InvitedReviewFunctionalexpressionofheterologousproteinsinyeast:insight - PDF document

InvitedReviewFunctionalexpressionofheterologousproteinsinyeast:insightsintoCasignalingandCa-transportingATPasesVan-KhueTonandRajiniRaoDepartmentofPhysiology,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland21205Ton,Van-Khue,andRajiniRao.Functionalexpressionofheterologousproteinsinyeast:insightsintoCasignalingandCa-transportingATPases.AmJPhysiolCellPhysiol287:C580–C589,2004;10.1152/ajpcell.00135.2004.— WHYYEAST?In1997,thehaploidbuddingyeastSaccharomycescerevi-becamethersteukaryoticorganismwithacompletelysequencedgenome.Ithas6,200genesorganizedintoacompactgenome,essentiallyfreeofintrons.Thesegenesand Addressforreprintrequestsandothercorrespondence:R.Rao,Dept.ofPhysiology,TheJohnsHopkinsUniv.SchoolofMedicine,725N.WolfeSt.,Baltimore,MD21205(E-mail:rrao@jhmi.edu).AmJPhysiolCellPhysiol287:C580–C589,2004; 0363-6143/04$5.00Copyright2004theAmericanPhysiologicalSocietyhttp://www.ajpcell.org fundamentallysimilartothoseinhighereukaryotes.Thees-sentialcomponentsofthecellularCasignalingmachinery,aswenameit,areconserved,includingCachannelsandtransporters,Casensors,andsignaltransducers.Amajordifferenceisthatyeastlackstheredundancyofmultipleisoformsandthecomplexityofsplicevariantsthatarecharacteristicofmammaliancells.Instead,thereareadenumberofgenesthatmaybedeletedindividuallyorincom-bination,withpredictableeffects.Figure1summarizeskeypathwaysandproteinsinvolvedinCaentry,signaling,andexitfromthecytosol.Attheplasmamembrane,high-afnityCauxismediatedbyacomplexconsistingofCch1,ahomologofthemammalianvoltage-gated-subunitofCaandMid1,aputativestretch-activatedCachannel(29,37,64).ThesechannelshavebeenimplicatedinCaassociatedwithexposuretomatingpheromone,endoplasmicreticulum(ER)stress,anddepletionofCastoresintheER/secretorypathway(aresponsesimilartocapacitativeCaentry;Ref.10).Calcineurinisknowntoinhibitchannelactiv-ity,possiblythroughdirectdephosphorylation.Thereisexper-imentalevidenceforatleastoneotherCauxpathwaythatremainstobeidentied,postulatedtomediatelow-afuxandcell-cellfusionduringthelatephaseofthematingprocess(58).Inyeast,thevacuoleservesasamajorstoreforCa,forthepurposesofbothdetoxicationandsignaling.Ahomologofthemammaliantransientreceptorpotential(TRP)-likechannel,Yvc1,hasbeenshowntoreleasefromthevacuoleintothecytosolinresponsetohyperosmoticshockandtheantiarrhythmicdrugamiodarone(33,65,98).BothmechanicalactivationofYvc1andCareleasehavebeenreportedinresponsetoosmoticupshock(98).Interestingly,yeastinositol1,4,5-trisphosphate)receptorchannelshavenotbeenidentied,correlatingwithlowlevelsofIPinyeast(seesignalingpathwaysanditisnotknownhowCaisreleased,ifatall,fromtheER/Golgistores.pumpsandtransporters.Inyeastandothereukaryotes,activetransportofCafromthecytosoltointracellularstoresorextracellularspaceoccursbyATP-drivenpumpsandcation-coupledexchangers.Inmetazoans,thewell-knownsarco(en-do)plasmicreticulumCa-ATPases(SERCA)sequesterCaintotheERandplayaprominentroleincytosolicCahomeostasis.AlthoughtherearenoSERCAhomologsin,CalevelsintheERarecontrolledbyaGolgi-localizedpump,Pmr1,presumablywhileenroutetotheGolgi(76).YeastPmr1(forplasmamembraneATPaserelatedwastherstmemberofthefamilyofsecretorypathway-ATPases(SPCA)tobeidentied(71).Itmediateshigh-nityCa(andMn)transportundernormalphysiologicalconditionsandservesadualroleinmaintainingcytosolicionhomeostasisaswellasinsupplyingtheGolgilumenwithCaandMnforproteinsorting,processing,andglycosylation(25).Pmr1homologsarenowknowntobeubiquitousinallhighereukaryotes,withthenotableexceptionofplants,andmuchofwhatweknowaboutthestructureandphysiologicalroleofthesetransporterscomesfromstudiesinyeast.Morerecently,thesestudieshaveextendedtoSPCAhomologsfromhumans(5,27,83),Caenorhabditiselegans(13,87),andrats(Ref.69;reviewedinRef.93,94).AmoredetailedreviewofinsightsintothisnovelclassofCapumpsispresentedlater.PlasmamembraneCa-ATPases(PMCA)constituteathirdbranchofthissuperfamilyofrelatedpumps,representedinyeastbyPmc1.AlthoughclearlyrelatedinsequencetothePMCAfamily,yeastPmc1lacksdomainsinvolvedinregula-tionbycalmodulinandacidicphospholipidstypicalofthemammalianmembersandlocalizestothevacuolarmembraneinsteadofthecellsurface.However,detoxicationofCavacuolarsequestrationistopologicallyequivalenttotransportintotheextracellularspace,andPmc1-likehomologsarecom-monlyfoundintheacidicvacuolesofotherfungiandproto-zoans.Transcriptionalactivationofoccursinthepres-enceofhighextracellularCa(19)orintheabsenceofPmr1(53)andisdependentoncalcineurin.Becausecalcineurin-dependenttranscriptionalactivationisaprocessconservedacrosskingdomsfromfungitomammals,itwillbeofinteresttodeterminewhethercompensatorychangesinexpressionlevelsofmammalianCapumpsandexchangersalsooccurbysimilarpathways.AnothermajorplayerinvacuolarCasequestrationisVcx1,aCaexchangerdrivenbytheprotonelectrochemicalgradientsetupbythevacuolarH-ATPase(20,55,67).Vcx1activityisapparentlyinhibitedbycalcineurin(20).Thusviabilityofadoublenullmutantofcanbeimprovedbytheadditionaldeletionoftheregulatorysubunitofcalcineurin(),resultinginupregulationofVcx1activity.Theresultingtripledeletionstrainisdevoidofendogenous-ATPasesandservesasanidealhostfortheheterologousexpressionofdiverseCapumps,asdiscussedinmoredetaillaterinthisreview.MutantslackingoneormoreoftheCa Fig.1.CasignalingandtransportpathwaysinSaccharomycescerevisiaeentersthecytosolviatheplasmamembranechannelcomplexCch1/Mid1orthevacuolartransientreceptorpotential-likechannelYvc1inresponsetodiverseenvironmentalcuessuchasendoplasmicreticulum(ER)stress,os-moticshock,ormatingpheromone.Themolecularpathwaysleadingfromactivationofcalmodulinhavebeenwellcharacterized.Caandcalmodulinactivatetheproteinphosphatasecalcineurin,whichinturnactivatesthetranscriptionfactorCrz1/Tcn1,leadingtoitsnucleartranslocationandthetranscriptionoftargetgenes.CaisprimarilyclearedfromthecytosolbyP-typeATPasesintheGolgi(Pmr1)andvacuole(Pmc1),andbythevacuolarexchangerVcx1.InvitedReview HETEROLOGOUSEXPRESSIONINYEASTAJP-CellPhysiolVOL287SEPTEMBER2004 transporterscanalsobeusedforassessingthecontributionofdifferentpathwaysinvolvedincellularCa(33,43).signalingpathways.ManydownstreamtranscriptionalandtranslationaleventsinyeastarecontrolledbytheCamediatedactivationofcalmodulin(CaM).Inyeast,asinotherorganisms,calmodulinisessentialforlife(21).Ayeastpro-teomemicroarrayof5,800puriedproteinshasidentied6knownand33additionalpotentialbindingpartnersforcalmod-ulin,consistentwitharoleforthisessentialproteinindiversecellularprocesses(99).CalmodulinparticipatesinCapendentstressresponsepathwaysthroughactivationofyeastCaMkinasesCmk1andCmk2(57)andthephosphatasecalcineurin(22).DephosphorylationbycalcineurinresultsinnucleartranslocationofCrz1/Tcn1,analogoustomammalianNFATc,andtranscriptionalactivationofmorethan160targetgenesinvolvedincellwallandlipidsynthesis,ionandsmallmoleculetransport,vesicletrafcking,andothersignalingproteins(22).Liketheprototypiccalmodulin,thereareotherEF-hand-containingCaregulatoryproteinsinyeast.Yeastfrequenin(Frq1),ahighlyconservedorthologoftheneuronalsensor-1,functionsastheCa-sensingsubunitofPik1,aphosphatidylinositol4-kinasethatisessentialforvesicularckinginthelatesecretorypathway(75).AdditionofglucosetostarvedcellsleadstoacytosolicCawithinminutesthatappearstobemediatedbyaphosphoino-sitide(PI)-specicphospholipaseCviaGprotein-coupledreceptors(GPCR)(80).Inmammaliancells,PIturnoverleadstoactivationofproteinkinaseCbydiacylglycerolandintra-cellularCareleasebyIP.Curiously,inyeast,IPappearstoberapidlyconvertedtopolyphosphates(IP,IP,andIP),andalthoughtherearenoknownIPreceptorchannels,geneticdisruptionofIPkinasesenhancestheCasignal(81).andcelldeath.Inkeepingwithitsdiversecellularfunctions,theroleofCainapoptosisislikeadouble-edgedsword.DisruptionofCahomeostasisisawell-establishedsignalforprogrammedcelldeathinmammaliancells,andmanyapoptoticeffectorssuchascalcineurinandcalpainsshow-dependentactivation(39,88).ProlongedcytosolicCaoverloadand/orcontinuousCauxtriggeredbydepletionoftheERCapoolcancausemassiveCauptakeintothemitochondrialmatrix.ThislargeincreaseinCalevelthentriggerstheopeningofthepermeabilitytransitionporeontheoutermitochondrialmembrane,resultinginthereleaseofandproapoptoticfactorsintothecytosol.Con-versely,Caalsocanfunctionasanantiapoptoticsignalinneurons(30,34,63).Althoughdeathbyapoptosisiswidelyconsidereduniquetometazoans,thereisnewevidencethatunicellularorganisms,includingS.cerevisiae,alsoundergoprogrammedcelldeathinresponsetodiverseinsults.Notsurprisingly,asinmammaliancells,Cacaninuencethisprocessinyeast.Thuscalcineurin-dependentCauxinresponsetoERstressprotectsagainstcelldeath(11),whereasthenovelantifungalagentamiodaronetriggersmassiveCauxfromexternalandinternalstores,leadingtotheappear-anceofapoptoticmarkersand,eventually,celldeath(33)(unpublishedobservation).Markersofprogrammedcelldeathinyeastareremarkablysimilartothoseinmammaliancells;theyincludelossofmembraneasymmetryandphosphatidyl-serineexternalization,reactiveoxygenspeciesgeneration,DNAfragmentation,lossofmitochondrialmembranepoten-tial,andcytochromereleasefrommitochondria(50).How-ever,theyeastapoptoticpathwayappearstobelesssophisti-catedthanthatofmetazoans.Recently,theyeastsystemhasbeensuccessfullyexploitedtostudymammalianproteinsin-volvedinapoptosis,suchasscreeningforBaxinhibitors(15)orcharacterizationoftheinteractionbetweentheproapoptoticBaxandBidwiththemitochondria(68).GivenourthoroughknowledgeofyeastCahomeostasis,ageneticdissectionof-mediatedapoptoticpathwaysisnowfeasible.Insummary,despitesomeobviousdifferences,anoverallconservationinCasignalingpathwaysandinCaporterswithhighereukaryotesmakesyeastasimpleandelegantexperimentalmodelsystem.HETEROLOGOUSEXPRESSIONINYEASTTheartofheterologousproteinexpressioninS.cerevisiaehasbeenrenedovermanyyears,beginninginthe1980s.Asamicrobialexpressionsystem,offersmoreadvantagesthanEscherichiacoli,sinceexogenousproteinsmaybetargetedtomembrane-boundcompartmentsthatarereadilyisolatedforbiochemicalexperiments(23,78,83).Italsosurpassesculturedinsectandmammaliancelllineswithsimpleandinexpensivegrowthconditionsandashortercellcycle.High-throughputscreensbasedongeneticfeaturesotherthanfunctionalcomplementationarealsofeasible.Forexam-ple,theabilitytoformwhiteandredcoloniesonrichmedium,whichdependsrespectivelyonthepresenceorabsenceof,genesencodingenzymesintheadenine-biosynthesispathway,wasthebasisofascreenformammalianprioninhibitors(4)andfortruncationmutationsinhumangenes(41).Otherscreensrelyonpathwayconservationsbe-tweenyeastandmammals.Theyeastpheromone-sensingpath-wayisaGPCRtransductionsystemwhosehomologsareabundantlyrepresentedinmammals.ItisthuspossibletoengineerhumanGPCRs,particularlyorphanreceptors,intoyeastdevoidofbackgroundGPCRforhigh-throughputscreens(17,91).Promotersandplasmids.Oneadvantageoftheyeastsystemistheavailabilityofstrongandconstitutivepromotersthatdrivetheexpressionofproteinssuchasplasmamembrane-ATPase(),glyceraldehyde-3-phosphatedehydrogenase(),phosphoglyceratekinase-1(),alcoholde-hydrogenase-1(),andpleiotropicdrug-resistantpump).Ofthese,thepromotersareexcep-tionallystrongandcandirecthigh-levelexpressionreachingupto10%ofplasmamembraneproteins.Inthepresenceofthemutantallele,encodingaconstitutivelyactivetran-scriptionfactor,isevenmoreheavilytranscribed(61).Besidesstrongpromoters,induciblepromotersallowcarefullytimedexpressionofproteins,especiallythosewhosepresenceistoxictocellgrowth.Thesepromotersinclude(inducedbygalactose),(inducedbylowextracellularinorganicphosphate),andtandemheatshock(inducedbytemperatureelevationto37C)(57).Itisworthnotingthatthepromotersarealsoexceptionallyusefultoturnoffgeneexpressionfollowingatransfertoglucoseme-dium.Promotersthatdirectvariableexpressioninresponsetoatitratableinducerincludethemethionine-responsivepromotersandcopper-dependenters.Anyofthesepromotersmaybeclonedintomulticopy(2InvitedReview HETEROLOGOUSEXPRESSIONINYEASTAJP-CellPhysiolVOL287SEPTEMBER2004 orsinglecopy()plasmidstogiveanadditionallevelofcontrolinexpressionlevel.Theplasmidscarrynutritionalmarkers(,andothers)forselectioninyeastandantibioticresistance()forpropagationinbacteria.Afteramplicationinbacteria,plasmidsareintroducedintothecorrespondingyeastauxotrophsbymethodsessentiallysimilartobacterialtransformationandcanbestablymaintainedindropoutmedia.Hoststrains.Notonlyindividualgenesbutalsomembersofanentiregenefamily,aswellastheregulatorsthatcontroltheirexpression,maybesystematicallydeletedwithrelativeeaseinyeast.Thisallowsheterologousexpressioninawell-controlledsystemdevoidofbackgroundcontaminationfromendogenousproteinsofsimilarfunction.Forexample,sevenpleiotropicdrug-resistanttransporters,,and,togetherwiththeiractivatingtranscriptionfactors,,havebeensimulta-neouslydeleted,renderingtheresultantstrainexquisitelysen-sitivetodrugs(70).Mutantsthatchangelipidcompositionoftheplasmamembrane,suchasthemutantdefectiveinergosterolbiosynthesis,alsosensitizeyeasttoavarietyofdrugs,presumablybyincreasingtherateofpassivedrugdiffusion(26).Suchmutantsareextremelyvaluableinstudiesinvolvingheterologousexpressionofmultidrugresistance(MDR)proteinsfromhumanorpathogenicmicroorganismsandinscreensformutationsconferringdrugresistance.Inanotherexample,upto18hexosetransportersresponsibleforglucoseuptakehavebeenknockedout(90),enablingscientiststounderstandthesugar-transportingnetworkinyeastandtoexpressanyputativesugartransporterfromotherorganismstoinvestigateitsfunctions.Similarly,ourlaboratoryhaspio-neeredtheapplicationofayeasttriplemutantlackingthetwoaswellastheregulatorysubunitofcalcineurin,,ashostfortheunambiguousbiochemicalanalysisofplasmid-encodedCapumps(74).Multiplegenedeletioninyeastmostlyreliesontheefofhomologousrecombinationtotargetchromosomallociandonthesubsequentcrossingofvariousmutantstoproducethedesiredcombination.Asaresult,itispossibletocreateanentirelynewdesignerstrainthatservesalmostanyexperi-mentalneed,providingthatsimultaneousdeletionofmanygenesdoesnotkillthecell.ProteinsthatarehighlysensitivetoproteolysiscanbeexpressedinastrainlackingthemastervacuolarendopeptidasePep4(44),whichcontrolstheactivationofothervacuolarhydrolases(92).Heterologousexpressioninstrainscarryingtemperature-sensitive()allelesofgenescanbeemployedifthecorrespondingnullmutantisinviable.Inaninnovativeexampleofthisapproach,astraincontainingthealleleof,wasexploitedtoenrichforsecretoryvesiclesduringa2-htemperatureshiftto37C(60).Theaccumulatedvesiclescanbereadilyisolatedingoodyieldandserveasasourceofhomogenouslyorientedright-side-outvesiclesfortransportassays.Thisstrategyhasbeenadaptedfortheexpressionofavarietyoftransportproteinsinyeast,includingmammalianaquaporins(18),Naglucosesymporter(28),multidrugtransporters,andothermembersoftheABCsuper-family(72).AsimilarapproachcouldbeusedtoenrichmembranesoftheERorGolgiortoenlargetheprevacuolarcompartment.Thereexistamultitudeofmutantsblockedatdifferent,discretestepsinthesecretoryorendocyticpathways.Forexample,mutantsshowingdefectsinexitfromER),Golgi(),orprevacuolarcompartment/lateendosome()orinendocytosis()maybeusedtofollowthebiosyntheticpathwayofatargetproteinfrombiogenesistodegradation.Functionalcomplementation.Assoonasautomatedse-quencingbecamecheaperandmorereliable,inthelate1990swewitnessedanexplosionofgenomesequencingprojects,resultinginthediscoveryofnovelgenesofunknownfunction.ThisiswheretheS.cerevisiaesystemdemonstratesitsmostpowerfulfeature,functionalcomplementation:essentiallytherestorationtowild-typephenotypesby(yourfavoritegene)inayeaststrainlackingtheputativehomolog.Functionalcomplementationisalsoofconsiderablepharmaceuticalinter-estbecauseitallowscell-basedhigh-throughputscreensfornaturalorsyntheticligands,specicallysmall-moleculeinhib-itors.Another,oftenoverlooked,advantageoffunctionalcomplementationisthatmutationsinmayberapidlyscreenedforloss-of-functionphenotypesinstructure-functionanalysis.Thisservestoprioritizethemoredifcultbiochem-icalstudiesbyscreeningoutmutationsthathavelittleornoeffectonthebiologicalfunctionoftheprotein.Suchscreensaresignicantlyharderorimpossibletoperforminotherheterologousexpressionsystemsbecauseoflongercellcycles,complicatedgrowthconditions,and/orendogenousback-groundactivities.Figure2summarizestheprocessfromiden-cationofanovelgenetotheuseofyeastforcomplemen-tationstudiesandbeyond.Whenagenomesequencingorchromosomalmappingprojectyieldsanovelgene,therststepistosearchforputativehomologswhosefunctionshavebeenstudiedand/ordocumentedindatabases,inthiscase,theSGD.Onceaputativehomologisidentiedonthebasisofsequencesimilarity,canbeclonedintoayeastexpressionvectorandtransformedintothestrainwithadeletioninthecorrespondingyeastgene().Multiplehomologsalsomaybedeletedbyhomologousrecombination.Mostlikely,hasbeenextensivelycharacterizedanditsphenotypesindifferentgrowthconditions(nutrients,drugs,salts,ortem-perature)documentedintheSGD.Alternatively,ifessential,oneoftwostrategiesmaybeemployedtoreplaceitwiththeheterologousgene.Thepromoterofmaybereplacedbytherepressiblepromoterbyhomologousrecombination.Theresultingstrainisviableongalactosemediumandwillnotgrowuponshifttoglucosemediumunlessfunctionalcomplementationisachievedbyplasmid-encoded.Anotheroptionisaplasmidshufingtechniquethatselectsforlossoffromaplasmidinmediumsupplementedwith5-uoroorticacid(5-FOA),atoxicanalogofuracilthatislethaltoyeastonlyinthepresenceofthegene.Again,viabilityismaintainedifcansuccessfullyreplacetheessentialyeastgene.Functionalcomplementationyieldstherstexperimentalcluetothebiologicalroleofanovelgene,whichcanbesubsequentlyconrmedbybiochem-icalcharacterization.Itisimportanttonotethatthereneednotbesequencesimilaritybetweenanditsyeastcounterpartforfunctionalcomplementationtobeappliedsuccessfully.Forexample,theKuptakedefectoftheyeastmutantpreventsgrowthinK-limitedmedium.Functionalcomplementationofthisgrowthlimitationhasbeensuccess-fullyexploitedforexpressioncloning,mutagenesis,andtraf-InvitedReview HETEROLOGOUSEXPRESSIONINYEASTAJP-CellPhysiolVOL287SEPTEMBER2004 ckingstudiesofavarietyofunrelatedKchannelsfromplantsandanimals(2,6,79).Inlaboratoriesaroundtheworld,manytypesofgenesfrombacteria(56),protozoa(7,12,31),fungi(100),plants(48),andanimals(83)havebeenexpressedinandshowntocomplementdeletionphenotypesofyeastmutants.Thereferencesgivenhereareaveryfewexamplesofthevastnumberofarticlespublishedonthesubjectoffunctionalcomplementationstudiesinyeast.SECRETORYPATHWAYC-ATPASEDEFECTIVEINHAILEY-HAILEYDISEASE:LESSONSFROMYEASTResearchfromseverallaboratoriesontheemergingfamilyofGolgi/secretorypathwayCa-ATPases,aswellasonputativemembersoftheSERCAandPMCAfamilies,illustratetheutilityofyeastinpredictingthecellularroleandmechanismofanovelprotein.IdentiÞcationofATP2C1asanovelgene.Disease(HHD),orbenignfamilialpemphigus,isanintraepi-dermalblisteringdisordercharacterizedbyacantholysisandimpairedkeratinocyteadhesion.Inheritedasanautosomaldominantdisease,theunderlyingcellularmechanismofHHDhadeludedscientistsfordecadesuntilsimultaneousreportsfromtwogroupsshowedthataffectedindividualshadmuta-tionsmappingtoanovelgene,designated(36,77).Sequenceanalysisrevealedthatthegeneproductofsharedhighidentity(49%)withyeastPmr1,theGolgi/secre-torypathwayCa-ATPase,hencethename(human)hSPCA1.Uptothispoint,Pmr1wastheonlySPCAmembertobeextensivelycharacterized.InactivationofinmissortingandmisprocessingofproteinsalongthesecretorypathwayandtoN-andO-linkedglycosylationdefects(3,25).TheseobservationswereconsistentwithdepletionofluminalGolgistoresandcouldbeseparatelycorrectedbyhighlevelsofextracellularCaandMn,respectively(25).Inaddition,mutantshaveelevatedrestinglevelsofCainthecytosolandcannoteffectivelymaintaincellularCahomeostasis(33,35,62).ThesephenotypesarestrikinglysimilartoobservationsinkeratinocytesisolatedfromHHDpatientsthatshowhigherlevelsofrestingcytosolic,defectsinrecoveryfromaCaload(36),andsignicantdecreasesinluminalCa(5).ThusthemoleculardefectsinHHDmaybelinkedtoeitherinsufcientCaandMntheGolgiordefectivecytosolicCaclearance,ormorelikely,both.InadequateglycosylationorprocessingofdesmosomalproteinsduringtransitthroughtheGolgimayleadtodefectivecellsurfaceadhesion.Itisalsoknownthatdesmosomalas-semblyandturnoverareexquisitelydependentonCathatalterationsincytosolicCasignalingmightleadtoproteininternalizationfromplasmamembraneanddesmosomedisruption(45).ItisinterestingtonotethatDarierdisease,arelatedblisteringdisorderalsocharacterizedbylossofkeratinocyteadhesion,iscausedbymutationsintheSERCA2Capumpand,likeHHD,showsautosomaldominantinheritance(73).PuttingATP2C1intoyeast.Thegenewasclonedintoamulticopyyeastexpressionvectorcarryingaselectableauxotrophicmarker(),undercontrolofthepromoterforphosphoglyceratekinase-1()andanNH-terminalHisepitopetag.Thechoiceofapromoterofmoderatestrengthwasbasedonourearlierunpublishedobservationthatconstitutivehigh-levelexpressionofyeastfromtheverystrongpromoterwaslethal.TheplasmidwastransformedintothetriplenullmutantstrainK616(),lackingendogenousCapumps.Ofthemanyphenotypesofnullmutantsreported,themostusefularehypersensitivitytogrowthinmediumsupplementedwithBAPTAorMn,aswehaveshownthatthesecorrelatewellwithCaandMntransportactivity,respectively.Chelationofextracellulardiva-lentcationsbyBAPTA(12mM)starvesthesecretorypath- Fig.2.Strategiesinheterologousexpressionandcharacterizationofanovelgeneinyeast.owchartoutlinesstepsleadingfromtheidenticationofyourfavoritegene(toheterologousexpressioninyeastandsub-sequentfunctionalstudies.WT,wildtype.Seetextfordetails.InvitedReview HETEROLOGOUSEXPRESSIONINYEASTAJP-CellPhysiolVOL287SEPTEMBER2004 way(mostlikely,theER)ofCaandpreventsyeastgrowth.Expressionofahigh-afnityCapumprescuesthecellsfromstarvation.ItwasobservedthathSPCA1fullycomplementedtheBAPTAhypersensitivityofK616,asdidheterol-ogousexpressionofmammalianPMCAandSERCApumps(83).MnhypersensitivityofthemutantresultsfromthetoxicityofexcessivecellularMn;forexample,MnknowntoincreasetheerrorrateofDNApolymerasesandtointerferewithMg-bindingenzymes(9).ExpressionofanityMnpumpeffectivelycompartmentalizesthetoxicionandrescuescellgrowth.Interestingly,hSPCA1,butnotrabbitSERCA1orhumanPMCA4,wasseentocomple-menttheMn-sensitivegrowthdefectofthestrain(83).EarlierstudiesdemonstratedavirtualabsenceofATP-dependent,protonophore-insensitivetransportactivityinGolgivesiclespuriedfromK616(74);thisallowedtheunambiguousbiochemicalcharacterizationofhSPCA1trans-portactivityinyeast.SimilartoyeastPmr1,hSPCA1trans-portedCawithhighafnity(M)andwasinsensitivetoinhibitionbythapsigargin,thediagnosticinhib-itorofSERCA.MnwasaneffectiveinhibitorofhSPCA1butonlypoorlyinhibitedtransportactivityofSERCAandPMCA.InconjunctionwiththeMn-tolerantphenotypesdescribedabove,thesedatasuggestthathigh-afnityMntransportisauniquepropertyofthesecretorypathwaypumps.uorescentprotein(GFP)-taggedhSPCA1localizedtotheGolgiinyeast,demonstratingtheelegantsimplicityyeteffectivenessoffunctionalcomplementation(83).Takento-gether,thesedataledustoconcludethathSPCA1isafunc-tionalorthologofyeastPmr1.Mutagenicanalysesinyeastandhumans.Notonlyisfunc-tionalcomplementationpowerfulinpredictingfunction,itisalsousefulininvestigatinghowmutationsaffectactivity.Withitself,alargenumberofsite-directedmutationsweremadeandrapidlyscreenedforhypersensitivitytoBAPTAand(51,89).Liquidgrowthassaysina96-wellformatareamenabletotestingarangeofinhibitorconcentrations,andgrowthcanbemonitoredbyusingamicroplatereaderforhigh-throughputscreeningofmutants.PhenotypescreeningofPmr1mutantsrevealedresiduesthatarecriticalforionselec-tivityandtransportintransmembranehelicesM4,M5,andM6(51,52).Subsequentinterpretationoftheseresultsinlightofrecenthigh-resolutioncrystalstructuresoftherabbitSERCA1pumpintwodifferentconformations(84,85)showsthatsidechainssensitivetosubstitutionprojectintothetransmembranetransportpathway.IonselectivitymutantsQ783AinM6andV335QinM4appeartoalterconformation-sensitivehelixpackingatthemembraneinterface(51).Insummary,theresultsfromphenotypescreeningofmutantsareremarkablyconsistentwiththecrystalstructureandconstituteexperimen-talvalidationofthehomologymodels.Figure3depictsthelocationofmissenseandnonsensemutationsinhSPCA1identiedinHHDpatients(16,24,36,47,77,96).Althoughchainterminationmutationsarescatteredthroughoutthelengthofthepolypeptide,themissensemuta-tionsarestrikinglyclusteredinregionsoftheproteinsthatarecriticalforfunction(Fig.3).TheseincludemembranehelicesM4,M5,andM6,whichconstitutetheCatransportpathway(Fig.3,),andthemajorcytoplasmicloop,whichconstitutesthephosphorylationandnucleotidebindingdo-mains.ThecarboxylicoxygenofD742directlycoordinatesandhasbeenshowntobeessentialforionbindingandtransportinPmr1(52).MutationofA304(M4)isalsolikelytodisruptionbindingbecausethebackbonecarbonylatthislocationcoordinatesCainSERCA(84).AsubsetofHHDmutationswasintroducedintohSPCA1andassessedforfunctionalcomplementationofBAPTA-and-sensitivephenotypesinK616.MutantsL341P,C411R,T570I,T709M,andD742Yfailedtocorrectthedefects(unpublishedobservations),consistentwiththediseasephenotypeinhumans.Surprisingly,mutantP201Lfullycom-plementedthenullphenotypesandwasindistinguishablefromthewild-typehSPCA1,suggestingthatP201Lmutationdoesnotdisruptpumpfunction.Althoughitremainsapossi-bilitythatsmalldecreasesinactivitymighthavebeenmaskedbyoverexpressionofthemutant,arecentbiochemicalanalysisofthismutantexpressedinCOS-1cellsalsofailedtorevealanydefectsinformationofthecatalyticphosphoenzymein-termediateorinsubcellularlocalization(27).Takentogether,datafromyeastandmammaliancellssuggestthatpatientswithP201Lharboradditionaldisease-causingmutationsinintronsorpromoterregionsof,assuggestedbyLietal.(47).MissenseHHDmutationstransformedintowild-typeyeastdidnotconferhypersensitivitytoBAPTAorMn.Therefore,weconcludedthatthesemutantsdonotexertadominantnegativeeffectontheendogenousPmr1pump.GiventhesignihomologybetweenhSPCA1andPmr1,anextensionofourndingstohumanswouldsuggestthatautosomaldominantinheritanceofHHDlikelyresultsfromhaploinsufciencyofandnotfromadominantnegativeinteractionwiththewild-typeallele.OtherCapumpsinyeast.Therapidaccumulationofcompletegenomesequenceshasledtoanurgentneedforfunctionaldata.FunctionalcomplementationinS.cerevisiaethemethodofchoiceforcharacterizationofnovelgenesfromorganismsrangingfromplantstoprotozoansandmammals.homologsfromotheryeasts(40,66,86)werefoundtocomplementtheS.cerevisiaepmr1mutant.Inaddi-tion,newlydiscoveredSERCA-andPMCA-typeCafromdiverseorganismsalsorestorewild-typephenotypesinyeastlackingendogenouspumpsandhavebeenbiochemicallycharacterized(23,31,48,49,78).HeterologousexpressionofrabbitSERCA1inyeasthasbeenusedtoproduceproteinforbiochemicalanalysisofsite-directedmutants(46).CHALLENGESINTHEYEASTMODELDespitetheadvantageswedescribe,thereremainexperi-mentalchallengestoheterologousproteinexpressionin.Onedifcultyisthatproteinglycosylationinyeastisdifferentfromthatinmammaliancells,soincaseswhereglycosylationisimportantforfunction,thispresentsaproblem.Inaddition,thebiosyntheticqualitycontrolmachineryinisfairlystringent,soexogenousproteinsmayberetainedintheER,possiblyduetodelaysordifcultiesinachievingcorrectfolding.Activationoftheunfoldedproteinresponseandproteosome-mediateddegradationfurtherde-creaseslevelsofthemisfoldedproteins,makinghigh-levelexpressionimpossibleinsuchcases(42).Occasionally,accu-mulationoftheheterologousproteinistoxic,resultinginslowgrowthorevendeathoftheyeasthost.Fortunately,inthesesituations,themethylotrophicyeastPichiapastoriscanbeInvitedReview HETEROLOGOUSEXPRESSIONINYEASTAJP-CellPhysiolVOL287SEPTEMBER2004 substitutedforhigh-levelproteinexpression.growtoaphenomenallyhighdensityonmethanol,andproteinexpressioncanbedrivenbytheinduciblealcoholoxidasepromoter.Additionalincreasesinexpressionlevelsmaybeachievedbyusingasyntheticgeneinwhichheterologouscodonsarereplacedwithpreferredyeastcodons(95,97).isreasonablyamenabletogeneticmanipulationandhastheabilitytoglycosylateproteins,albeitdifferentlyfrom(8,14).Together,thetwoyeastsystemscanbedifferentlyexploitedinmultipleways.maybethesystemofchoiceforgeneratinglargequantitiesofproteinforstructuralstudies,whereascanbeusedtogaininsightintoproteinfunction.Saccharomycescerevisiaeisastraightforwardeukaryoticmodelcellforheterologousexpression.Ithasafullyse-quencedgenome,deletionmutantsthatarereadilyavailable,andexperimentallyamenablegenetics.Manyyeastpathwaysareconservedacrosskingdoms.Bacterial,protozoan,plant,nematode,andmammalianproteinsareoftenfoundtofunc-tionallycomplementyeastknockouts,thusprovidingthe Fig.3.Hailey-Haileydisease(HDD)muta-tionsinhumanGolgi/secretorypathway-ATPase(hSPCA1).:topologicaldispositionofhSPCA1.Nonsense(chaintermination)mutationsidentiedinHHDpatientsarescatteredthroughoutthepolypeptide,whereasmissensemutationsclusterinthetransmembranehelicesknowntobecriticalfortransport(M4,M5,andM6)andinthecentralhydrophilicregionconsti-tutingthephosphorylationandnucleotidebindingdomains.D350isthesiteofcatalyticphosphorylationintermediate.:ho-mologymodeloftransmembranehelicesM4,M5,andM6ofhSPCA1basedonthecrystalstructureofrabbitsarco(endo)plas-micreticulumCa-ATPase(SERCA1)intheCa-boundconformation(84).ModelsweregeneratedinDeepview-SwissPDB-Viewer(GlaxoSmithKline)andrenderedwithPov-Ray3.5andMegaPOV1.0.ThelocationofthesingleCaisbasedoninformationfrommutagenesisofyeastPmr1(51)andtheequivalentresiduesinSERCA1.ResiduesshownarethosemutatedinHHD.:sideviewparalleltolipidbilayer.:viewfromcytosolicface.InvitedReview HETEROLOGOUSEXPRESSIONINYEASTAJP-CellPhysiolVOL287SEPTEMBER2004 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