Purification ofa Plant Mediator from Arabidopsisthaliana Identifies

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MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. 717 for>60%ofthegenome( Blancetal.,2000;Linetal., 1999;Mayeretal.,1999 ).Someduplicationshavebeen followedbyrearrangementsanddivergentevolution,but still,upto40%ofthe A . thaliana genesmightcomprise pairsofhighlyrelatedsequences. TheabilitytoUowerisauniqueandmostinteresting propertyofplants,anditislikelythatseveralofthe $ 700plant-speciTctranscriptionfactorsareinvolvedin thiscentralprocess.InductionofUoweringiscontrolled byenvironmentalfactorssuchastemperatureandlight ( LevyandDean,1998 ).OnepathwaythatcontrolsUower- ingisthewell-characterizedCONSTANS-dependentreg- ulationofUoweringtimethatspeciTcallyrespondsto changesindaylength.However,plantscanalsoperceive changesinlightqualitysuchasadecreaseintheratioof redtofar-redincominglightthatissensedbytheplant asawarningofcompetition.Thiselicitsaseriesofre- sponsesknownasthe 8 8shade-avoidancesyndrome 9 9 wherebystemselongateattheexpenseofleafexpansion, andUoweringisaccelerated( Ballare,1999;Hallidayetal., 1994 ).PhytochromeB(phyB)isoneoutofTvered/far-red lightphotoreceptorsin A . thaliana .Ofthese,phyBhasthe mostsigniTcantroleintheshade-avoidanceresponse, anditisknownthatphyBsignalstoPHYTOCHROME ANDFLOWERINGTIME1(PFT1)toregulatetheexpres- sionofFLOWERINGLOCUST( FT )inresponsetosubop- timallightconditions( Aukermanetal.,1997;Cerdanand Chory,2003;Devlinetal.,1998 ).PFT1wassuggestedto functionasatranscriptionalcoactivatorbasedonthe presenceofaglutamine-richregion,itsnuclearlocaliza- tion,anditsabilitytoactivatetranscriptioninyeastwhen fusedtotheLexADNA-bindingdomain.However,themo- leculardetailsofthephyB-PFT1-FTpathwayarenot known,anditisnotknownwhetherphyBactsdirectly onPFT1orviapromoter-boundtranscriptionalregulators. AninterestingTndingin A . thaliana wasrecentlyre- portedwhenSTRUWWELPETER(SWP)wasdescribed ashomologoustotheyeastandmetazoanMediator subunitMed14( Autranetal.,2002 ).The swp mutantin A . thaliana showsreducedcellnumbersinallaerialor- gans,changesinthewindowofcellproliferation,and perturbationsintheshootapicalmeristem,whichbe- comesgraduallydisorganized.Inaddition,constitutive upregulationofSWPalsointerferedwithproliferation anditwasthereforesuggestedthatthelevelsofSWP bothplayaroleinpatternformationatthemeristemand areimportantindeTningthedurationofcellproliferation duringleafdevelopment. RESULTS Identi5cationofthe A . thaliana MediatorHomologs toMed6,Med7,Med10,Med14,Med15,andMed21 byDatabaseSearches Weinitiallyperformedsimplesearchesofthe A . thaliana genomeforhomologstothe S . cerevisiae Mediatorsub- unitsandcouldthenidentify A . thaliana Med6(AtMed6) (24%identityand41%similarityovera191aminoacids longregion),AtMed7(33%/48%over188aminoacids), AtMed10(26%/50%over139aminoacids),AtMed14 (21%/41%over171aminoacids),AtMed15(26%/38% over132aminoacids,andAtMed21(24%/41%over 139aminoacids)(seethe SupplementalData available withthisarticleonline).Othershavealsoreportedsimilar resultsforadditionalsubunits,butthesequencehomolo- gieshavegenerallybeentoolowtomakeTrmconclusions aboutthepresenceofaplantMediatorcomplex( Boube etal.,2002 ).Inordertoobtaintoolsforfurtherbiochemical studies,weexpressedAtMed6andAtMed7in E . coli ,pu- riTedtherecombinantproteins,andusedthemtoproduce polyclonalantibodies. Copuri5cationofAtMed6andAtMed7 Forisolationofproteinsfrom A . thaliana cellsuspension cultures,weessentiallyfollowedthepreviouslydescribed procedureforisolationofwhole-cellproteinextractsand puriTcationofMediatorfrom S . cerevisiae ( Lietal., 1996 ; Figure1 A).FractionationonBioRex-70showed thattheAtMed6andAtMed7coeluted,thusindicating thataMediatorcomplexmightalsoexistin A . thaliana ( Figure1 B).However,theAtMed6andAtMed7proteins elutedatamuchhigherpotassiumacetateconcentration (1200mM)comparedtotheyeastMediatorcomplex (600mM).Thisindicatedadifferencebetweentheyeast and A . thaliana Mediatorbutalsoresultedinahigherpuri- Tcationrateofthelattercomplex.Wealsofoundthatthe majorityofPolIIinthe A . thaliana whole-cellextracteluted at600mMpotassiumacetate,butaminorfractioncould bedetectedinthe1200mMpotassiumacetateeluate ( Figure1 B). TheAtMed6andAtMed7proteinsalsocoelutedfrom thenextDEAE-Sephacelcolumn,butagainthe A . thaliana Mediatorhomologseluteddifferentlycomparedtothe yeastproteins(inthe200mMeluateinsteadofthe 550mMeluate; Figure1 C).BecausetheelutionproTles ofthe A . thaliana Mediatorhomologsresultedinahigher puriTcationratecomparedtoyeast,wedecidedtoafTnity purifytheanti-AtMed6antibodiestousethemforimmu- noprecipitationofapossible A . thaliana Mediatorcomplex directlyfromthe200mMpotassiumacetateeluateofthe DEAE-Sephacelcolumn.Westernblotanalysisshowed thatAtMed7partiallycoimmunoprecipitatedwithAtMed6 ( Figure1 D).However,wenoticedthatafractionofAtMed7 doesnotseemtointeractwithAtMed6.Thisisshown bothin Figures1 Band1Dandisalsosupportedbyresults describedbelow. Identi5cationofan A . thaliana MediatorComplex Proteinsboundtotheanti-AtMed6proteinASepharose beadswereresolvedbybothone-dimensional(1D)and two-dimensional(2D)gelelectrophoresis( Figures2 A and2B).Thepurposeofthesegelswasnottorevealindi- vidualproteinsbutrathertoseparatethecomponentsfor lateridentiTcationbymassspectrometry(cf.lanes1and2 in Figure2 A).Intotal,136bandsandspotsfromthe1D and2Dgelswereexcised,digested,andanalyzedby 718 MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. MolecularCell IdentiTcationofthe A . thaliana Mediator reverse-phaseliquidchromatography-electrosprayioni- zation-tandemmassspectrometry(LC-ESI-MS/MS)per- formedonacapillaryHPLCcoupledtoaQ-ToFmass spectrometer.Wefoundpeptidesmappingto19Mediator subunits( Table1 ),butonlyTveofthem(AtMed6,AtMed7, AtMed10,AtMed14,andAtMed21)wereannotatedas Mediatorhomologsbythe Arabidopsis Information Resource(TAIR)(see FigureS1 foralignmentsofthe aminoacidsequencesforallidentiTedMediatorsubunits). Sixteenofthe A . thaliana homologsaresharedyeastand Figure1.IsolationofMediatorfrom A . thaliana Whole-Cell ProteinExtracts (A)PuriTcationscheme.Thehorizontallinesindicatestepwiseelution withpotassiumacetateconcentrationsindicatedinmM. (B)ImmunoblotanalysisofBiorex-70fractionswithmonoclonalanti- body8WG16(torevealthelargestPolIIsubunit),andwith1 m g/ml and3 m g/mlofafTnity-puriTedanti-AtMed6andanti-AtMed7. (C)Sameasin(B),butonfractionsfromtheDEAE-Sephacelcolumn. (D)ImmunoprecipitationbyafTnity-puriTedanti-AtMed6antibodycou- pledtoproteinA-Sepharose.Afractionfromthe200mMpotassium acetateeluatefromtheDEAE-Sephacelcolumn(Load,150 m g)wasin- cubatedwithafTnity-puriTedanti-AtMed6antibodybeads.Equal amountsofproteinfromtheloadandsupernatant(Sup)andsixtimes moreproteinfromthepellet(Pel)wereanalyzedbyimmunoblotting withantibodiesagainsttheproteinsindicated. Figure2.Identi5cationofthe A . thaliana MediatorSubunits (A)1Dgelelectrophoresisof A . thaliana Mediator.Elutedproteinfrom theimmunoprecipitationdescribedin Figure1 Dwassubjectedto SDS-PAGE(12%)andvisualizedbycolloidalCoomassiestaining (lane1).Asareference,lane2containsthesameaslane1,except thatno200mMpotassiumacetateeluatefromtheDEAE-Sephacel columnwasadded.Themolecularmassesofstandardproteinsare indicatedtotheright(laneM).Numberstotheleftindicateprotein- containinggelslicessubjectedtoin-geltrypticdigestionandLC- ESI-MS/MS. (B)2Dgelelectrophoresisof A . thaliana Mediator.Elutedproteinfrom theimmunoprecipitationdescribedin Figure1 Dwasseparatedinthe Trstdimensionbyisoelectricfocusingfollowedbyseparationon SDS-PAGE(12%).Estimatesofmolecularmass(kDa)andp I areindi- catedontheordinateandabscissa,respectively.ImmobilizedpHgra- dientwaspH3 310.Numbersoutsidethecirclesindicatespotssub- jectedtoin-geltrypticdigestionandLC-ESI-MS/MS(See Table2 ). MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. 719 MolecularCell IdentiTcationofthe A . thaliana Mediator Table1.MediatorandRNAPolymeraseSubunitsof Arabidopsisthaliana Identi,edbyLC-ESI-MS/MS ProteinName Accession Number Numberof Peptides IdentiTed P max /P tot c Sequence Coverage (%) Protein Size (kDa) Mediator Module IdentiTed inSlice/ Spot b Med4At5g028504/103546Middle24 Med6At3g213504/63529Head10 Med7a d At5g032204/42719Middle22 Med7b d At5g035001/1 a 2319/50 e MiddleNotshown Med8At2g0307011/133258Head9 Med9At1g550804/41629Middle2 Med10a f At5g419101/2 a 1521Middle22 Med10b f At1g266651/1 a 721MiddleNotshown Med11At3g014357/76413Head1 Med14At3g0474016/3526186Middle12 Med15At1g1578012/1518146Tail10 Med16At4g0492016/2221135Tail14 Med17At5g2017014/153474Head12 Med18At2g223702/21123Head21 Med20At2g282301/1 a 525Head19 Med21At4g047802/44815/43 g Middle5 Med22a h At1g164305/87816Head3 Med22b h At1g079505/66117Head4 Med23At1g2323021/2517180NotidentiTed15 Med25At1g255403/3390NotidentiTed26 Med28At3g528602/64018NotidentiTed7 Med31At5g199101/1 a 623Middle23 DNA-directedRNApolymeraseAt3g576606/85188N.A. j 15 DNA-directedRNApolymeraseAt1g299406/87132N.A.14 DNA-directedRNApolymeraseAt1g608506/82342N.A.11 DNA-directedRNApolymeraseAt1g54250 and/or At3g59600 1/1 a 417N.A.27 DNA-directedRNApolymeraseAt2g295401/2 a 1614N.A.18 DNA-directedRNApolymeraseAt3g259402/33014N.A.3 DNA-directedRNApolymeraseAt5g410102/2316N.A.16 Med19a i At5g122302/21226HeadNotshown Med27 i At3g091803/3844NotidentiTedNotshown a Individualfragmentationspectraandsearchresultsareavailableuponrequest. b Theband/spotin Figure2 withthehighestsequencecoverage. c P max ,maximumnumberofuniquepeptides/spotorband.P tot ,totalnumberofuniquepeptidesfromallgels. d TheMed7aandbparalogsdifferin7outof168positionsintheaminoacidsequence(see FigureS2 ). e ThisgeneencodestwodifferentspliceformsofAtMed7b.UsingbothwesternblottingandLC-ESI-MS/MS,weonlyidentiTed peptidesmappingtotheshorterversion. f TheAtMed10aandAtMed10bparalogsdifferin46outof190positionsintheaminoacidsequence(see FigureS2 ). g TheN-terminalsequenceofAtMed21isprobablywronginTAIR.WeonlyidentiTedpeptidesmappingtotheshorterC-terminal version,whichsupportsthegenemodelpresentedinGenBank(accessionnumbergi:4115933). h TheAtMed22aandAtMed22aparalogsdifferin15outof155positionsintheaminoacidsequence. 720 MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. MolecularCell IdentiTcationofthe A . thaliana Mediator metazoanMediatorsubunits,andthreeofthem(Med23, Med25,andMed28)arespeciTctothemetazoanMedia- torcomplexes. Itiswellknownthatevolutionof A . thaliana involvedare- centwhole-genomeduplication,followedbysubsequent lossofgenes.Consequently,wefoundduplicatedgenes encodingparalogsforfour A . thaliana Mediatorsubunits: AtMed7,AtMed10,AtMed20,andAtMed22(see Table1 and FigureS2 ).Laterwealsofoundtwogenesthaten- codeparalogsofAtMed19(seebelow).Inallcasesexcept forAtMed19andAtMed20,ourmassspectrometryanaly- sesofthepuriTed A . thaliana MediatorcomplexidentiTed peptidesthatmaptobothparalogs,indicatingthatboth areexpressedandbuiltintoaMediatorcomplex,even thoughtheydifferinupto46outof189positionsinthe aminoacidsequence(e.g.,AtMed10aandb;see FigureS2 ).Finally,wealsoidentiTedseveralRNApoly- merasepeptidesinourpuriTed A . thaliana Mediatorfrac- tion( Table1 ),indicatingthatwehadisolatedthe A . thali- ana Mediatorintheformofaholoenzymecomplex. Eventhoughwefound A . thaliana homologstothe kinasedomainsubunitsMed12andMed13andseveral homologstobothCDK8andCycCbysearchingthe TAIRdatabase,wewereunabletoidentifypeptidesin ourpuriTedMediatorfractionthatmappedtoanyofthese subunits.Furthermore,wecouldnotTndhomologous sequencesorpeptidesmappingtothesharedyeastand humanMed1andMed19subunits;totheyeast-speciTc Med2,Med3,andMed5subunits;ortothemetazoan- speciTcMed24,Med26,Med27,Med29,andMed30 subunits. The A . thaliana Med14andMed25Subunits AreEncodedby SWP and PFT1 ,Respectively Mostinterestingly,ourbiochemicalpuriTcationofthe A . thaliana MediatorrevealedthatthepreviouslyidentiTed proteinsSWP( Autranetal.,2002 )andPFT1( Cerdanand Chory,2003 )arehomologoustothesharedyeast/meta- zoanMed14andthemetazoan-speciTcMed25 subunits,respectively( Figure3 A).SWPwasalready annotatedasapotentialMed14homologinTAIR,but ourbiochemicalpuriTcationconclusivelyidentiTesSWP asan A . thaliana Mediatorsubunit. OuridentiTcationofPFT1ashomologoustoMed25is uniqueandreliedonthebiochemicalapproach.Inmeta- zoans,Med25hasbeendescribedasbothArc92and ACIDanditwasidentiTedastheMediatorsubunitthat functionsastargetforthewell-studiedherpessimplex virusVP16transcriptionalactivationdomain( Mittler etal.,2003;Yangetal.,2004 ).TheN-terminalpartofthe metazoanMed25interactswithotherMediatorsubunits (aminoacids1 3226;blackboxin Figures3 Aand3B), andinlinewiththeseresultswefoundthattheN-terminal partofPFT1isthemostconservedregionoftheprotein. Furthermore,aBLASTsearchusingtheentirePFT1amino acidsequenceasqueryonlyidentiTedaminoacids13 3 192inhumanMed25(28%identities,42%positives). TheC-terminalpartofhumanMed25(aminoacids402 3 543;blueboxin Figures3 Aand3B)wasshowntointeract withtheVP16activationdomain.WeTndthatthispartis lessconservedbetweenmetazoansand A . thaliana ( Figure3 A).ThehomologiesintheCTDthatTrstseem apparentaremostlyduetothehighcontentofglutamine residuesinthisregion.However,acomparisonof Med25betweendifferentplantsrevealsahighlycon- serveddomainthatoverlapswiththepositionintheplant Med25sequencesthatcorrespondstotheVP16interac- tiondomaininhumanMed25(blueboxin Figure3 B),sug- gestingthatthisdomainofAtMed25mightinteractwith plant-speciTctranscriptionalregulatorproteins. Identi5cationofPlant-Speci5cMediatorSubunits Wealsofoundasetof13proteinsthatcouldnotbeiden- tiTedashomologstoanyyeastormetazoanMediator subunits( Table2 ).Severaloftheseproteinshavebeenim- plicatedintranscriptionalregulationorcontrolofUower- ing,anditisthereforepossiblethatsomeofthemare plant-speciTcMediatorsubunits. At1g44910 encodesaWWdomain-containingprotein, adomainthatisalsofoundintheUoweringtimecontrol proteinFCA( Quesadaetal.,2003 ).Italsoshowshomol- ogytoCA150,anuclearproteinthatisassociatedwith thehumanPolIIholoenzymeandthatisinvolvedinTat- dependenttranscriptionalactivationinHIV-1( Sune etal.,1997 ).At5g42060issimilartothetranscriptional coactivatorp15(PC4)familyprotein( GeandRoeder, 1994 ).PC4isahomologofSUB1inyeastthathasbeen showntointeractwithMed17intwo-hybridexperiments ( Itoetal.,2001 ).At5g64680showshomologytoTRIP12, whichspeciTcallyinteractswiththeligand-bindingdo- mainofthehumanthyroidhormonereceptor( Leeetal., 1995 ).TheclosesthomologofTRIP12inyeastisAsh1, aproteinthathasanegativeeffectonexpressionof HO andshowsgeneticinteractionswithMed21( Hallberg etal.,2006;MaxonandHerskowitz,2001 ).Wealsoiden- tiTedpeptidesmappingtotwoproteinsthathavebeen identiTedastranscriptionfactors.TheTrst,GT-3b (At2g38250),isamemberofafamilyofplant-speciTc trihelix(helix-loop-helix-loop-helix)DNA-bindingfactors thatarepredominantlyexpressedinUoralbudsandroots andbindashomodimersorheterodimerstoGTelements inseveralplantpromoters( Ayadietal.,2004;Parketal., 2004 ).GTelementswereinitiallyidentiTedinthepea rbcS-3Agenepromoterasalight-responsiveelement calledBoxII( Fluhretal.,1986 ).Thesecondtranscription factor,At4g25210,belongstoafamilyofplant-speciTc i Med19aandMed27arepresentedseparateandinboldheretoemphasizethattheseproteinswereidentiTedonlyinthesecond immunoprecipitationexperimentusinganti-At1g11760antibodies(datanotshown). j N.A.,notapplicable. MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. 721 MolecularCell IdentiTcationofthe A . thaliana Mediator Figure3.Identi5cationofPFT1asthe A . thaliana Med25Subunit (A)Mediatorhomologsequencesfrom A . thaliana , O . sativa (gi:115478458), H . sapiens (gi:42765934)and D . melanogaster (gi:21356325)werealigned usingT-coffeeandcoloredwithTeXshade.Red-shadedbackgroundindicatesidenticalaminoacids,orangeindicatesaminoacidsconservedin threeoutoffourspecies,andyellowindicatessimilaraminoacidsinthreeoutoffourspecies.AminoacidsconsideredsimilarwereF/Y/W,I/L/V/ M,R/K,D/E,G/A,S/T,andN/Q.TheblackboxesindicatearegioncorrespondingtothemappedMediator-interactingvonWillebrandfactortype A(VWA)domainofhumanMed25(aminoacids1 3226).TheblueboxesindicatearegioncorrespondingtotheminimalVP16interactiondomain ofhumanMed25composedfromtheresultspresentedby Mittleretal.(2003) and Yangetal.(2004) . (B)SequencealignmentsofMed25fromtheplants A . thaliana , P . trichocarpa ( http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html ), O . sativa , and P . patens ( http://www.jgi.doe.gov/sequencing/why/CSP2005/physcomitrella.html ).Theblackandblueboxesindicatetheregionsintheplant MediatorsequencesthatcorrespondtothesameregionsinthehumanMed25sequenceasindicatedin(A). 722 MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. MolecularCell IdentiTcationofthe A . thaliana Mediator transcriptionfactorsthatallcontainacommoncentral regionsimilartotheGLABROUS1enhancerbindingpro- tein(GeBP)( Curabaetal.,2003 ).Inaddition,theclosest humanhomologtoAt3g10690,topoisomeraseII b ,has recentlybeenshowntoberequiredforregulatedtran- scriptionbyinducingdouble-strandedDNAbreaks( Ju etal.,2006 ).Bydatabasesearches,wefoundthat At1g11760showsweakhomologytoyeastMed17and At1g31360isaRecQDNAhelicase( Hartungetal.,2000 ). WealsoidentiTedasetofproteins(At5g67240, At5g28540/At5g42020,andAt4g25630)intheimmuno- precipitatedfractionforwhichwewereunabletoTndclear connectionstotranscriptionorUowering.Finally,weiden- tiTedpeptidesmappingtotheuncharacterized,plant- speciTcproteinsencodedby At5g63480 , At3g23590 , and At2g48110 .Thelasttwogenesencodeproteinsthat arehighlysimilartoeachother(52%identity/68%similar- ityover1336aminoacids),andwethereforeinterpret themasbeingparalogs.However,becauseAt3g23590 andAt2g48110areidentiTedfromdifferentspotsand becauseweidentifypeptidesmappingtobothproteins weconcludethatbothparalogsarepresentinthepuriTed Mediator. Identi5cationofAt1g11760asan A . thaliana MediatorSubunit An a -At1g11760antiserumwasobtainedbyimmunizing rabbitswithapeptidefromtheCterminusofthe At1g11760protein.Westernblottingoffractionseluted fromthepreviouslydescribedBioRex-70columnshowed thatAt1g11760coelutedwithAtMed6andAtMed7inthe 1200mMpotassiumacetatefraction(Figures 1 Band 4 A). Similarly,wealsofoundthatAt1g11760,AtMed6,and AtMed7coelutefromtheDEAE-Sephacelcolumn(data notshown)andthatAt1g11760coimmunoprecipitates withAtMed6( Figure4 B).Inordertofurthercharacterize the A . thaliana Mediator,the a -At1g11760antibodies wereafTnitypuriTedandusedforimmunoprecipitation experimentsusingthe200mMpotassiumacetateeluate fromtheDEAE-Sephacelcolumn. Figure4 Cshowsthat bothAtMed6andAtMed7arecoimmunoprecipitated withAt1g11760. Proteinsboundtothe a -At1g11760antibody-protein ASepharosebeadswereresolvedby1DSDS-PAGE.In total30bandsfromthisgelwereexcised,digested,and analyzedbyreverse-phaseLC-ESI-MS/MSasdescribed above.Again,weidentiTed19 A . thaliana Mediator Table2.Additional Arabidopsisthaliana ProteinsIdenti,edinthePuri,edMediatorComplexbyLC-ESI-MS/MS ProteinName Accession Number TotalNumber ofPeptides IdentiTed P max /P tot c Sequence Coverage(%) Protein Size(kDa) IdentiTedin Slice/Spot b UnknownAt3g2359014/181514314 UnknownAt2g481104/5414215 PRP40-like/WWdomain-containingproteinAt1g449102/2210914 TopoisomeraseII-likeAt3g106902/2310413 SimilartoRNAexonucleasefamilyproteinAt5g672401/2 a 38813 RecQhelicaseAt1g313604/4679Notshown HSP70similartoyeastSSA1and3At5g28540/ At5g42020 2/487412 SimilartoGeBPAt4g252102/374025 GT-3bAt2g382503/3103420 Fibrillarin2At4g256301/1 a 3339 Thyroidreceptorinteractingprotein(TRIP)12-likeAt5g646804/421228 UnknownAt5g634801/3 a 15206 WeaksimilaritytoyeastMed17andPfamPF01648At1g117604/43216 d 4 Similartotranscriptionalcoactivatorp15(PC4)family protein(KELP) At5g420604/4451117 a Individualfragmentationspectraandsearchresultsareavailableuponrequest.TwoadditionalpeptidesmappingtoFibrillarin2 werealsoidentiTedinthesecondimmunoprecipitationusingtheanti-At1g11760antibodies(datanotshown). b Theband/spotin Figure2 withthehighestsequencecoverage. c P max ,maximumnumberofuniquepeptides/spotorband.P tot ,totalnumberofuniquepeptidesfromallgels. d Themolecularweightofthisproteinismostlikelyerroneouslyannotatedasbeing44kDainTAIR.InNCBI,weidentiTedafull- lengthmRNAencodingthisproteinwithamolecularweightof16kDa.Homologsofthisproteininotherplantsarealsotypically inthe15kDarange.PeptidesweidentiTedthatmappedtothisproteinwereexcisedfrombandsandspotsinthe15kDarange. MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. 723 MolecularCell IdentiTcationofthe A . thaliana Mediator Figure4.Identi5cationofAt1g11760as an A . thaliana MediatorSubunit (A)ImmunoblotanalysisofBiorex-70fractions (asin Figure1 B)withafTnity-puriTedanti- bodiesraisedagainstaC-terminalpeptide fromAt1g11760. (B)Immunoprecipitationbytheanti-AtMed6 antibody.Afractionfromthe200mMpotas- siumacetateeluatefromtheDEAE-Sephacel columnwasincubatedwithafTnity-puriTed anti-AtMed6antibodybeadsasdescribedin Figure1 D. (C)Immunoprecipitationfromthe200mMpo- tassiumacetateeluatefromtheDEAE-Sepha- celcolumn.Proteinfromthisfraction(120 m g foreachimmunoprecipitation)wasincubated withantibodybeads.Thesupernatantwasre- moved,thebeadswerewashed,andimmuno- precipitatedproteinwaseluted.Proteinfrom theload(Load)andeachoftheanti-AtMed6, anti-At1g11760,andanti-GSTimmunoprecipi- tationpelletswasanalyzedbyimmunoblotting withantibodiesagainsttheproteinsindicated totheright.Sixtimesmoreproteinfromthe pelletswasusedinboth(B)and(C). (D)Venndiagramsillustratingtheoverlapbe- tweenconservedMediatorproteins(left)and nonconserved(right)identiTedintheimmuno- precipitationusinganti-AtMed6(solidlines)or anti-At1g11760(dashedlines)antibodybeads. Proteinhitsthatareconsideredparalogsare onlycountedonce. (E)Summaryofcommonandspecies-speciTc Mediatorsubunitsin S . cerevisiae,H . sapiens , and A . thaliana .GrayTeldsindicateconserved Mediatorsubunits,whiteTeldsrepresentsub- unitsthatareabsentinMediatorfromeach species,andblackTeldsrepresentspecies- speciTcsubunits.Accessionnumbersto thedifferent A . thaliana Mediatorsubunitsare indicated. 724 MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. MolecularCell IdentiTcationofthe A . thaliana Mediator homologsintheimmunoprecipitatedfraction.Ofthese,17 wereidenticaltotheMediatorproteinsthatcoimmunopre- cipitatedwiththeAtMed6protein( Table1 and Figure4 D). Inaddition,wealsofoundpeptidesmappingtotwoaddi- tional A . thaliana Mediatorsubunits:AtMed19(At5g12230) andAtMed27(At3g09180)( Table1 ;inboldstyle).How- ever,inthisexperimentwewereunabletoTndpeptides mappingtoAtMed7andAtMed21.Itisknownthat Med7andMed21interactandformasubcomplexinthe yeastMediator,andtheymightthereforebelostasasub- complexfromMediatorinthispuriTcation( Kangetal., 2001 ).Thisisalsoinlinewiththeresultsshownin Figure1 , whereonlyafractionofAtMed7iscoimmunoprecipitated withAtMed6.Intotal,theseresultsshowthatAt1g11760is abonaTdeMediatorsubunitspeciTcfor A . thaliana . AccordingtothesuggestednewuniTednomenclature forMediatorproteins,At1g11760shouldthereforebe namedMed32. Identi5cationofSixUnique A . thaliana Mediator Subunits InadditiontotheMediatorsubunithomologs,ourimmu- noprecipitationusingthe a -At1g11760antibodyandthe followingLC-ESI-MS/MSanalysisagainidentiTed peptidesthatmaptosixofthepotentialplant-speciTc Mediatorsubunitsthatcoimmunoprecipitatedwiththe anti-AtMed6antibody.Therefore,sixofthe14proteins thatwereidentiTedaspotentialMediatorsubunitsin Table 2 wereagainidentiTedinthissecondimmunoprecipitation ( Figure4 D).Becausetheseproteinscoimmunoprecipitate withtwoindependentMediatorsubunits,weconsidered themasuniqueplantMediatorsubunitsandaccordingly wesuggestthattheyarenamedMed32 3Med37 (At1g11760,Med32;At3g23590/At2g48110,Med33a/ Med33b;At1g31360,Med34;At1g44910,Med35; At4g25630,Med36;andAt5g28540/At5g42020,Med37; see Table1 and Figure4 E).Oftheseproteins,Med32, Med33a,andMed33bareallencodedbygenesthatare onlyfoundintheplantkingdom. DISCUSSION MediatorwasoriginallyidentiTedasacentraltranscrip- tionalcoregulatorinyeast( Flanaganetal.,1991;Kelleher etal.,1990;Kimetal.,1994 ),butitisnowevidentthatit playsthesameimportantrolealsoinhighereukaryotes suchas S . pombe , C . elegans ,and D . melanogaster , andinhumancells( Bourbonetal.,2004 ).Itispuzzling thattherehasbeennoreport,toourknowledge,ofaMe- diatorcomplexinplantssofar,butouridentiTcationhere ofMediatorisolatedfrom A . thaliana givesanexplanation tothisconundrum.WeTndthattheplantMediatorsub- unitsgenerallyshowverylowsequencehomologytothe correspondingsubunitsfrombothyeastandmetazoans, eventhoughmostoftheyeastandmetazoansubunits arepresentalsoinplants.Thislowhomologyisnotso surprisingconsideringthatthe A . thaliana genomeis estimatedtoencode $ 1700transcriptionfactors,ofwhich $ 700arespeciTcforplants( RiechmannandRatcliffe, 2000 ).Sincetheseplant-speciTctranscriptionfactors willexecutetheirfunctionthroughMediator,itisrather expectedthattheplantMediatorsubunitsshoulddiffer fromtheyeastandhumanMediatorsubunits. Intotal,weidentify21conserved A . thaliana Mediator subunitsthroughbiochemicalpuriTcationandLC-ESI-MS/ MS.WewereunabletoTnd A . thaliana homologstothe conservedMed1subunit;theyeast-speciTcMediator subunitsMed2,Med3,andMed5;andthemetazoan- speciTcMed24,Med26,Med29,andMed30subunits.It thereforeseemslikethe A . thaliana Mediatorcontains nearlyallsubunitsthatconstitutetheheadandmiddle domains,whichinyeastarelocatedmostproximalto thePolII.However,itlackshomologstomostofthesub- unitsthatconstitutetheverytipofyeastMediatortail domain,whichhasbeensuggestedtointeractwithpro- moter-boundregulatoryproteins(activatorsandrepres- sors).Thus,byanalogy,wehypothesizethatatleast someofthemetazoan-speciTcMediatorsubunits (Med24,Med26,Med29,andMed30)constitutethetail domainofthemetazoanMediator.Alternatively,these subunitscouldbelocatedinotherdomainsofMediator, butmostlikelytheyshouldbeexposedonthesurfaceof thecomplex.Thetaildomainisthepartthatisleast conservedbetweentheyeastandmetazoanMediator, anditisthereforelikelythatthe A . thaliana Mediatoralso comprisesaplant-speciTc,taildomain.Insupportofthis idea,weidentifysixnewplant-speciTc,Mediatorsub- unitsthatwenameMed32 3Med37(see Figure4 Efor acomparisonoftheyeast,human,and A . thaliana Media- torsubunits). InbothafTnitypuriTcationsofMediatorpresentedhere, weTndthatthepreviouslydescribedMed7-Med21dimer isabsentfrom,oronlypartiallyassociatedwith,the A . thaliana Mediator.Oneexplanationfortheseresultscould bethattheantibodiesusedforimmunoprecipitationmight interferewiththeinteractionbetweenMed7/Med21and therestofMediator.Thisisapossibleexplanationinthe immunoprecipitationexperimentusinganti-AtMed6anti- bodies,sinceithasbeenshownthatthereisadirect bindinginteractionbetweentheyeastMed6subunitand theMed7-Med21dimer( Baumlietal.,2005 ).However, theresultswepresenthereshowingthattheMed7-Med21 dimeriscompletelyabsentfromMediatorimmunopuriTed usinganti-AtMed32antibodiesargueagainstthesug- gestedhypothesisthatMed7-Med21wouldformaUexible hingethatconnectstheheadandmiddledomainsof Mediator.IfMed7-Med21wouldformsuchahinge,itis difTculttoexplainthatourpuriTed A . thaliana Mediator thatlacksbothMed7andMed21wouldstillcontainsub- unitsfromboththemiddleandheaddomains. InadditiontoMediatorsubunits,wealsofoundRNA polymerasesubunitsinourpuriTed A . thaliana Mediator fraction.Itisthereforenotsurprisingthatwewereunable toTndpeptidesmappingtothekinasedomainsubunits CDK8,CycC,Med12,andMed13inourpuriTedMediator fractiondespitethefactthatweTndseveralpotential MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. 725 MolecularCell IdentiTcationofthe A . thaliana Mediator homologstotheseproteinsusingBLAST.Inyeast,the kinasedomainisonlyassociatedwithfreeMediatorand notwithMediatorboundtoPolII( Samuelsenetal., 2003 ).Interestingly,thegenethatweidentifyasthe A . thaliana Med12homologisreportedasCRYPTIC PRECOCIOUS( CRP ).CRPhasbeenreportedtobein- volvedintheregulationofUoweringtime,anda crp mutant enhancestheexpressionoftheUoweringtimegene FT .A knockoutmutantof CRP withlate-Uoweringphenotype hasbeendescribed(Y.Kobayashietal.,2002,TAIR, abstract, http://www.arabidopsis.org/servlets/TairObject? type=publication&id=501708911 ). Avariantofthedivisionofcoreandperipheralpartsof Mediatorisalsofoundwithinonesinglesubunit,namely Med25.WeshowthattheN-terminaldomainofMed25, whichinteractswithcoreMediatorinhumancells,iscon- servedalsoin A . thaliana .However,amoreC-terminal domainofhumanMed25interactswiththetranscriptional activatorVP16.Thisdomainisconservedbetweenmeta- zoansbutisnotconservedin A . thaliana .Instead,the correspondingC-terminalregionisconservedbetween differentplants( Figure3 B).Thus,itseemslikeMediator hasevolvedtoadapttospecies-speciTctranscriptional regulatorsintwoways,eitherbyincorporatingcompletely newsubunitsorbyexpressingsubunitscomposedof oneconserved,Mediator-interactingdomainandone species-speciTc,regulator-interactingdomain. Itisinterestingthatweidentifypeptidesthatmapto differentparalogsoftheAtMed7,AtMed10,AtMed19, AtMed20,andAtMed22subunitsinourpuriTed A . thaliana Mediator.BecausewepuriTedthe A . thaliana Mediator fromacellsuspensionculture,weareunabletodetermine whetherdifferentparalogsarepresentinthesamecom- plexesorwhethertheynormallyareevenexpressedin thesamecellorcelltype.ItispossiblethatMediatorcom- plexescontainingdifferentparalogsdifferinfunctionand thatdifferentparalogsareexpressedindifferenttissues orindifferentdevelopmentalstages.Tissue-orcell- speciTcfunctionsforMediatorhavebeenreportedprevi- ously.Forexample,Med1hasbeenidentiTedascritical forthedevelopmentoftheplacentaandforthenormal embryonicdevelopmentoftheheart,liver,eye,vascular, andhematopoieticsystems( Itoetal.,2000;Crawford etal.,2002 ).GeneticablationofMed24resultsincardiac hypoplasia,narrowbloodvessels,andabnormalCNSde- velopment( Itoetal.,2002 ).Finally,Med12isrequiredfor developmentofthebrain,neuralcrest,andkidney( Hong etal.,2005 )andbothMed12andMed13arerequiredto establishnormalcellafTnitydifferencesattheanterior- posterioranddorsal-ventralcompartmentboundariesof thewingdiscin Drosophila ( Janodyetal.,2003 ).These resultscouldbeinterpretedasifdifferentcelltypesor tissuescontaindifferentcompositionsofMediatorsub- units,andvarioussubunitcompositionswerealsoorigi- nallyreportedfordifferentpuriTcationsofmammalianMe- diators.Evenifthispossibilityisnotcompletelyruledout, therecentidentiTcationofacommonsetof32mammalian subunitsinsixdifferentmammalianMediatorpreparations usingMudPITrathersuggeststhatMediatorcomposition issimilaroridenticalinmostcelltypes( Satoetal.,2004 ). Plantshaveevolvedsophisticatedsystemstosense changesinlightqualitythroughthecombinedactionof asuiteofphotoreceptors.Amongthese,thered/far-red- absorbingphytochromes(phyA 3phyE)arethebestchar- acterized( Aukermanetal.,1997;Devlinetal.,1998 ).In A . thaliana ,PFT1isinvolvedinaphyB-dependentpath- wayresponsiblefortheregulationofUoweringtimeinre- sponsetochangesinlightquality.Thispathwayappears tobecompletelydifferentfromthewell-characterized CONSTANS-dependentregulationofUoweringtimethat speciTcallyrespondstochangesindaylength.However, bothpathwaysleadtotheinductionoftheUowering activator FT geneinleaves. PFT1wasoriginallysuggestedtofunctionasatran- scriptionalcoactivatorbasedonthepresenceofagluta- mine-richregioninitsaminoacidsequence,itsnuclear localization,anditsabilitytoactivatetranscriptioninyeast whenfusedtotheLexADNA-bindingdomain( Cerdanand Chory,2003 ).Itwas,however,unclearhowPhyB,PFT1, andFTarelinked.OurpuriTcationofMediatorfrom A . thaliana andouridentiTcationofPFT1asAtMed25 thusprovideanessentiallinkintheseriesofeventslead- ingfromacytoplasmicphotoreceptortotheinductionof genesthatpromoteUowering.Therefore,wesuggest thatPFT1isthetargetinMediatorforaCONSTANS- independentactivationpathwayof FT transcription.This pathwaycouldeitherbedirectfromphyBtoPFT1,orvia transcriptionalregulatorsboundtothe FT promoter ( Figure5 ). OuridentiTcationofSWPasasubunitofMediatorin A . thaliana isinterestinginseveralaspects.Itisshown thatmutantscarryingloss-of-functionmutationsinSWP havereducedcellnumbersinaerialorgansandoverex- pressionofSWPleadstoanincreasednumberofsmall Figure5.AProposedModelforAtMed25/PFT1Functionin theLight-QualityPathwaythatRegulatesFloweringTime Theredtofarred(R/FR)ratioofincominglightissensedbyphyBin leaves.Thissignalistransmittedtothe A . thaliana Mediatorcomplex, eitherbydirectactionontheAtMed25subunitorindirectlyviapro- moter-boundtranscriptionalregulatoryproteinstoaffecttranscription oftargetgenes,hererepresentedby FT . 726 MolecularCell 26 ,717 3729,June8,2007 ª 2007ElsevierInc. MolecularCell IdentiTcationofthe A . thaliana Mediator cellsinclusters( Autranetal.,2002 ).Itwastherefore proposedthatSWPplaysanimportantroleindeTning thedurationofcellproliferationin A . thaliana .Inyeast, Med14wasoriginallyidentiTedasResistanttoGlucose Repression(Rgr1).Interestingly,itwasalsoreportedthat eventhoughthe rgr1 mutantcellsdidnotshowcell-cycle phenotypes,theyshowedallofthemorphologicalabnor- malitiesassociatedwithcdcmutations( Sakaietal.,1990 ). Inmetazoans,itwasfoundthatdeveloping C . elegans embryoshasabroadMed14requirementforbothtran- scriptionandphosphorylationofSer-2andSer-5ofthe PolIICTD( Shimetal.,2002 ). OurbiochemicalpuriTcationofMediatorfrom A . thaliana issigniTcantinseveralaspects.First,theiden- tiTcationofSWPandPFT1asAtMed14andAtMed25, respectively,emphasizesthecentralimportanceof Mediatorinintegrationofimportantsignalingpathways inplantcells.Italsoprovidesatestablehypothesisof howcellsrespondtoexternalstimuli(lightquality)atthe molecularlevelbyregulationofexpressionoftargetgenes. Ourresultsshowthattheheadandmiddledomainsof Mediatorareconservedinalleukaryotes.However,our identiTcationofsix A . thaliana -speciTcMediatorsubunits indicatesthatMediatorsindifferentorganismscontain uniquedomainsthatarespeciTcallyadaptedtointer- actwithorganism-speciTctranscriptionalactivatorsand repressors. EXPERIMENTALPROCEDURES A . thaliana CellSuspensionCultureandProteinPuri5cation A . thaliana cellsuspensioncultures(ecotypeColumbia)weregrownin MSAR( Konczetal.,1994 ).Cellsweremaintainedbyweeklysubcultur- ingof7.5mlsaturatedcultureinto42.5mloffreshMSARandgrownat 24 Cundercontinuouslightonagyratoryshakerat120rpm.They werethenharvestedbyvacuumTltration,frozeninliquidnitrogen, andstoredat À 80 C.Whole-cell A . thaliana proteinextractswereiso- latedessentiallyaccordingtoamethodusedforpreparationofwhole- cellextractsfromyeast( Lietal.,1996 ).BrieUy,450gof A . thaliana cells wasdisruptedusingaFreezer/MillModel6850(SPEXcertiprep)and thehomogeneouscellpowderwasdissolvedin250mlof3 3 lysis buffer.Aftercentrifugation,theclearsupernatant(6.3gofprotein) wasappliedtoa350mlBiorex-70column(Bio-Rad).Thecolumn waswashedwith700mleachofbuffersA-0.15,A-0.3,andA-0.6, andtheAtMed6andAtMed7subunitsofMediatorwereelutedwith bufferA-1.2.Thiseluate(225ml,184mg)wasdialyzedandbound batch-wiseto25mlDEAE-SephacelequilibratedwithbufferB-0.1 for45minat4 C.TheDEAE-Sephacelwaspackedinacolumnand washedwith25mlofbufferB-0.1,andtheAtMed6andAtMed7sub- unitsofMediatorwereelutedwith50mlB-0.2.ProteinfromtheB-0.2 peak(20ml,12mg)wasfrozeninliquidnitrogenandstoredat À 80 C. TrypticDigestionandPeptideAnalysiswithNanoLC-MS/MS In-geltrypticdigestsperformedinasimilarmannertothatdescribed previously( Pandeyetal.,2000 )wereanalyzedbyreverse-phaseLC- MS/MSperformedonacapillaryHPLCcoupledtoaQ-TOFmass spectrometer(CapLCQ-TOFUltima,WatersCorporation)( Lovgren etal.,2004 ).TheacquisitionofMS/MSspectrawasperformedwith anautomateddata-directedswitchingbetweentheMSandMS/MS modesusingtheinstrumentsoftware(MassLynxV4.0SP4).Thethree mostabundantsignalsofasurveyscan(400 31500m/zrange,0.87s scantime,and0.13sinterdelay)wereselectedbychargestate,and collisionenergywasappliedaccordinglyforsequentialMS/MSfrag- mentationscanning(50 32000m/zrange,0.9sscantime,0.1sinter delay).Accuratemassdeterminationoftheionfromdodecamethyl cyclohexasiloxane(C 12 H 36 O 6 Si 6 ,m/z445.1206)( Schlosserand Volkmer-Engert,2003 )wasusedforanoffsetcalibrationoftheMS channelbycombining30scans(scans30 359)ofthebackground signal.Conversionofrawdatatopeaklistsfordatabasesearching wasperformedwiththeProteinLynxGlobalServersoftware(V2.0.5). ProteinIdenti5cationandDataAnalysis ProteinswereidentiTedbyalocalversionoftheMASCOTsearchpro- gram(v2.1.04;MatrixScienceLimited, http://www.matrixscience. com )andtheMascotDaemonapplication(V2.1.6)usingan A . thaliana taxonomydatabasefromTIGR(version5;27,855entries),andsup- portingsearcheswereperformedwithinProteinLynx.Thefollowing settingswereusedforthedatabasesearch:trypsin-speciTcdigestion withonemissedcleavageallowed,carbamidomethylatedcysteineset asTxedmodiTcation(onlyfor2Dgels),andoxidizedmethionineinvari- ablemodiTcation,peptidetolerancesetto40ppmandfragmenttoler- ancesetto0.08Da.PeptideswithMascotionscoreexceedingthe thresholdforstatisticalsigniTcance(p<0.05)wereselected.Single peptidesmatchingaproteinweremanuallyreprocessed,inspected, andeitherinterpretedasvalidordiscarded.Additionalsearchesin theNCBIdatabase(downloadedAugust28,2006,3,952,414entries) werealsoperformedtoidentifycontaminations(keratin)andimmuno- globulinusedintheextraction. Identi5cationofHomologsandProteinAlignments AllproteinsidentiTedwerescannedagainsttheNCBIdatabaseusing iterativepsi-blastbyincludinghomologsfromtheplants O . sativa , M . trunculata ,and/ortheslimemold D . discoideum ( Altschuletal., 1997 ).AlignmentsandvisualizationsweredoneusingtheT-coffee ( Notredameetal.,2000 )andTeXshadeprograms( Beitz,2000 ). AdditionalMethods Methodsforcloning,expressionandpuriTcationofrecombinantpro- teins,antibodyproduction,afTnitypuriTcationofantibodies,2Dgel electrophoresis,andimmunoprecipitationareprovidedinthe Supple- mentalData onthe MolecularCell website. SupplementalData SupplementalDataincludetwoTguresandSupplementalExperimen- talProceduresandcanbefoundwiththisarticleonlineat http://www. molecule.org/cgi/content/full/26/5/717/DC1/ . 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