取得学位：博士(薬学），学位授与番号：博甲第1034号，学位授与年月日：平成20年3月22

Hasina Akhter

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取得学位：博士(薬学），学位授与番号：博甲第1034号，学位授与年月日：平成20年3月22日othe

Hasina, Akhter

60381

Kanazawa University Repository for Academic Resources

氏名学位の種類学位記番号学位授与の日付学位授与の要件学位授与の題目ＨＡＳＩＮＡＡＫＨＴＥＲ博士（薬学）博甲第１０３４号平成２０年３月２２日課程博士(学位規則第４条第１項）CharacterizationofArfGAP1andFmGERWFmGER8interactionbyquantitativeyeasttwo-hybridanalysis（定量的酵母ツーハイブリッド法によるArfGAP1とFinGER7/ＦｍＧＥＲ８の相互作用の解析）中村暢宏（自然科学研究科・准教授）中西義信（医学系研究科・教授)，松永司（自然科学研究科・教授)，横井毅（医学系研究科・教授)，山下克美（自然科学研究科・准教授）論文審査委員（主査）論文審査委員（副査）AbstractByusmgaquantitativemethodfOryeasttwo-hybridanalysis，wetriedtoidentifymteractmgreglonsofAjrfGAP1,aGTPaseactivatmgprotcmfbrArLandFInGER7andFinGER8,familymcmbersoffivespantransmembraneproteinslocalizingintheGolgiapparatusandERnleN-teminalregion(1-152)containingaGTPaseactivationdomain,↑andtheC-temmnalregion(256-406)ofArfGAP1wasdispensableandthecentralregion(153-257)reportedtofUnctionfOrsensmgmembranecurvatureandalsofOrtargetingtotheGolgiapparatuswasnecessaryandenoughfOrtheinteractionwithHnGER7orFHnGER8・Ａｍｕｔationof211thtryptophanofArfGAP1toalaImnethatdisruptstheGolgitargetmgandlipidcurvaturesensmgabolishedthcinteractionwithFinGER7orFinGER8suggestingtherolcofHnGER7andFinGER8fOrthetargctingofArfGAP1totheGolgiapparatus・meseverereductionoftheinteractionwasobservedbydeletionoftheN‐tenninal31stto41stresiduesofFinGER7showingtheimportanceofthisrcgion(31-41）fOrthemtcraction・DeletionofanypartsoftheC-tennmaltransmembranesegmentscompletelyabolishedtheinteraction・ReplacementofthecytoplasmicreglonorthetransmembraneregionofHnGER7byFinGER8didnotrecreatetheoriginalhigller-1evelmteractionsuggestmgthatthecytoplasmicreglonandthetransmembraneregioncooperatcfbrthemteractionwithArfGAP1．-548-IntrOductionTheGolgiapparatusissituatedatthecentcrofthevesiculartransportpathwayltrecelvessecretoryandtransmembraneproteinsfromthcER,processes，sortsandsendsouttothefinaldestmations・TransportvcsiclesarcproducedbyCOPII，ＣＯＰＩａｎｄｃｌａｔｈｒｉｎｃｏａｔａｔｔｈｅＥＲ，ｔｈｅＧｏｌｇｉａｐparatusandplasmamembrane，respectively・CoatprotemsafterrecruitmentontothemembranebyactivatedArfl/Sarl，defmnflatmembranesintosphericaｌｂｕｄｓｔｈａｔｆｉｎａｌｌｙｐｍｃｈｏｆｆｔｈｅｍｅｍｂranefOrmingtransportvesicles・ActivationｏｆＡｒｆｉｓｍｅｄｉａｔｅｄｂｙＡｒｆｓｐｅｃｉｆｉｃｇｕanlnenucleotideexchangefactor(GEF)IWosubfamiliesoflargeArfGEFsoperateattheGolgicomplex,BIGｓ(BrefeldminhibitedGEFs)andGBF1(GolgispecificBrefeldinAresistancefactorl).InvesiculartransportmediatedbyＣＯＰＩｃｏａｔ,theGTPaseactivityofArflisstimulatedbyArfGAP1，ａＧ１[Paseactivatmgprotem,thatcatalyzcstheprerequisitestepofuncoatingofvesicles・ArfGAPsconstitutealargefanlilyofproteinscontammgahigj11yconServe。(fromyeasttomammals)catalyticdomamof~l30residues(GTPaseactivationorGAPdomam)AcentralregionofArfGAP1(residuel9Z-231)temedasALPSdomainactsasalipid-paCkingsensorandhelpsanchorArfGAP1atthesurfaceofthehigl11ycurvedmcmbrane,ｔｌｍｓａｌｌｏｗｍｇｄ[PhydrolysisonArfl・Ypt/rabsareknowntoregUlatevesiclebuddingand/orfUsionatthevariousstepsofvesiculartransportpathway・Ｉｎｍａｍｍａｌｓ，rablregulatesERtoGolgiandintra-Golgiproteintransportwhereasrab6isinvolvedintransportprocessesbetweencndosomesandtrans-Golginetwork(TGN)andGolgitoERtransport・hyeast,YptlpisthougjlttoplayakeyrolemthetetheringofER-derivedtransportvesiclestothcGolgiapparatus・YiplpisanYPtlp-interactingprotcinrequiredfOrtransportthroughtheearlysecretorypathwayandYiplplikeFinGERfamilyproteinshasbeenidentifiedinhumanswhicharefive-spantransnlembraneproteinslocalizmginthcERandGolgi・EightfamilymemberswerefOundi、human(FmGER1~FinGER8）andtheyareamexpressedwidelyinvarioustissues・ItwasreportedpreviouslythattheGolgiapparatusisfragmenteｄｂｙｔｈｅｏｖｅｒｅｘｐｒｅｓｓｉｏｎｏｆＦｉｎＧＥＲ１，２，３，４ａｎｄ７－５４９－suggestingtheirfUnctionmthemamtenanceoftheGolgistructureand/OrthetransportbetweentheERandtheGolgiapparatus・YiplpisaputativeyeastorthologJｌｅｏｆＦｉｎＧＥＲ４ａｎｄＦｉｎＧＥＲ５・ＹｉｆｌｐｉｓｔｈａｔｏｆＦｍＧＥＲ３，FinGER7，ＨｎＧＥＲ８、YiplpandYiflpareessentialyeastgeneproductslocalizedintheGolgiapparatusThelossoffUnctionoftheseprotcinscausesablockofthcERtoGolgitransportandacculnulationofERmembranesII1eseproteinsinteractwithYptlpandYPt31p,suggestmgtheirmvolvementinvesicledoCkingandfUsion・Supportmgthispossibility,ｉｔwasreportedtllatYiplpandYiflpwerenecessarytofOrmvesiclescompetentfOrfUsionwiththeGolgiapparatus、Ontllccontrary，itwasalsoreportedthatYiplpfUnctionsmvesiclebuddmgfromtheERbutnotinthefUsionwiththeGolgiapparatus,ThcrefOre,itremainsobscurewhetherYiplpandYiflpfUnctionmvesiclebuddmgand/orfilsionsteps、ResultｓＴｏｅｘｐｌｏｒｅｔｈｅｆｉｍｃｔｉｏｎｏｆＦｉｎＧＥＲｐroteinsinthesecretorypathway，itwastriedtoidentifytheirinteractmgpartner(s).Aquantitativemcthodofyeasttwo-hybridanalysiswasdevelopedandusmgthisapproach,theinteractionofalltheFinGERswithrabl,rab6，ＧＢＦ1,ArfGAP1waschecked・NosigmificantlevelofinteractionwasdetectedfOrallthoscFinGERswithrabl,ｒａｂ６ａｎｄＧＢＦ１（unpublishedobscrvation).Interestingly，ArfCAP1showedreproducibleinteractionsignalwithHnGER7andFmGER8(Ｈｇl)．伽獅０釦加釦伽切０帥２２１１｛の一言。〉澄冨石二個①．ＲＦ２Ｆ３Ｆ４Ｆ５Ｆ６Ｆ７Ｆ８ＮｏｎFigurelSpecifHcinteractionofArfCAP1witllFinGER7/FinGER8Quantitativeyeasttwo-hybridanalysiswasperfOrmedandthemeanactivitiesoftriplicateassaysareshown(bars)withSD(errorbars)．-550-」■AＤＩ了■ＡＤｐＢＤⅡ玉□＿＿●－日＿＿＿＿＿＿＿＿＿ロ‐＿＿＿＿■■ 可一 ■Inthedeletionmappmgexperiments,thercgloncontainingthel53rdto257thresiduesfOundnecessaryandsufficicntfOrthemteractionofArfGAP1withFinGER7orFinGER8(Ｆｉｇ２).since△N192-AC231（ALPSdomainonly),△136ArfUAP1(withoutthcGAPdomam)didnotshowinteractionwitheitherFmGER7orFinGER8・TherefOre，GAPdomainisdispensablcandthcALPSregiononlyisnotenoughfOrtheinteractionALPSaswellas～４０residuesupstreamofALPSand二OresiduesdownstreamofALPShaveapivotalrolemthisprotem-proteinmteraction,AmutationofZ11thtryptophanofArfGAP1toalaninethatdisruptstheGolgjtargetmgandlipidcurvaturesensingabolishedtheinteractionwithHnGER7orFinGER8suggestingtheroleofFinGER7andFinGER8fOrthetargetmgofArfGAP1totheGolgiapparatus．ＣＡ ＢResiduesO１００２００３００４００GaIActivity(Units）GaIActivity(Units）0２５０５０００２００４００ＡＡ鰄醐翻麺辨麺馴剛棚醐柵州州岼》》“』剛》棚Ｍ》Ｆ・△ＡＡＡＡＡＡ△△ＡＡ△△Figure21nteractionofArfCAP1mutantswitlnFinGEIMFinGER8(A)SchematicreprcsentationofArfGAP1mutants､AGTPaseactivationdomain(GAP）ａｎｄＡＬＰＳｄｏｍａｍｓａｒｅｓｈｏｗｎｍｔｈｅｔｏｐｆＵlllengthArfGAP1，theW211AmutationisindicatedbytheletterA.(B)Quantitativcyeasttwo-hybridanalysiswaspcrfOnnedandtherawactivityisshown・TriplicatcassayswereperfOnnedandthemeanactivitiesareshown(bars)ｗｉｔｈＳＤ(errorbars).(C)Thcresultsaftersubtractionofthcbackgroundactivityareshown・Thepropagationoferror(SD)wascalculatedandisshown(errorbars)．TheresultofdeletionanalysisofHnGER7showedscverereductionoftheinteractionbydeletionoftheN-terminal31stto41stresiduesimplyingtheimportanceofthisreglon(31-41)forthemteractionwithArfGAP1(Fig3).DcletionofanypartsoftheC-tenninal-551-凸凸占＝＝凸ｺﾏ --ヨ÷－－－門ヨ甲一ヨＩ:＝＝＆エョ:1１山里三増．、二･2．ｚ１．Ｆヨ■ヨ-１■F７口F８凸酋占~一四丁■■■ 、=＝肩r、ヨ子劃０回二二丁＝＝字Ｆ‐丁■■閉囚■■画司‐’■F７口F８□Ｎｏｎtransmembranesegmentscompletelyabolishedtheinteractionsuggestingtheimportance0oftransmcmbraneregionfOrtheinteraction．Ａ ＢResidueslOO２００GalActivity(UnIts）300０１００２００３００４０００ＮｏｎＦｕｌｌＡＮ１１ＡＮ２１ＡＮ２５ＡＮ３１ＡＮ４２ＡＮ５９；■■■■■■■■■■△N113i■■■■■■■AN137■■■■■■ＡＣ136￣；４．１“■凸－１△…■■■て￣．AC22gＡＣ１３６ＡＣ１６４△Ｃ１ｇ６ＡＣ２２ｇＡＣ２５５Figue3InteractionofFinGER7deletionmutantswithArfCAP1(A)SchematicrepresentationofHnGER7mutants､PutativetransmelnbrancsegmentsareshowninthetopfUlllengthFinGER7.(B)Quantitativeyeasttwo-hybridanalysiswasperfOnncdasabove・TriplicateassayswcreperfOmedmorethanthreetimesandthemeanactivitiesareshown(bars)withSD(errorbars)．ItispossiblethatdeletionofanypartsofthetranslnembraneregloncouldpreventFinGER7fromproperlyinsertmgintothemembrane，takeaproperconfOnnationortopology，orbetransportedtothecorrectsubcellulardestinationfOrthcinteractionTherefbre，ｉｔｗａｓｔｈｅｎｔｒｉｅｄｔｏｒｅｐｌａｃｅｔｈｅｗｈｏｌetransmembranedomainofFInGER7witllthatofotherFinGERproteins(FinGER8,FinGER5,FinGER3)butreplacementoftheputativecytoplasmicreglonorthetransmembraｎｅｒｅｇｉｏｎｏｆＦｉｎＧＥＲ７ｂｙＦｉｎＧＥＲ８ｄｉｄｎotrecreatetheoriginalhigher-1evelinteractionsuggestingtheco-operationofcytoplasmicandtransmembranedomainsfbrtheinteractionwithArfGAP1(Fig4)．-552-Ⅳ 画十ヨ400350300（圏這。｝声潭三】ｃｍ－ｍｃZ5０ＺＯＯ1501００5００FＷＦ８Ｆ８/F７Ｆ７/Ｆ５Ｆ５ﾉF７ＦｱﾉＦ３Ｆ３/F７F８F７Figure41nteractionofchimericmuｔａｎｔｓｏｆＦｉｎＧＥＲ７ａｎｄＦｉｎＧＥＲ８ｏｒＦｉｎＧＥＲ５ｏｒＦｉｎＧＥＲ３ｗｉｔｈＡｒｆＧＡＰ１Ｐｕｔａｔｉｖecytoplasmic（1-111）ortransmembrmeregions（l1z-293)ofFinGER7wereswappedwithhomologousreglonsofFmGER8(1-126,127-311)orFmGER5(1-112,113-257)ｏｒＦｍＧＥＲ３（1-138,139-350).Quantitativeyeasttwo-hybridanalysiswasperfOrmedasabove・rriplicateassayswereperfOrmedandthemeanactivitiesareshown(bars)withSD(errorbars)．CmcuusimItistherefOreimplicatedthatFinＧＥＲ７ａｎｄＦｉｎＧＥＲ８ａｒｅｉｎｖｏｌｖｅｄｉｎｔｈｅCOPIvesiclefOnnationorcargosortingintoaCOPIvcsicle・hthisaspect，thefactthatFinGER7mtcractswithArfGAP1atthelipidcurvaturesensmgdomamoverlappingwiththeGolgitargetingdomamappealsfOritssignificance.ＩtispossiblethatHnGER7andHnGER8controltheactivityofArfGAP1tomodulatetheactivationofArflleadingtothcstimulationormhibitionoftheCOPIcoatasscmblyonGolgimembranes．-553-1１ 】８ＵＯｆＴと と学位論文審査結果の要旨杢憂磯は曇-A｣b腿鵬鬮一三山!z俸間11Ｍ蝋輸送の調節機構塗賜堕な應武魚ニー≧塗-国_的≧上払鴦のj調節機撹Ｉ昌闇ねゐ棗溌示唆責iiLnM菖仙良雄三一三izk蓑佳間1三局産｡)二豆複数回膜貫通j’z惣沈質nコ為…特1二ｘ聾Q胆Ｚ.と．n】QLQ煕旦.に着貝上玉壜ﾛｰﾆｰ拠負、fit:王と雪担冥佳思式ゑ三.裟三：之質i董瀧案.具.同定_Lm-壹_負1二證堕相互f雇用部位A【２分壬生物鴬的ｊｉ曜抗塗丘ｎ迄-Ｍ１重迄量輿.灘農璽烹:Ｍ:ZLz-E麓塗思い五相互lElH念壬Ｑ深』索塗征、鳶と.二畳塾蝉iQAEL迄互璽煕肛.と.Ej型QEBM〕し画友嬉特異的1二相互』(E閉式員公壬_≧｣Lご巴冒EL同定式向:ムーム1三成委ｔＬ迄GL-典fq4AE]L1主銚.ｴ山地hzx三以扁11Ｍ１型ｑｒ亙鰕.鱒.JqUmQpR交換I室ｌ王五jji?-j9L､…三iZkL乏俸血旦Ｑ－ｃＱＨ轍j菫｣上胞QjIi2fjjii上輸送少胞｣Ｍ〕し翼ｚｆ:i之質!pL選択的蚊、込晶'二関ね負臺zlSj報責嶌itLn.)ゑ異…とJ1二薮、盃､_泰研究z'ﾐら_五in餌Rz-≧…E趣Q邸昼丞Ａｌｆｆｑ１ＬＥＬ上想真土j巨思式ゑ息と.に主２衷ＭＭｆ１ｌｆｌＥ三－三'と乏撚間1Ｍ〕砲輸送Ｑ調範應働式…≧n.画期飽なril熊j)1tﾐ顛初堕天.唄旦鉦上な、為一志迄ｊ髪f蔓も互扁ｆＭｚ:此z-E法の壬縫塗改L良LL定量蜘二jij案jlr式魚臺塗ｺﾖｴ熊1烏燃馳QE肌と_jIm｣qInIiMK典｣p△ｕと狽亙能思式畳音肱点撤莨上迄鳥点典迭貝1量値式ゑ輿…………………－－－…………－－………………＿以よ、二と.窪一ら灸-率疏窺の成暴健合麓iの当該jilI鍵i:i蕊の裳fl萱1ﾖA)L髪i展!≦太鳶≦_寄長式量典Ｑ上煮ヱ旦拠墨圭麟!=b-1薬裳)…Qji量苫菖E1呈値式量_≧捌衷豊狐迄具….……………_…－－－－…＿…-554-

Characterization of ArfGAP1 and FinGER7/FinGER8 interaction by quantitative yeast two-hybrid analysis

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定量的酵母ツーハイブリッド法によるArfGAP1とFinGER7/FinGER8の相互作用の解析

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