1991 A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein (1).pdf

AGolgiRetentionSignal inaMembrane spanning DomainofCoronavirusElProtein AnnM SwiftandCarolynE Machamer DepartmentofCellBiologyandAnatomy TheJohnsHopkinsUniversitySchoolofMedicine Baltimore Maryland 21205 Abstract TheElglycoproteinfrom anavian coronavirusisamodelproteinforstudyingretention intheGolgicomplex Inanimalcellsexpressingthe proteinfromcDNA theElproteinistargetedtocis Golgicisternae Machamer C E S A Mentone J K Rose andM G Farquhar 1990 Proc Natl Acad Sci USA 87 6944 6948 Weshowthat the firstofthethreemembrane spanningdomainsofthe Elproteincanretaintwodifferentplasmamembrane proteinsin theGolgiregionoftransfectedcells Both thevesicularstomatitisvirusGproteinandthealpha subunitofhumanchorionicgonadotropin anchoredto themembranebyfusionwiththeGproteinmem brane spanningdomainandcytoplasmictail werere tainedintheGolgiregion oftransfectedcellswhen S ORTINGofnewlysynthesizedproteinsintheexocytic pathwayisafundamentalproblemincellbiologywhich hasreceiveda great dealofattentioninrecentyears Secretedand plasmamembraneproteinsfollowacommon pathwaythroughthecell fromtheER throughtheGolgi complex to thecellsurface 32 ResidentproteinsoftheER andtheGolgicomplexarespecificallyretained Although muchisknownregardingthesignalsequence mediated translocationofproteinsacrossthemembraneof theER 46 lessisknownregardingthetraffickingofproteinsonce they have enteredthispathway Onecurrenthypothesisinvolves theideathatproteins des tinedforconstitutivesecretion orinsertionat theplasma membranearetransportedbydefaultwiththebulkflowof lipid 35 Proteinsdestinedforlysosomesorsecretory granules incellswhichperformregulatedsecretion are directedbyspecificsignalsoncethey havetraversedthe Golgicomplex Thishypothesisrequiresthatresidentpro teinsoftheERandGolgicomplexhavespecificsignalsthat causetheirretentionintheappropriatecompartment Evi denceisaccumulatingtosupportthisidea Atripeptide whichispresumedtolackanysignalsfortransportis secreted rapidlyfromcellsanddefinestherateof bulkflow 47 Retentionsignalsforbothsolubleandmembrane boundERproteinshavebeenidentified 14 30 31 The mannose 6 phosphatemodificationonlysosomalhydrolases isrecognizedbyareceptorin theGolgicomplexwhichtar getsthese proteins tolysosomes 20 TheRockefellerUniversityPress 0021 9525 91 10 19 12 2 00 TheJournalof CellBiology Volume115 Number1 October199119 30 theirsinglemembrane spanningdomains werere placed withthefirstmembrane spanningdomainfrom El Singleaminoacidsubstitutionsinthissequence released retentionofthechimericGprotein aswell as amutantElproteinwhichlacksthesecondand thirdmembrane spanningdomains Theimportantfea tureofthe retentionsequenceappearstobethe un chargedpolarresidueswhichlineonefaceofapre dictedalphahelix Thisisthefirstretentionsignal to bedefinedforaresidentGolgiprotein Thefactthatit ispresentina membrane spanningdomainsuggestsa novelmechanismofretentioninwhichthemembrane compositionoftheGolgicomplexplaysaninstrumen talroleinretainingitsresidentproteins TheGolgicomplexplaysacentralroleintheprocessing andsortingofnewlysynthesizedproteins reviewedinrefer ence9 Itscharacteristicmorphology stacksofflattened saccularmembranes andcentrallocation peri orjuxtanu clear in thecellmaybeimportantforthesefunctions Four Golgisubcompartmentshavebeendefinedfunctionally cis medial trans andtrans Golginetwork Newlysynthesized proteinsarethoughttomovevectoriallythroughtheGolgi complexsubcompartmentsvia vesiculartransport fromthe cis to thetrans sideofthestack EndogenousGolgiproteins suchas the glycosyltransferasesandglycosidasesthatarein volvedintheprocessingofasparagine linkedoligosaccha ridesareeachenrichedinaspecificsubcompartment 8 It hasbeensuggestedthatthisarrangementallowssequential andorderlyprocessingofglycoproteinsas they arevectori allytransportedthroughtheGolgicomplex Inadditiontoitsrolein protein processing theGolgicom plexisinstrumentalincorrect sorting ofproteintraffic Lysosomalhydrolases regulatedsecretoryproteins and proteinsdestinedfor the apicalorbasolateralplasmamem branedomainsinpolarizedkidneyepitheliaaresortedinthe trans mostcisternaeofthe Golgi thetrans Golginetwork 15 43 Thecis sideof theGolgicomplexmayalsobein volvedinsorting sinceescapedERresident proteinsmust beseparatedfromthosethataretransportedforward 34 Wehavebeenstudyingthe sortingofnewlysynthesized proteins in the exocyticpathwayusingamodelGolgiprotein theElglycoproteinofthe aviancoronavirusinfectiousbron 19 on April 3 2015jcb rupress orgDownloaded from Published October 1 1991 chitisvirus IBV TheElproteinconsistsofashort gly cosylatedamino terminaldomain threemembrane span ningdomains andalong carboxy terminalcytoplasmicdo main TherestrictedintracellularlocalizationoftheElpro tein incoronavirus infectedcellsisbelievedtodirectvirus assemblyatintracellularmembranes 45 WhencDNAis expressedinanimalcellsintheabsenceof the otherviral proteins theIBVElproteinistargeted to cis Golgimem branes 29 Deletionofthefirstandsecond or thesecond andthirdofthe threeElmembrane spanning domainsshowed thatthefirstmembrane spanningdomainwasapparentlyre quiredforintracellularretention 26 Weshowherethatthe firstmembrane spanningdomainisaGolgiretentionsignal sinceitisbothnecessaryandsufficientforGolgiretention UnlikeERretentionsignalsthathavebeenidentifiedat the carboxy terminiof proteins oneitherthelumenalor cyto plasmicsideoftheERmembrane thisGolgiretentionsig nalisburiedin themembrane Ourresultssuggestthe novel possibilitythatthemembranecompositionofGolgisubcom partmentsmayplayanimportantrolein retaining resident proteins inthisorganelle MaterialsandMethods CellsandTransfection COS 7andHeLacellsweremaintainedinDMEwith5 FBS COS 7cells 1 Abbreviationsusedinthispaper IBV infectiousbronchitisvirus VSV vesicular stomatitisvirus TheJournalofCellBiology Volume115 1991 Figure1 Themlsequencefunctionsas anormalmembrane spanningdomain intheVSVGprotein A Schematic representationoftheElprotein VSVG andthechimericproteinGml Loca tionsofN linkedoligosaccharides are marked B HeLacellsexpressingei therG lanes 1 4 orGml lanes5 8 werelabeledwith 35S cysteinefor30 minandanaliquotof eachcelllysate wasimmunoprecipitated withantibody totheectodomainofGprotein aVSV thecytoplasmictail ceCTG oroneof twoconformation specificmAbs II and114 Microsomalmembranesfrom transfectedHeLacellslabeledfor10 minwereincubatedwith lanes10 and 12 or without lanes9and11 trypsin solubilized andimmunoprecipitated withpolyclonalantiVSVserum Sam pleswereelectrophoresedandthegel wasfluorographed platedin35 mmdishes 70 confluent weretransfectedwith anSV 40 basedexpressionvectorusingDEAE dextranasdescribed 28 Elexpres sionwasanalyzed44 hposttransfection ForexpressionusingthevacciniaT7 system HeLacells 70 confluent wereinfectedwiththerecombinant vacciniavirusvTF7 3encodingT7RNApolymerase 10 atamultiplicity ofinfectionof20 After adsorptionfor30 minat37 C theinoculumwas replacedwith0 75 nilofserumfreemediumcontaining4Wgofavector pAR2529 encodingtheappropriategenebehindtheT7promoterand10pl ofthecationiclipid TransfectACE BethesdaResearchLaboratories Gaithersburg MD andreference37 Expressionwasanalyzedbymeta boliclabelingstartingat4 hpostinfection MutagenesisandProductionofChimericProteins Formostofthemutations theKunkelmethodofoligonucleotide directed mutagenesis 21 wasused TheEl Am2 3 andGmlgeneswerecloned intotheM13vectormp8 andsinglestrandsproducedinEscherichiacoli RZ1032 dut ung TheexceptionwasproductionofthechimeraGml which wasproducedbydomainreplacementusingtheoligonucleotide 5 CAGTAGT IGGAAAAGCTATAATTTATTTATAACTG CATTCTTGTTGTTCT TAACCATAATACTTCAGTATGGCTATGCAACCCGG GTTGGTATCCATC 3 usingsinglestrandedGtemplatefromE coliJM103andscreeningplaques bydifferentialhybridization Thefollowingoligonucleotideswere usedfor mutationofEl withmutatednucleotides underlined NIZZ 5 AAAGAGTATATCTTATTTATAACTG 3 T133 5 GTTGTTCTTAATTATAAATACTTCAG 3 QI37 5 CATAATACTTATATATGGCTATGC 3 mlins 5 ACTGCATTCTTGATAATATTGTTCTTAACC 3 and LQ30 5 CTGCATTCTTGCAGTTCTTAACCA3 NIZZTI33wasproducedusingbothN122andT133oligonucleotidesas 20 on April 3 2015jcb rupress orgDownloaded from Published October 1 1991 Figure2 GmlisretainedintheGolgiregionoftransfectedcells COScellsexpressingeitherGorGmlwerefixedandstainedbydouble labelindirectimmunofluorescencemicroscopy SurfaceGproteinwasdetectedbystainingwithrabbitantiVSVserumfollowedbya Texas red conjugatedsecondantibody InternalGproteinwasdetectedafterpermeabilizationwith amonoclonal anti Gantibodyanda fluorescein conjugatedsecondantibody Leftpanelswerephotographedwiththefluoresceinfilter and thoseontherightarethesame fieldphotographedwiththerhodaminefilter Bar 10 Am primersforsecondstrandsynthesis Thesesameoligonucleotideswereused tocreatethemutationsintheEldeletionmutant Gm2 3 Gmlinswaspro ducedwiththemlinsoligonucleotide butGm1QIwasobtained onlyafter alongeroligonucleotide 5 CTTAACCATAATACTTATCTATGGCTATGCAACCC 3 wasused T4DNApolymerase Biolabs wasusedforsecondstrandsynthesis and thedouble strandedmoleculesweretransfectedintoE coliNM522 Single strandedDNAfromthreetosixplaqueswassequencedusingthe dideoxyprocedure Sequenase USB to select thedesiredmutations The mutatedgeneswereexcisedfromthedouble strandedreplicadveform DNAandsubclonedintoboththeSV 40expressionvectorpJC119 44 and theT7expressionvectorpAR2529 10 AllgeneralrecombinantDNA techniqueswereasdescribed 41 Themembrane spanningdomainofamwasreplacedwitheithertheIBV Elmlorm3domainusingrestrictionsitesinthecodingsequence Tocreate am1G aBamHItoRsalfragment encodingtheasubunit wasfilledinwith theKlenowfragmentofDNApolymeraseI digestedwithXhol andligated withaHpalItoBamHIfragment encodingthemldomainandGtail from Gmlwhichwaspreparedsimilarly Tocreateam3G thesamea encoding fragmentdescribedabovewasligatedwithaDraltoBamHIfragmentfrom Aml 2 encodingthem3domain andaBamHItoXhoIfragment encoding theGtail fromtheGmutantTMB whichhasaBamHIsiteintroduced atnucleotide1483 reference 36 IndirectImmunofluorescenceMicroscopy COS 7cellsgrown oncoverslipswerefixed permeabilized andstained 44hposttansfectionessentiallyasdescribed 26 27 FordetectionofEl SwiftandMachamerGolgiRetentionSignal and mutantElproteins anaffinity purifiedrabbitanti peptideantiserum recognizingtheCOOH terminusof Elwastheprimaryantibody 1 40 N5 gg ml followedbyTexasred conjugated affinity purifiedgoatanti rabbit IgG 1 500 JacksonImmunoResearchLaboratoriesInc Avondale PA FordetectionofGproteinand mutantGproteinsby doublelabeling non permeabilizedfixedcellswerefirststainedwitharabbit antiVSVserum 1 200 followedbyTexasred conjugated affinity purifiedgoatanti rabbit IgG Afterpermeabilizationwith0 5 Triton X 100 internalGproteinwas detectedbystainingwithamonoclonalanti Gantibody 11 4pg ml refer ence23 followedbyfluorescein conjugatedaffinity purifiedgoatanti mouseIgG 1 200 JacksonImmunoResearchLaboratoriesInc Cellsex pressingthechimericamproteinswerestainedwithanaffinity purifiedrab bitanti peptideantibodywhichrecognizestheGcytoplasmictail 1 20 ref erence29 followedbytheTexasred conjugatedsecondantibodydescribed above CellswerevisualizedwithaNikonMicrophotmicroscope Nikon Inc GardenCity NJ equippedwithepifluorescenceilluminationanda Nikon60 xoilimmersionplanapochromatobjective Photographswere takenwithTri XPanfilm EastmanKodakCo Rochester NY andpro cessedwithDiafinedeveloper Accufine Inc Chicago IL RadiolabelingandImmunoprecipitation COS 7cellsexpressingElandmutantElproteins orGandmutantGpro teinswerelabeled 44hposttransfection Elproteinswerelabeledfor1or 2hin0 5mlcysteine freeDMEwith100ACi 358 cysteine 1 300Ci mmol AmershamCorp ArlingtonHeights IL Cellswereharvestedim mediately or after a 3 hchaseinregulargrowthmediumcontainingathree foldexcessofunlabeledcysteine Cellswerelysedindetergentsolution 50 mMTris pH8 0 1 NP 40 0 4 deoxycholate 62 5mMEDTA and0 13 TIU mlaprotinin andElproteinsimmunoprecipitatedusingtheanti 21 on April 3 2015jcb rupress orgDownloaded from Published October 1 1991 peptideserum andfixedStaphylococcusaureus Calbiochem Behring Corp SanDiego CA asdescribedpreviously 26 ForanalysisofGproteins HeLacells 4hpostinfection orCOS 7cells 44hposttransfection wereincubatedincysteine freemediumfor10min andthenlabeledfor30 minin0 5mlcysteine freemediumcontaining50 ACi S cysteine Cellswereharvestedimmediately oraftervarioustimes ofchaseasabove Cellswerelysedasabove andGproteinsimmunoprecip itatedwitheither 3Al ofapolyclonalrabbitantiVSVserum 3Alofarabbit anti peptideserumwhichrecognizestheGcytoplasmictail 27 orwith 2jIofmAbsIlor114 23 To showthatGmlspannedthemembrane HeLa cellswerelabeledfor10min scrapedfromthedish dounced50timeswith atight fittingpestle andtreatedwith orwithout100jig mlTPCK trypsin Boehringer MannheimBiochemicals Indianapolis IN for60minat0 C PMSFwasaddedto50mM microsomesweresolubilizedindetergentso lution asabove andGproteinsimmunoprecipitatedwiththepolyclonal antiVSV serum Elproteinswereelectrophoresedin 15 polyacrylamidegelscontaining SDS andGproteinswereelectrophoresedin10 gels 22 Markerpro teinswere C methylatedstandardmolecularweightmarkers Amer shamCorp Labeledproteinsweredetectedbyfluorography 2 AnalysisofOligosaccharides EloligosaccharideswereanalyzedafterElproteinsintransfectedCOS 7 cellswerelabeledfor2 handchasedfor 3h S aureuspelletswereeluted andaliquotsweretreatedwithendoH 0 1mU ICNRadiochemicals Ir vine CA N glycanase peptide N glycosidaseF 0 05mU Genzyme Corp Boston MA orbufferalone usingtheprotocoldescribedprevi ously 29 TheJournalof CellBiology Volume115 1991 ForGproteins thekineticsofoligosaccharideprocessing weredeter minedincellslabeledfor10minfollowedby variouschase times Im munoprecipitatesweretreatedwithendoH 0 1mU asdescribed 28 Fluorogramswerequantitatedbydensitometry TrfmerAssay Figure3 Gmlforms anoligomerlarger thanatrimer HeLacellsexpressingei therGorGmlwerelabeledfor10min andlysedimmediately oraftera 20min chaseinunlabeledcysteine Lysates werecentrifugedin5to20 continuous sucrosegradients andgradientfrac tionswereimmunoprecipitated with antiVSVserum seeMaterialsand Methods Aportion 20 of eachcell lysatewasimmunoprecipitateddirectly and runinthefarright handlaneofeach gel Althoughapparentlyamonomeraf tersynthesis Gml formedalarge 15S aggregateduringthechase OligomerizationoftheGmlproteinwasanalyzed byvelocitygradientcen trifugation insucroseessentially asdescribed 7 Continuous5to20 su crosegradientswerepouredovera0 25ml60 sucrosecushioninSW50 1 tubes Allsolutionswerein20mMTris 30mMMES pH5 8 1 Triton X 100 100mMNaCl HeLacellsexpressingeitherGorGmlwerelabeled with 35S cysteine for10minandharvestedimmediatelyorafter20min ofchaseinunlabeledcysteine Lysateswereloadedontopofthegradients andspunat47 000rpmfor16h Fractions 0 33ml werecollected immu noprecipitatedwithantiVSVantibody andelectrophoresedtodetermine thelocationofGproteininthegradient Results RetentionofaPlasmaMembraneProtein Inanearlierstudy 26 wefoundthatdeletionofthefirst andsecondmembrane spanningdomainsofElresultedin amutantprotein Aml 2 whichwasefficientlytransported totheplasmamembrane However whenthesecondand thirdmembrane spanningdomainsweredeleted Am2 3 22 on April 3 2015jcb rupress orgDownloaded from Published October 1 1991 themutantproteinwasretainedintheGolgiregionoftrans fectedcells Bothmutantproteinswereinserted intoand spannedthemembraneproperly Theseresultssuggested thateithertheGolgiretentionsignalwasinthefirstmem branespanningdomain ml orthatthe deletion creating Oml 2disruptedaretentionsignalelsewherein themole cule Todistinguishbetweenthesetwopossibilities we askedwhethermlcouldretainaproteinnormallytrans portedto theplasmamembrane TheGprotein ofvesicularstomatitisvirus VSV istrans portedrapidlyandefficientlyto theplasmamembranein transfectedcells andmuchisknownaboutitsfoldingand oligomerization 7 Thesinglemembrane spanningdomain of theGproteinwasreplacedwiththatofmlfromIBVEl Fig 1A Thedomainreplacementwasperformedpre ciselyusingoligonucleotide directedmutagenesis Thechi mericGprotein calledGml wasexpressedtransientlyin SwiftandMachamerGolgiRetentionSignal Figure4 Themldomain butnot the m3domain retainsanotherplasma membraneproteinintheGolgire gion A TransfectedCOScellsex pressingam am1G oram3Gwere fixed permeabilized andstainedfor indirectimmunofluorescencemi croscopy withananti peptidewhich recognizestheGcytoplasmictail andaTexasred conjugatedsecond antibody B Theaminoacidse quences singlelettercode are shownforthetransmembranedo mainsofVSVGprotein andboth themlandm3domainsoftheIBVEl protein Bar 10Am COScellsusingaSV 40 basedvector 28 orinHeLacells usingavacciniavirusT7RNApolymeraseexpressionsystem 10 Gmlwasrecognizedbypolyclonalantibodies toboth theGectodomainandthecytoplasmictail andbytwomAbs whichrecognizeconformation sensitiveepitopes Fig 1B lanes5 8 Inaddition Gmlspannedthemembranesince thecytoplasmictailwassusceptibleto trypsin digestionin microsomalmembranes Fig 1B lane 12 Theseresultsin dicatedthatmlfunctionedas apropermembrane spanning domaininGml andthatthechimericproteinwasnot grosslymisfolded Gmlwasnottransportedtotheplasmamembrane how ever Indirectimmunofluorescencemicroscopydemonstrated thatGmlwasabsentfromthecellsurfacebutpresentina juxtanuclear regionconsistentwithGolgilocalization Fig 2 Inaddition thetwoN linkedoligosaccharidesaddedto GmlwerenotprocessedtoanendoH resistantformas they 23 on April 3 2015jcb rupress orgDownloaded from Published October 1 1991 wereonwild typeGprotein Aftera10minpulselabel wild typeGproteinbecameendoHresistantwithahalf time ofabout20min whereasGmlwasendoHsensitive evenafter4hofchase seeFig 10 Thissuggestedthatthe Gmlproteinwasretained in apre medialGolgicompart ment likethewild typeElprotein Gproteinhasbeenshowntoformanoncovalentlyas sociatedhomotrimerbeforeitsexitfromtheER 6 We testedtheoligomericstructureofGmlonsucrosegradients afterapulse chaselabel Fig 3 Aftera 10minlabel wild typeGproteinwas 50 trimer 8S and50 monomer 4S consistentwiththeresultsofDomsetal 6 7 After 20 minofchase alltheGproteinwasfoundinthe8Strimer peak Althoughapparentlyamonomerafterthe10minla bel Gmlpelletedafterthe20minchase Othercentrifuga tioncond