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).FluorescentlytaggedABCG11orABCG12islocalizedtotheplasmamembraneandrescuescuticularlipidde cienciesoftheknockoutmutantsinstablytransformedlines(Pighinetal.,2004;Birdetal.,2007).Consistentwitharoleincuticularlipidexport,bothgenesarehighlyexpressedinthestemepidermis,wherewaxandcutinsynthesisandsecretionareextremelyhigh(Pighinetal.,2004;Suhetal.,2005;Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).However,ABCG11isalsoexpressedintissuesinwhichcuticularlipidsarenotsynthesized(e.g.,emerginglateralroots),suggestingthatitmayplayrolesbeyondcuticularlipidexport(Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).Indeed,abcg11mutantsdisplaypleiotropicphenotypes,includingdwar sm,lossofapicaldominance,andsterility,whileabcg12mutantsarephenotypicallynormalexceptfortheirglossystems(Pighinetal.,2004;Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).Epidermalcellsofbothabcg11andabcg12mutantsaccumulatelipidicinclusions,presumablyduetotheaggregationofwaxmoleculesthataresynthesized,butnotexported,fromthesemutants(Pighinetal.,2004;Birdetal.,2007;Panikashvilietal.,2007).abcg11abcg12doublemutantshavethesamelevelsofresidualwaxaseitherofthesinglemutants,suggestingthatABCG11andABCG12actinthesamepathwayorcomplexincuticularwaxexport(Birdetal.,2007).Basedonthepleiotropicphenotypesofabcg11mutants,wehypothesizedthatdifferentdimerizationcombinationsofABCGtransporterscouldaccountforthemultiplefunctionsoftheABCG11half-transporter(Birdetal.,2007).Inthisstudy,ABCG11/ABCG12heterodimersandABCG11homodimersweredemonstratedusingbimolecular uorescencecomple-mentation(BiFC),whileABCG12homodimerswerenot.Het-erodimerizationbetweenABCG11andABCG12wasfurther

FlexiblePairingofABCGTransporters3067

supportedbythebehavioroftheABCtransportersduringtheirbiosynthesisandsecretioninepidermalcells,whichactivelysynthesizeandsecretecuticularlipids.ThedifferentdimercombinationsdemonstratedbyBiFCcanaccountforthebe-haviorofthesehalf-transportersduringtheirtraf cking.ABCG12wasretainedinlipidicinclusionsintheabsenceofABCG11,andtheseinclusionswerecontiguouswiththeER.Thisstudyem-phasizesthe exibilityofthehalf-transportersystemofABCGtransporters,inwhichspeci cdimercombinationsperformspecializedfunctionsanddifferentcombinationsofABCGtrans-porterscanperformdifferentfunctions.

RESULTS

ABCG11andABCG12FormaHeterodimer,andABCG11CanHomodimerize

GiventhatABCG11andABCG12arebothhalf-transporters(Verrieretal.,2008)andthatthedoublemutantphenotypeindicatedthattheylikelyactinthesamepathwayorcomplexinwaxexport(Birdetal.,2007),theirphysicalinteractionwastestedinvivousingBiFC(Figure1).Thisplant-basedsystemwasusedbecauseithasprovendif cult,bothinourhandsandasdocumentedbyothers(GeislerandMurphy,2006;YangandMurphy,2009),toexpressplantABCtransportersinSaccharo-mycescerevisiae,thuslimitingyeastprotein–proteininteractionstudies.IntheBiFCsystem,enhancedyellow uorescentprotein(EYFP)issplitintotwohalves:N-terminal(nYFP)andC-terminal(cYFP),whicharefusedtotheproteinsofinterest.Cotransfor-mationoftheseconstructsintoatransientexpressionsysteminArabidopsisleafmesophyllprotoplastsallowsreconstitutionofYFPifthetwoproteinsareinclosephysicalproximity,therebyallowingdetectionofYFPsignal(Citovskyetal.,2006).Asigni cantproportionofprotoplastscotransformedwithcYFP-ABCG11andnYFP-ABCG12,orconversely,withcYFP-ABCG12andnYFP-ABCG11,displayedbrightyellow uores-cenceintheplasmamembrane,indicatingthatthesetwoproteinsarecapableofformingaheterodimerinvivo(P<0.05)(Figures1C,1D,and1I;seeSupplementalFigure1online).Thisalsocon rmsthatABCGBiFCconstructswitheithertheC-orN-terminalpiecesofYFParecapableofgeneratingyellow uorescence,regardlessofthecombinationused.

WhencYFP-ABCG11andnYFP-ABCG11werecotrans-formedintoprotoplasts,yellow uorescencewasobservedintheplasmamembraneofasigni cantproportionofthesecells,indicatingthatABCG11canalsohomodimerize(P<0.05)(Figures1A,1B,and1I).Bycontrast,whencYFP-ABCG12andnYFP-ABCG12werecotransformed,onlyasmallproportionofprotoplasts uorescedfaintlywhenimagedunderthesameconditions(Figures1E,1F,and1I;seeSupplementalFigure1online).Todeterminewhetherthissignalwasduetoagenuine,low-levelhomodimerizationofABCG12orwhetheritwasanartifactofcolocalizationoftwohalf-YFPconstructstotheplasmamembrane,eachoftheABCGBiFCconstructswerecotrans-formedwithBiFCconstructsforcomponentsofaplasmamem-brane–localizedrootnitratetransportercomplex(ArabidopsisNRT2.1orNRT3.1)(Yongetal.,2010),whicharenotpredictedto

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Figure1.ABCG11andABCG12HeterodimerizeandABCG11Homo-dimerizesintheBiFCSystem.

Onlyprotoplastswithintactplasmamembranes,shownwithbright- eldlightmicroscopy(A,C,E,G),weretestedforthepresenceofyellow uorescence,indicatingprotein–proteininteractionduetoassemblyofsplitYFP,shownwithconfocalmicroscopy([B],[D],[F],and[H]).CotransformationofcYFP-ABCG11andnYFP-ABCG11intoprotoplastswithintactplasmamembranes(A)generatesyellow uorescence(false-coloredgreen)attheplasmamembrane,surroundingchloroplastauto- uorescence(false-coloredmagenta)inconfocal(B).CotransformationofcYFP-ABCG11andnYFP-ABCG12alsogeneratesyellow uores-cenceattheplasmamembrane([C]and[D]).However,cotransforma-tionofcYFP-ABCG12andnYFP-ABCG12([E]and[F])orcYFP-ABCG11andNRT3.1-nYFP([G]and[H])generatedonlyfaintyellow uorescenceinalowproportionofprotoplasts.Resultswerequanti edasthepercentageof uorescentprotoplasts,relativetothenumberof uores-centprotoplastsinthepositivecontrol(full-lengthYFP)(I).n=8independentexperiments(totalof>1300protoplastsscored),barsrepresentSE,*denotesstatisticallysigni cantdifferencesbetweensamples(P<0.05),andbars=10mm.Allprotoplasts(exceptfortheoverexpressingYFPcontrol)wereimagedatthesameexposuretimeanddetectorgaintoallowforcomparisonsofrelative uorescence.

interactwithABCGtransporters.ProtoplastscotransformedwithNRT2.1-cYFPandeithernYFP-ABCG11ornYFP-ABCG12orwithNRT3.1-nYFPandeithercYFP-ABCG11orcYFP-ABCG12displayedthesamefaint uorescenceascYFP-ABCG12withnYFP-ABCG12(Figures1Eto1I;seeSupplementalFigure1on-line).Theseproportionsof uorescentprotoplastswerenotsigni cantlydifferentfromtheproportionobservedwhenthetwoABCG12constructswerecotransformed(P>0.35).Thus,theselowproportionsoffaintly uorescingprotoplastsarelikely

backgroundsignalduetochanceassociationsbetweenproteinsthatareplasmamembranelocalizedbutdonotlegitimatelyinteract.

ABCG11Traf ckingtothePlasmaMembraneIsIndependentofABCG12

StudiesofABCGhalf-transportersinmammaliancellsindicatethatbothpartnersofaheterodimermustbepresentforthedimertotraf ctotheplasmamembrane(Grafetal.,2003,2004).TotestwhetherABCG11orABCG12cantraf cnormallyonlyinthepresenceoftheirpartnersandtoputtheseinteractionsintothecontextofcuticularlipidsecretionintheepidermis,stablytransformedplantlinesexpressing uorescentlytaggedABCGs(Pighinetal.,2004;Birdetal.,2007)werecrossedintothesingle(abcg11)anddouble(abcg11abcg12)knockoutmutants.YFP-ABCG11successfullytraf ckedtotheplasmamembraneinthepresenceofitswild-typeABCG12partner(i.e.,inthesingleabcg11mutantbackground;Figure2A).PlasmamembranelocalizationofYFP-ABCG11wasvisualizedastwodistinctsignalsfromneighboringcellsliningtheadjacentcellwall,whichwaslabeledwithpropidiumiodide(Figures2Band2C).IntheabsenceofABCG12(inabcg11abcg12doubleknockoutmutantbackground),YFP-ABCG11wasalsolocalizedtotheplasmamembrane(Figure2E),whereitcolocalizedwiththeplasmamembranemarkerFM4-64(Figures2Fand2G).YFP-ABCG11localizationinthedoublemutantbackgroundwascon rmedusingtransmissionelectronmicroscopy(TEM)immunogoldla-belingofcryo- xed,freeze-substitutedstemepidermalcells(Figure2D),probedwithananti-green uorescentprotein(GFP)antibody.Theplasmamembraneofepidermalcellswaslabeledbyanti-GFPintransgenicplantlinescarryingYFP-ABCG11(Figure2H),butnosignalabovebackgroundwasdetectedinwild-typecontrolplantsorinsamplesprobedwithoutaprimaryantibody(seeSupplementalFigure2online).TherewasnoevidenceofpolarlocalizationofYFP-ABCG11instemepidermalcellsusingthishigh-resolutionTEMtechnique,incontrastwithpreviousreportsusingconfocalmicroscopy,wheresignalin-tensitiescanbedistortedbyopticalconditionsordissection(Panikashvilietal.,2007).TheseresultsdemonstratethatABCG11isabletotraf ctotheplasmamembraneevenintheabsenceofABCG12.ThisindicatesthatABCG11mayhetero-dimerizewithotherABCGhalf-transportersthatarepresentinthestemepidermis,suchasABCG18andABCG19(Suhetal.,2005).Alternatively,ABCG11mayformahomodimerinvivo,aninterpretationthatisconsistentwiththeBiFCdata.

ABCG12Traf ckingtothePlasmaMembraneIsDependentonABCG11

IfABCG12formsanobligateheterodimerwithABCG11,thenABCG11ispredictedtoberequiredfornormaltraf cofABCG12totheplasmamembrane.Therefore,GFP-ABCG12waslocal-izedinabcg12andabcg11abcg12mutantstoinvestigateitstraf ckinginthepresenceandabsenceofitsABCG11partner,respectively.Inthesingleabcg12knockoutmutants,GFP-ABCG12signalwasdetectedattheplasmamembrane(Figure3A),adjacenttothecellwall,visualizedwithpropidium

iodide

FlexiblePairingofABCGTransporters3069

Figure2.Traf ckingofABCG11tothePlasmaMembraneIsIndependentofABCG12.

Inabcg11mutants([A]to[C]),YFP-ABCG11islocalizedtotheplasmamembrane(A),asshownbycounterstainingwithpropidiumiodide(B)andmerge(C).TEMofthewild-typestemillustratesepidermalcellmorphology(D).Inabcg11abcg12doublemutants([E]to[H]),YFP-ABCG11isalsolocalizedtotheplasmamembrane(E),whereitcolocalizeswithFM4-64(F)inthemerge(G).TEMimmunogoldlocalizationwithanti-GFPcon rmsYFP-ABCG11localizationtotheplasmamembrane(H).Thewhiteboxin(G)highlightsanareathatisrepresentativeofthe eldofviewin(H).Circleshighlightgoldparticles,andarrowheadsdenotetheplasmamembrane.Bars=10mminconfocalimages([A]to[C]and[E]to[G]),5mmin(D),and200nmin(H).

(Figures3Band3C).ImmunogoldTEMlabelingofthesingleabcg12mutantexpressingtheGFP-ABCG12constructrevealedthatanti-GFPsignalwaspredominantlyattheplasmamem-brane,withlittleintracellularandcellwallbackgroundlabel(Figure3D;seeSupplementalFigure2online).However,intheabcg11abcg12doublemutantbackground,GFP-ABCG12sig-nalwasdetectedinareticulatenetworkresemblingtheERandinlargeaggregationsinthemiddleofthecells(Figure3E).ThissignalhadalowlevelofcolocalizationwithFM4-64(Figures3Fand3G),whichisnotconsistentwithplasmamembranelocal-ization.InTEM,anti-GFPlabelingwasconcentratedinthelargeaggregationsofsheet-likeinclusionsthathavebeendetectedwithinthecytoplasmofabcg11andabcg12mutants(Figure3H;Pighinetal.,2004;Birdetal.,2007;Panikashvilietal.,2007).GFP-ABCG12retentionintheERintheabsenceofABCG11isconsistentwiththepredictionthatABCG12dimerizesonlywithABCG11inthestemepidermalcellsandthat,asinmammals,ABCGhalf-transporterdimerformationisrequiredfortraf ckingtotheplasmamembrane(Grafetal.,2003,2004).

AccumulationofGFP-ABCG12intheabcg11abcg12mutantinclusionscouldbeduetoanonspeci cERstressresponseand/orageneraltraf ckingdefectinabcg11mutants.ImmunogoldTEMlabelingforanotherplasmamembrane–localizedprotein,thePIP2aquaporin(Botsetal.,2005),detectedPIP2intheplasma

membraneofwild-typeandabcg11mutantcells(Figures4Aand4B).However,nolabelingabovebackgroundwasdetectedintheinclusions(Figure4C;seeSupplementalFigure3online).Thiscon rmsthatthefailureofGFP-ABCG12totraf ctotheplasmamembraneinabcg11mutantsisspeci ctoABCG12andthattheinclusionsarenotageneralsinkforplasmamembrane–localizedproteins.Similarly,RT-PCRanalysisofgenestypicallyupregu-latedbytheunfoldedproteinresponse(MartinezandChrispeels,2003;Kamauchietal.,2005)con rmedthatthepresenceofinclusionsdoesnotinducesigni cantunfoldedproteinresponse(seeSupplementalFigure4online).Thesedataagreewitharecentmicroarrayanalysisofgeneexpressionchangesinabcg11mutants(Panikashvilietal.,2010).Therefore,GFP-ABCG12re-tentionininclusionsisnotduetoageneralproteinormembranetraf ckingdefectinabcg11mutants.

MembraneInclusionsinabcg11MutantsAreContiguouswiththeER

BecauseGFP-ABCG12wasretainedintheERandintheinclusionsofabcg11abcg12doublemutants,therelationshipbetweentheseinclusionsandtheERwasinvestigatedfurther.TEMwasusedtoexaminethemorphologyofabcg11stemepidermalcellswhereinclusionsprotrudedintothevacuole

of

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Figure3.Traf ckingofABCG12tothePlasmaMembraneIsDependentonABCG11.

Inabcg12mutants([A]to[D]),GFP-ABCG12islocalizedtotheplasmamembrane(A),asshownbycounterstainingwiththecellwalldyepropidiumiodide(B),merge(C),andTEMimmunogoldlocalizationwithanti-GFP(D).Inabcg11abcg12doublemutants([E]to[H]),GFP-ABCG12isretainedwithintheERandinlipidicinclusionsofthesemutants(E)andfailstocolocalizewiththeplasmamembranedyeFM4-64(F)inthemerge(G).TEMimmunogoldlocalizationwithanti-GFPcon rmsGFP-ABCG12localizationtoinclusions(H).Whiteboxesin(C)and(G)highlightanareathatisrepresentativeofthe eldofviewin(D)and(H),respectively,circleshighlightgoldparticles,andarrowheadsdenotetheplasmamembrane.Bars=10mminconfocalimages([A]to[C]and[E]to[G])and200nminTEM([D]and[H]).

epidermalcells(Figure5A).ERmembranesareoftencloselyassociatedwiththeinclusions,eitherlayeredbetweeninclu-sions,oratthetipsoftheinclusions(Figures5Band5C).Bycontrast,therewasnorelationshipbetweentheGolgiapparatusandtheinclusions(Figure5B).

Tocon rmtheassociationoftheERwiththeinclusions,ERwaspositivelyidenti edwithestablishedmarkersinabcg11mutants.Inwild-typeepidermalcells,theERmarkerGFP-HDEL(Batokoetal.,2000)labeledtheERnetwork,includingthecorticalER(Figure6A).Inabcg11mutantstemepidermalcells,GFP-HDELlabeledlargeaggregationsinthecenterofthesecells,inadditiontothenormalERnetwork(Figure6E).ThispatternisstrikinglysimilartothelipidicinclusionslabeledwithNileReddetectedinabcg12mutants(Pighinetal.,2004).Inabcg11mutants,NileRedstainedasimilarpatterntoGFP-HDEL(Figures6B,6C,6F,and6G),suggestingthattheseERaccu-mulationsarelipidrichandmaybecomposedofcuticularlipidsthataresynthesized,butnotsecreted,inabcg11mutants.

SincetheERwasoftenlayeredbetweeninclusions,thelive-cellimagingwithGFP-HDELmightre ecttrappingofERmembranesbetweeninclusions,ratherthanexpansionoftheERmembranestoforminclusions.Todifferentiatebetweenthesepossibilities,TEMimmunogoldlabelingwasemployed

withanticalreticulin,anestablishedmarkerfortheER(Coughlanetal.,1997).Inwild-typecells,anticalreticulinlabeledtheelectron-lucentERlumen(Figure6D;seeSupplementalFigure5online).Inabcg11mutants,anticalreticulinlabeledbothERlumenandinclusions(Figure6H;seeSupplementalFigure5online).Thiscon rmsthattheinclusionsthemselvescontainbona deERmarkers.Together,thesedatasuggestthatinabcg11mutants,lipidsareretainedininclusionsthatareasso-ciatedwithER-derivedmembranes.DISCUSSION

Speci cdimerpairingsofABCGtransportershavebeenhy-pothesizedtoallowmultiplefunctionsforasinglegeneproductinbothArabidopsiscuticularlipidexportbyABCG11andABCG12(Birdetal.,2007,Bird,2008)andinDrosophilaeyepigmentaccumulationbyWHITE,BROWN,andSCARLET(SullivanandSullivan,1975;Sullivanetal.,1979;Mackenzieetal.,2000;Evansetal.,2008).However,thesedimercombina-tionshavebeenhypothesizedbasedonsequenceanalysisandmutantphenotypesandhavenotbeendirectlytested,untilnow.Here,physicalinteractionsbetweenArabidopsisABCG11andABCG12areexperimentallydemonstratedbyBiFC.BiFC

data

FlexiblePairingofABCGTransporters3071

Figure4.Traf ckingofPIP2tothePlasmaMembraneIsIndependentofABCG11.

Inwild-typestemepidermalcells,thePIP2aquaporinislocalizedtotheplasmamembrane(A).PIP2isalsolocalizedtotheplasmamembraneinabcg11mutants(B)andisnotretainedwithintheabcg11mutantinclusions(C).Circleshighlightgoldparticles,arrowheadsdenotetheplasmamembrane,andbars=200

nm.

indicatewhichpairingsarephysicallypossible,assumingthesegeneproductsareexpressedinthesamecellatthesametime.ToplaceABCGtransporterdimerizationinthecontextoftheepidermalcellduringlipidsecretion,biosynthetictraf cofABCGtransportersfromtheERtotheplasmamembranewasexam-inedinthesecells.Thebehaviorofthesehalf-transportersduringtheirbiosynthesisandtraf ckingareconsistentwiththedifferentdimercombinationsdemonstratedbyBiFC.Theconsistencybetweenthesetwodatasets,togetherwithfunctionaldatadeducedfrommutantphenotypes(Pighinetal.,2004;Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007),leadtothemodelsproposedbelowforspeci cdimeriza-tionpairingsofABCGtransportersindiversefunctions.ABCG11UndergoesFlexibleDimerizationandPerformsMultipleFunctions

abcg11mutantsdisplaypleiotropicphenotypes,includingre-ducedsurfacewaxandcutinmonomers,stuntedgrowth,organfusions,reducedfertility,andreducedapicaldominance,indi-catingthatABCG11isinvolvedinprocessesinadditiontowaxexport(Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007;Ukitsuetal.,2007).Furthermore,theexpressionpatternofABCG11extendsbeyondthatofABCG12totissuesinwhichneithercutinnorwaxisbeingsynthesized(e.g.,emerginglateralroots)(Suhetal.,2005;Tou ghietal.,2005;Birdetal.,2007;Luoetal.,2007;Panikashvilietal.,2007).Basedonmutantpheno-typesandexpressionpattern,weproposethatABCG11actsasageneralistABCGtransporter,pairingwithdifferenthalf-trans-portersindifferenttissuestotransportstructurallydiversesub-strates.Consistentwiththis,ABCG11wasabletoexittheERandtraf ctotheplasmamembraneindependentlyofABCG12,indicatingthatitcandimerizewithotherABCGhalf-transportersinepidermalcells.Additionally,homodimerizationofABCG11wasdemonstratedinBiFCassays.TheseresultsareconsistentwiththemodelinwhichABCG11homodimerizestoexportcutinprecursorsfromthestemepidermis(Birdetal.,2007).Deter-miningwhetherABCG11functionsincutinexportasahomo-dimerwillrequirefurtherexperiments.Whileanalysisofseveralcutinbiosynthesismutantshasrevealedgenesrequiredfor

cutin

Figure5.MembraneInclusionsinabcg11MutantsAreCloselyAssociatedwiththeER.

Stemepidermalcellsofabcg11mutantsaccumulateinclusionsthatprotrudeintothelargecentralvacuole(A).Theseinclusionsarecloselyassociatedandinterspersedwiththeelectron-lucentER([B]and[C]);however,themorphologyoftheGolgiapparatusseemsunaffected(B).Arrowsin(B)and(C)highlightcloseassociationsbetweenERandinclusions.Bars=5mmin(A),500nmin(B),and200nmin(C).

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Figure6.MembraneInclusionsinabcg11MutantsAreContiguouswiththeER.

Inwild-typestemepidermalcells([A]to[D]),GFP-HDELlabelstheERnetwork(A),andNileRed(alipidicdye)faintlylabelscellularmembranes(B),whichpartiallycolocalizewithGFP-HDELinthemerge(C).TEMimmunogoldlabelingwithanticalreticulincon rmsthepresenceofthisepitopeinthewild-typeelectron-lucentER(D).Inabcg11mutants([E]to[H]),GFP-HDELlabelsthereticulateERnetworkaswellaslargeaggregationsinthecenterofthecells(E).NileRedsignalindicatesthattheseinclusionsarelipidic(F),andGFP-HDELandNileRedoverlapininclusionsinthemerge(G).TEMimmunogoldlabelingwithanticalreticulinshowsanaccumulationofthisERepitopeininclusions(H).Whiteboxesin(C)and(G)highlightanareathatisrepresentativeofthe eldofviewin(D)and(H),respectively,andcircleshighlightgoldparticles.Bars=10mminconfocalimages([A]to[C]and[E]to[G])and200nminTEM([D]and[H]).

monomerbiosynthesisandassembly,thenatureofcutinpre-cursorsthatareexportedforassemblyinthecuticleisnotknown(Pollardetal.,2008).Thisgapinourunderstanding,aswellastheproblemthatwaxconstituentsaresolidatbiologicallyrelevanttemperatures,makesassessmentofthepossiblesubstratesofABCG11challenging.BothBiFCandtraf ckingdatademon-stratethatABCG11iscapableofpromiscuouspairingwithmultipleABCGhalf-transporters.ItispossiblethatABCG11dimerizeswithmultiplepartnerstoformavarietyoffulltrans-porterstoperformdiversefunctions,ashasbeenhypothesizedintheDrosophilaWHITE/BROWN/SCARLETmodel.Indeed,ABCG11hasbeenimplicatedinfunctionsasdiverseasmain-tenanceofapicaldominance, owerdevelopment,embryogen-esis,androotsuberinexport(Panikashvilietal.,2010).Thisgivesrisetoamodelinwhichthepleiotropiceffectsofabcg11mutationareduetothelossofthisvarietyoffulltransporters.ABCG12Speci callyFormsHeterodimerswithABCG11forWaxExport

IncontrastwiththebroadexpressionpatternofABCG11andthepleiotropicphenotypesofabcg11mutants,mutantsinabcg12displayonlywax-relatedphenotypes,andexpressionofABCG12isenrichedinthestemepidermiswherewaxisactivelybeingsynthesizedandsecreted(Pighinetal.,2004;Suhetal.,2005;Tou ghietal.,2005).ThereisaprecedentforstrictABCGheterodimerizationinMedicagotruncatulaarbuscularmycorrhi-zalsymbiosis(Zhangetal.,2010).Duringarbusculeformation,twoABCGhalf-transportergenes,STUNTEDARBUSCULE(STR)andSTR2,arehighlyexpressed.STRandSTR2hetero-dimerizeandareincapableofforminghomodimers.Theiriden-ticalmutantphenotypesandexpressionpatternssuggestthat,likeABCG12,thesetwoABCGhalf-transportersarespecialists(Zhangetal.,2010).ConsistentwiththehypothesisthatABCG12isaspecialist,GFP-ABCG12wasonlyabletoexittheERandmovetotheplasmamembraneofepidermalcellsinthepresenceofABCG11,indicatingthatABCG12formsanobligatehetero-dimerwithABCG11instemepidermalcells.ThisissupportedbytheBiFCinteractiondata,whichindicatethatABCG12canheterodimerizewithABCG11butisincapableofformingahomodimer.Therefore,thewaxphenotypesoftheabcg11andabcg12singlemutantsandtheabcg11abcg12doublemutantsarearesultofdefectsinthis

heterodimer.

WaxesThatAreNotExportedinABCGMutantsAccumulateinER-DerivedInclusions

Inabcg11orabcg12mutants,membranouslipidicinclusionsaccumulateintheepidermalcells(Pighinetal.,2004;Birdetal.,2007;Panikashvilietal.,2007).Here,wedemonstratethattheinclusionsinabcg11mutantscontainmarkersoftheERbutnotmarkersoftheplasmamembrane.ItispossiblethatERexpan-sionisastressresponsetosequesterinsolublecuticularlipids.Similarly,inyeast,theERincreasesinsizetoaccommodatedefectiveproteins,independentoftheunfoldedproteinre-sponse(Schucketal.,2009).Alternatively,theinclusionscouldre ectthenatureoflipidtraf cduringcuticularlipidsecretion.Alloftheenzymesthathavebeencharacterizedinwaxsyn-thesishavebeenlocalizedtotheERmembranes(Zhengetal.,2005;Greeretal.,2007;Bachetal.,2008;Lietal.,2008).Ifintermediatesecretorycompartments,suchastheGolgiapparatus,arerequiredforwaxsecretiontothecellsurface,thenwaxaccumulationinthesecompartmentswouldbeex-pectedtooccurwhentransportisblocked.However,GolgiandplasmamembranemorphologyappearedunaffectedinTEMofabcg11knockoutmutants.Whilethesubcellularcom-partmentsinvolvedinwaxsecretionarenotknown,accumu-lationofwaxesonlyintheERisconsistentwithwaxmovingdirectlyfromtheERtotheplasmamembranevianonvesicularlipidtraf c,byanalogytoyeastlipidtransportsystems(LevineandLoewen,2006).

Insummary,ArabidopsisABCG11iscapableof exibledi-merizationtoformeitheraheterodimerorahomodimerattheplasmamembraneinvivo.Bycontrast,ABCG12formsanobligateheterodimerwithABCG11,andheterodimerizationisrequiredfornormaltraf ckingtotheplasmamembrane.BasedonthesedataandonpreviousABCGstudies,therearetwoemergingparadigms: exiblepairingofoneABCGgeneproductwithmultiplepartnerstoperformdiversefunctionsandformationofobligateABCGheterodimersforspecializedfunctions.TheseparadigmshavebeenformerlydeducedbutnotexperimentallytestedinthecanonicalWHITE/BROWN/SCARLETcomplexinDrosophila.Inmammaliancells,casesofbiologicallyrelevant exibleABCGtransporterpartneringhavenotbeendescribed,althoughseverallinesofevidencehintthatthisispossible(Grafetal.,2002;Cserepesetal.,2004).ThisstudydemonstratesthatmechanismsofABCGtran-sporterdimerizationareconservedacrossbiologicalkingdoms,despitethedivergentfunctionalrolesplayedbythesetrans-porters.

METHODSPlantMaterial

SeedlingsweresownonATmediaandgrowninanenvironmentalgrowthchamberat218C,70to80%humidity,and24hlight(80to100mEm22s21)forroughly10dbeforetransfertoSunshineMix5soil.Thecer5-2,wbc11-3,cer5-2+CER5pro:GFP-CER5,andwbc11-3+35Spro:YFP-WBC11lines(allintheColumbia-0[Col-0]background)havebeenpreviouslydescribed(Pighinetal.,2004;Birdetal.,2007),andCol-0seedscarryingthe35Spro:GFP-HDELERmarkerwereagenerousgiftfromHugoZheng(Batokoetal.,2000).

FlexiblePairingofABCGTransporters3073

BiFCTransgeneConstructionandProtoplastTransformationABCG12cDNAwasampli edfromtheGFP:CER5vector(Pighinetal.,2004),andXmaIandEcoRIsiteswereaddedusingtheprimersABCG12.P3andABCG12.P4(seeSupplementalTable1online).ABCG11cDNAwasampli edfromYFP:WBC11(Birdetal.,2007),andXmaIandEcoRIsiteswereaddedusingtheprimersABCG11.P3andABCG11.P4(seeSupplementalFigure6online).TheresultingproductswereligatedintotheEcoRIandXmaIsitesofpSAT4-cEYFP-C1-BandpSAT4-nEYFP-C1(Citovskyetal.,2006)andveri edbysequencing.ControlBiFCvectorscontainingcomponentsoftheArabidopsisthaliananitratetransportmachinerywereagenerousgiftfromZ.KoturandA.Glass(Yongetal.,2010).PlasmidDNAwaspuri edfromEscherichiacoliculturesusinganendotoxin-freeplasmidmaxiprepkit(Qiagen).ProtoplastsfromCol-0leaveswerepreparedandtransformedasdescribed(Tiwarietal.,2006)with10mgofeachvectorusedineachprotoplasttransformation.Thenumberof uorescentprotoplastswasscoredoutofthetotalnumberofliveprotoplasts(i.e.,thosewithanintactplasmamembranewhenviewedwithbright- eldmicroscopy)andpresentedasapercentagerelativetothenumberof uorescentprotoplastsinthepositivecontrol(pSAT6+35Spro:EYFP)asanestimationoftransformationef ciency.Datafromconversetransformations(e.g.,cYFP-ABCG11+nYFP-ABCG12andcYFP-ABCG12+nYFP-ABCG11)weregrouped.Tomeettheassump-tionsofanalysisofvariance(WhitlockandSchluter,2009),datafromeightindependentexperimentswerenaturallog(ln)transformedandcorrectedforvariationsintransformationef ciencyamongexperimentsbysub-tractingthemeantransformationef ciencyofthatexperimentfromeachvalue.MeanswerecomparedusinganalysisofvarianceandaTukeypost-hoctestusingSPSS(IBM).ConfocalMicroscopy

TransformedprotoplastsweremountedinWIsolution,ordissectedstemsegmentsfromthetop3cmfromtheshootapicalmeristem(wherecuticlesynthesisandsecretionisthehighest;Suhetal.,2005)weremountedindistilledwaterandimmediatelyimaged.Stemsegmentswerestainedwith1mg/mLpropidiumiodide(Sigma-Aldrich)or10mMFM4-64(MolecularProbes)for10min.ImageswerecollectedusingaZeiss510MetascanheadonaZeissAxiovert200MwithaZeissAxioCamHRmCCDcameraorusingaQuorumWaveFXspinning-diskscanheadonaLeicaDMI6000microscopewithaHamamatsuImagEMCCDcamera.OntheZeissmicroscope,GFPwasdetectedusinga488-nmlaserwitha505-to530-nm lter,YFPwasdetectedusinga514-nmlaserwitha535-to580-nm lter,andpropidiumiodideandFM4-64weredetectedusinga514-nmlaserwitha600-to650-nm lter.OntheQuorumsystem,GFPandYFPweredetectedusinga491-nmlaserwitha528-to566-nm lter.ToallowcomparisonoftherelativebrightnessbetweendifferentBiFCtreatments,allprotoplasts(excepttheYFPoverexpressingpositivecontrol)wereimagedunderidenticalconditions,includingdetectorgainandexposuretime.ImageswereprocessedusingVolocity(Improvision)orImageJ,andAdobeIllustrator.

High-PressureFreezing,TEM,andImmunogoldLabeling

Stemtissuefromthetop1to3cmfromtheshootapicalmeristemwasfrozenin0.2MsucroseinBsampleholders(TedPella)usingaLeicaHPM-100high-pressurefreezer.Freezesubstitution,resinin ltration,sectioning,poststaining,andimagingwereperformedasdescribed(McFarlaneetal.,2008).Immunolabelingofhigh-pressurefrozenmaterialwasperformedasdescribed(McFarlaneetal.,2008).Primaryantibodieswere1/50polyclonalanti-GFP(A6455fromMolecularProbes);1/20polyclonalanti-calreticulin,generatedagainstcalreticulinfromcastorbean(RicinuscommuniscvHale),agenerousgiftfromSeanCoughlan(Coughlanetal.,1997);and1/50polyclonalanti-PIP2,generatedagainst

3074ThePlantCell

PIP2fromtobacco(NicotianatabacumcvPetitHavanaSR1),agenerousgiftfromRalfKaldenhoff,(Botsetal.,2005).Secondaryantibodywas1/10010nmgold-conjugatedgoat-anti-rabbit(TedPella).ImageswereprocessedusingImageJandAdobeIllustrator.GeneExpressionAnalysis

TotalRNAwasextractedusingTRIzolreagent(Invitrogen)fromstemsegments1to3cmfromtheshootapicalmeristemorfrom10-d-oldseedlingsgrowninliquidATmediaplus1%sucrose,withandwithout5mMDTT(aspositiveandnegativecontrolsfortheunfoldedproteinresponse,respectively).cDNAwassynthesizedfrom15mgofRNAusinganoligodT18primerandSuperScriptIIIreversetranscriptase(Invitrogen).RT-PCRwasperformedfor20cyclesusing1mLofcDNAwithintron- anking,gene-speci cprimersforHSP90.7,BiP1/BiP2(notspeci ctoeithergenealone,sincetheseare97%identical),CALNEXIN1,CALRE-TICULIN2,andPDI-LIKE9(seeSupplementalTable1online).cDNAlevelswerenormalizedusingprimersfortheUBC10ubiquitinconjugatingenzymegenefor20cycles.PCRproductswerevisualizedusingSYBR-Safe(Invitrogen).

AccessionNumbers

SequencedatafromthisarticlecanbefoundintheArabidopsisGenomeInitiativeorGenBank/EMBLdatabasesunderthefollowingaccessionnumbers:At1g17840(ABCG11),At1g51500(ABCG12),At5g53300(UBC10),At4g24190(HSP90.7),At5g28540(BiP1),At5g42020(BiP2),At5g61790(Calnexin1),At1g09210(Calreticulin2),andAt2g32920(PDI-like9).

SupplementalData

Thefollowingmaterialsareavailableintheonlineversionofthisarticle.SupplementalFigure1.ControlsforBiFCProtein–ProteinInteractionAssay.

SupplementalFigure2.ControlsforAnti-GFPImmunogoldTEM.SupplementalFigure3.ControlsforAnti-PIP2ImmunogoldTEM.SupplementalFigure4.TheUnfoldedProteinResponseIsNotSigni cantlyUpregulatedinabcg11MutantswithInclusions.SupplementalFigure5.ControlsforAnticalreticulinImmunogoldTEM.

SupplementalTable1.AListofPrimersEmployedinThisStudy.

ACKNOWLEDGMENTS

WethanktheABRCforprovidingseedstocksandBiFCvectors,SeanCoughlanforanticalreticulin,RalfKaldenhoffforanti-PIP2,ZoricaKoturforthenitratetransporterBiFCconstructs,HugoZhengfortheGFP-HDELconstruct,theUniversityofBritishColumbiaBioimagingFacilityfortechnicalassistance,andLjerkaKunst,MathiasSchuetz,andTeagenQuilichiniforhelpfuldiscussionsandcommentsonthemanu-script.ThisworkwasfundedbyCanadianNaturalSciencesandEngineeringResearchCouncilDiscoveryGrantstoD.A.B.andA.L.S.andbyaCanadaGraduateScholarship-D3toH.E.M.

ReceivedJuly5,2010;revisedAugust27,2010;acceptedSeptember3,2010;publishedSeptember24,2010.

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Arabidopsis ABCG Transporters, Which Are Required for Export of Diverse Cuticular Lipids,

Dimerize in Different Combinations

Heather E. McFarlane, John J.H. Shin, David A. Bird and A. Lacey Samuels PLANT CELL 2010;22;3066-3075; originally published online Sep 24, 2010;

DOI: 10.1105/tpc.110.077974

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