ThePlantCell,Vol.22:3066–3075,September2010,http://ã2010AmericanSocietyofPlantBiologists

ArabidopsisABCGTransporters,WhichAreRequiredforExportofDiverseCuticularLipids,DimerizeinDifferentCombinations

HeatherE.McFarlane,aJohnJ.H.Shin,aDavidA.Bird,http://ceySamuelsa,1

aDepartmentbMount

ofBotany,UniversityofBritishColumbia,Vancouver,Canada,V6T1Z4RoyalUniversity,Calgary,Canada,T3E6K6

ATPbindingcassette(ABC)transportersplaydiverseroles,includinglipidtransport,inallkingdoms.ABCGsubfamilytransportersthatareencodedashalf-transportersrequiredimerizationtoformafunctionalABCtransporter.Differentdimercombinationsthatmaytransportdiversesubstrateshavebeenpredictedfrommutantphenotypes.InArabidopsisthaliana,mutantanalyseshaveshownthatABCG11/WBC11andABCG12/CER5arerequiredforlipidexportfromtheepidermistotheprotectivecuticle.TheobjectiveofthisstudywastodeterminewhetherABCG11andABCG12interactwiththemselvesoreachotherusingbimolecular uorescencecomplementation(BiFC)andproteintraf cassaysinvivo.WithBiFC,ABCG11/ABCG12heterodimersandABCG11homodimersweredetected,whileABCG12homodimerswerenot.FluorescentlytaggedABCG11orABCG12waslocalizedinthestemepidermalcellsofabcg11abcg12doublemutants.ABCG11couldtraf ctotheplasmamembraneintheabsenceofABCG12,suggestingthatABCG11iscapableofforming exibledimerpartnerships.Bycontrast,ABCG12wasretainedintheendoplasmicreticulumintheabsenceofABCG11,indicatingthatABCG12isonlycapableofformingadimerwithABCG11inepidermalcells.EmergingthemesinABCGtransporterbiologyarethatsomeABCGproteinsarepromiscuous,havingmultiplepartnerships,whileotherABCGtransportersformobligateheterodimersforspecializedfunctions.

INTRODUCTION

ATPbindingcassette(ABC)transportersareuniversalcompo-nentsofcellsfromallkingdomsandplaydiverseroles,includinglipidtransport.ManyABCtransportersareencodedasfullyfunctionalunits,consistingoftwoATPbindingcassettesandtwotransmembranedomains.ABCGfamilymembersthatareen-codedashalf-transportersmustdimerizetoformafull,functionalABCtransporter.InArabidopsisthaliana,theABCGgenefamilycontains28genesannotatedashalf-transporters(Verrieretal.,2008).ABCGhalf-transportershavebeenimplicatedinthetrans-portofabscisicacid(Kuromorietal.,2010)andexportofkana-mycin(MentewabandStewart,2005),sporopollenin(Quilichinietal.,2010;Xuetal.,2010),andcuticularlipids(Pighinetal.,2004;Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).Whetherthesehalf-transportersfunctionashomodimersorheterodimersisnotknown.

AmodelofABCGhalf-transportersformingdifferenthetero-dimerstoperformmultiplefunctionshasbeenproposedinothereukaryoticsystems.InDrosophilamelanogaster,WHITE,SCAR-LET,andBROWNgenesareinvolvedineyepigmentaccumu-lationandWHITEisalsoinvolvedincGTPtransport(Sullivanand

correspondencetolsamuels@interchange.ubc.ca.

Theauthorresponsiblefordistributionofmaterialsintegraltothe ndingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(http://)is:http://ceySamuels(lsamuels@interchange.ubc.ca).versioncontainsWeb-onlydata.

http:///cgi/doi/10.1105/tpc.110.077974

1AddressSullivan,1975;Sullivanetal.,1979;Mackenzieetal.,2000;Evansetal.,2008).MutantphenotypeshaveindicatedthatWHITEisrequiredforaccumulationofbothguanine-derived(red)orTrp-derived(brown)eyepigments,astheeyesofwhitemutantslackanypigmentation(Sullivanetal.,1980;Ewartetal.,1994).Similarly,brownandscarletmutantsindicatedthatthesehalf-transportersarerequiredforaccumulationofguanine-derivedorTrp-derivedeyepigments,respectively(Ewartetal.,1994).ThisimpliesthatWHITEandBROWNdimerizetotransportguanine-derivedpigments,whileWHITEandSCARLETdimerizetotransportTrp-derivedpigments.Whetherthesegeneproductsphysicallyinteractinvivoandwhethertheydirectlytransportthesesubstrateshasnotbeentested.

ABCGtransportersinmammalsappeartoformeitherobligateheterodimers,suchastheABCG5/ABCG8,orhomodimers,suchasABCG2(Tarretal.,2009).WhilebothABCG5andABCG8caninteractpromiscuouslywithotherABCGtrans-porterstestedinvitro(Grafetal.,2003),mouseABCG5andABCG8mustdimerizetoexittheendoplasmicreticulum(ER),undergoposttranslationalmodi cationintheGolgi,andtraf ctotheirusualplasmamembranelocation(Grafetal.,2002,2003).IfeitherABCG5orABCG8isexpressedindividually,orifhetero-dimerizationisdisruptedbyapointmutationineithertransporter,bothtransportersareretainedintheERanddegraded,indicatingthatformationoftheABCG5-ABCG8heterodimerisaprerequi-siteforplasmamembranelocalization(Grafetal.,2002,2004).ThesestudiessuggestthatonlyasubsetoftheABCGtrans-porterdimersthatmaybedetectedbyimmunoprecipitationarecapableofexitingtheproteinmaturationmachineryintheERand

thatERexitcanbeusedasanassessmentofthebiologicalrelevanceoffunctionaldimers.

TwoABCGhalf-transporters,ABCG11andABCG12,havebeenimplicatedintheexportoflipidsfromtheepidermistothecuticle,whichsealsandprotectstheaerialtissuesoftheplantbody(Bird,2008).Thecuticleisatoughcross-linkedcutinpolyesterscaffoldcomposedofhydroxy-C16andC18fattyacidsandglycerol(Pollardetal.,2008),surroundedbyandcoveredwithahydrophobicwaxmixturedominatedbyvery-long-chainfattyacidderivatives(Jetteretal.,2006;Samuelsetal.,2008).ABCGhalf-transportersrequiredforaccumulationofbothcutinandwaxatthecellsurfacehavebeenidenti edbymutantanalysisinArabidopsis.abcg11/wbc11/desperado/cof1mutantsdisplayreducedcutinandwaxlevels(Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).Detailedanalysesofthesechemicalphenotypesrevealedthatallwaxandcutinconstituentsweredecreasedinthesemutants.AssumingthatABCG11candirectlytransportcuticularlipids,thesedataimplythatABCG11hasabroadsubstratespeci cityforavarietyofstructurallydiversecuticularlipids(Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).ThecloselyrelatedABCG12/CER5transporterisrequiredforwax(Pighinetal.,2004),butnotcutin,export(Birdetal.,2007).abcg12mutantshaveareductiononlyinwaxcompo-nents,suggestingthatABCG12hasanarrowersubstratespec-i citythanABCG11(Pighinetal.,2004).FluorescentlytaggedABCG11o …… 此处隐藏：41867字，全部文档内容请下载后查看。喜欢就下载吧 ……