccharol/vce.v(c*reisiac.Mol.CellBiol.6:4274-4280.24.Lynch,D.R.,andS.H.Snyder.1986.Neuropeptides:multiplemolecular-forms,metabolicpathways.andreceptors.Annu.Rev.Biochem.55:773-799.25.Maniatis,T.,E.F.Fritsch,andJ.Sambrook.1982.Molecularcloning:alaboratorymanuial.ColdSpringHarborLaboratory.ColdSpringHarbor.N.Y.26.Nakano,A.,D.Brada,andR.Schekman.1988.AmembraneglycoproteinSecl2p.requiredforproteintransportfromtheendoplasmicreticulumtotheGolgiapparatusinyeast.J.CellBiol.107:851-863.27.Novick,P.,S.Ferro,andR.Schekman.1981.Orderofeventsintheyeastsecretor-ypathway.Cell25:461-469.28.Pelham,H.R.B.1988.EvidencethatluminalERproteinsaresortedfromsecr-etedproteinsinapost-ERcompartment.EMBOJ.7:913-918.29.Pelham,H.R.B.,K.G.Hardwick,andM.J.Lewis.1988.SortingofsolubleERproteinsinyeast.EMBOJ.7:1757-1762.29a.Rossier,J.,E.Barres,J.C.Hutton,andR.J.Bicknell.1989.RadiometricassayforcarboxypeptidaseH(EC3.4.17.10))andothercarboxypeptidaseB-likeenzymes.Anal.Biochem.178:27-31.30.Rothman,J.E.1987.ProteinsortingbyselectiveretentionintheendoplasnmicreticulumandGolgistack.Cell50:521-522.31.Rothstein,R.J.1983.One-stepgenedisruptioninyeast.Meth-odsEnzvmol.101:202-211.32.Schoellman,G.,andE.Shaw.1963.Directevidenceforthepresenceofhistidineintheactivecenterofchymotrypsin.Biochemistry2:252-255.33.Shaw,E.,NI.MkIares-Guia,andW.Cohen.1965.Evidenceforanactive-centerhistidineintrypsinthroughuseofaspecificreagent.1-chloro-3-tosylamido-7-amino-2-heptanone.thechlo-romethylketonefromN'-tosyl-L-lysine.Biochemistry4:2219-2224.34.Singh,A.,E.Y.Chen,J.NI.Lugovoy,C.N.Chang,andR.A.Hitzman.1983.Saccharolmvcescerevisiaecontainstwodiscretegenescodingforthealpha-factorpheromone.NucleicAcidsRes.11:4049-4063.35.Steiner,D.F.,P.S.Quinn,S.J.Chan,J.Marsh,andH.S.Tager.1980.Processingmechanismsinthebiosynthesisofproteins.Ann.N.Y.Acad.Sci.343:1-16.36.Tipper,D.J.,andK.A.Bostian.1984.Double-strandedribo-nucleicacidkillersysteminyeasts.Microbiol.Rev.48:125-156.37.Vernet,T.,D.Dignard,andD.Y.Thomas.1987.Afamilyofyeastexpressionvectorscontainingthephageflintergenicregion.Gene52:225-233.38.Wagner,J.C.,andD.H.Wolf.1987.Hormone(pheromone)processingenzymesinyeast:thecarboxyterminalprocessingenzymeofthematingpheromoneat-factor,thecarboxypepti-daseysc(x.isabsentina-factormaturationdefectiveke.xImutantcells.FEBSLett.221:423-426.39.Wickner,R.B.,andJ.M.Leibowitz.1976.TwochromosomalgenesrequiredforkillingexpressioninkillerstrainsofSaccha-romyce.scerevisiwie.Genetics82:429-442.MOI..CELLI.