Regulation of GLUT4 glucose transporter trafficking

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1 Yale University EliScholar A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine Regulation of GLUT4 glucose transporter trafficking Leah McNally Follow this and additional works at: Recommended Citation McNally, Leah, "Regulation of GLUT4 glucose transporter trafficking" (2010). Yale Medicine Thesis Digital Library This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar A Digital Platform for Scholarly Publishing at Yale. For more information, please contact elischolar@yale.edu.

2 RegulationofGLUT4glucosetransportertrafficking AThesisSubmittedtothe YaleUniversitySchoolofMedicine InPartialFulfillmentoftheRequirementsforthe DegreeofDoctorofMedicine By LeahJenniferMcNally 2010

3 2 Abstract REGULATIONOFGLUT4GLUCOSETRANSPORTERTRAFFICKING.LeahMcNallyand JonathanBogan,DepartmentsofInternalMedicineandCellularBiology,Yale UniversitySchoolofMedicine,NewHavenCT. Infatandmusclecells,insulinstimulatesglucoseuptakebycausingthe translocationofglut4glucosetransportersoutofintracellularmembranesandto theplasmamembrane.impairedglut4translocationresultsininsulinresistance, andcontributestothepathogenesisoftype2diabetes.yet,howinsulinsignaling andproteintraffickingpathwaysintersectremainspoorlyunderstood.in3t3 L1 adipocytes,datasupportamodelinwhichtug( tether,containingaubxdomain, forglut4)bindsglut4andretainsitintracellularlyintheabsenceofinsulin. InsulinthensignalsthereleaseGLUT4fromTUG,whichmobilizesGLUT4tothecell surface.howandwheretugretainsglut4intracellularlyremainsunknown. PreviousdatashowthattheTUGcarboxylterminusisrequiredforintracellular retentionofglut4,butisdispensableforbindingtoglut4itself.thereforewe hypothesizedthatthisregioninteractswithanunidentified,intracellular anchoring protein.here,wetestedifgcc185maybethissought afterprotein,to whichtuglinksglut4inunstimulatedcells.gcc185isagolgimatrixproteinthat capturesvesiclesarrivingatthetrans Golginetwork(TGN)fromendocyticor biosyntheticpathways.ittethersthevesiclestothetgnmembraneandpromotes theirfusionatthetgn.ithaspreviouslybeensuggestedthatglut4mayby retainedbyanintracellularcycleoffusionandbuddingatthetgninunstimulated

4 3 cells.totestthehypothesisthattugcooperateswithgcc185tofacilitatesucha cycle,weperformedcoimmunoprecipitationexperiments.wefoundthattug interactedwithgcc185incotransfected293cells.importantly,thisinteraction requiredthetugcarboxylterminus,aspredictedforthe anchoring protein.the TUG GCC185interactionwasconfirmedinreciprocalcoimmunoprecipitation experiments.mutagenesisidentifiedaparticularresidueintugthatislikely involvedinthisinteraction,whichmaybemodifiedtocontrolthebindingoftug andgcc185.asecondprojectwaspromptedbytheobservationthatubc9is anotherproteinthatbindsglut4andpromotesitsaccumulationininsulinresponsivestoragevesicles.becauseubc9isaconjugatingenzymeforthe ubiquitin likeprotein,sumo,wetestedthehypothesisthattugisatargetofsumo modification.however,nodatawereobtainedtosupportthishypothesis.in summary,ourdatashowthatgcc185andtuginteract,andsupportamodelin whichgcc185participatesintargetingglut4tovesiclesthataremobilizedacutely byinsulintocontrolglucoseuptake.

5 4 Acknowledgements Iwouldliketothankthefollowingpeopleanddepartmentsfortheirhelpand support: TheBoganLab: JonathanBogan JamesCresswell MingmingHao WhitneyHarris MichaelLöffler CharisseOrme BradRubin EmilyStoops MyThesisCommittee: LloydCantley TomMelia RaymondRussell OfficeofStudentResearch JohnForrest DonnaCarranzo MaeGeter NIDDK

6 5 TableofContents TitlePage..1 Abstract.2 3 Acknowledgements 4 Introduction 6 16 SpecificAims..17 Methods Results Discussion References 35 40

7 6 Introduction Ithasbeenestimatedthatby2030,thenumberofpeoplesufferingfromtype 2diabeteswillreachsome366million(1).Itisalsopredictedthattype2diabetes willincreasinglybecomeaworldwidephenomenon,withnationsthattraditionally hadlowdiseaserates(e.g.thoseinafricaandsoutheastasia)seeingasteeprisein diseaseprevalenceoverthecomingyears.therefore,understandingthe pathophysiologyofthediseaseandidentifyingnewtargetsfortherapy,have becomeincreasinglyimportant. BloodGlucoseHomeostasisandtheGLUT4Transporter Underphysiologicalconditions,bloodglucoseconcentrationsareverytightly controlled,sothatvariationisminimizedpost prandiallyorintimesoffasting.the mainhormonethatregulatesbloodglucoseconcentrationisinsulin,whichboth suppresseshepaticglucoseproductionanddrivesglucoseuptakeintoadiposeand musclecells(2).adipocytesandmyocytescontainglut4glucosetransporters, whichcontaintwelvemembrane spanningdomainsand,togetherwithglut1,are themainglucosetransporterpresentinthesetissues.glut4proteinsare facilitativetransportersthatareinsertedintotheplasmamembraneinresponseto insulinsignaling(3,4,5).thisdistinguishesthemfromglut1proteins,whichare presentattheplasmamembraneevenatthebasalstate.theirinsertionintothe membranecanoccurrapidlybecausethetransportersaretargetedtoandstoredin specific,intracellularvesicles termedglut4storagevesicles(gsvs) within unstimulatedcells.afterinsulinhasfinishedsignaling,glut4isendocytosedand sentbackthroughtherecyclingpathwayintogsvs,toawaitthenextstimulus(6).

8 7 Thus,theprocessisreversible.ThenumberofGLUT4transporterspresentinthe plasmamembranecontrolstheoverallrateofglucoseuptakeintofatandmuscle cells.inaddition,insulinappearstobothincreaseexocytosisofglut4andto slightlydecreaseitsendocytosis(7). Clearly,GLUT4playsakeyroleinmaintainingglucosehomeostasis.One couldhypothesizethatdefectsinglut4oritsmovementtotheplasmamembrane areatleastpartiallyresponsibleforthehyperglycemiaoftheinsulinresistantand type2diabeticstate.however,oneofthefirstquestionsthatwasaskedand answeredinregardstothishyperglycemiawaswhetheritwaschieflyduetofaulty movementofglucoseacrosstheplasmamembrane(suggestingaroleforglut4)or tofaultyutilizationofglucoseonceinsidethesecells.studiesutilizingnmrstrongly supportedthehypothesisthattheproblemlayinthetransportofglucoseintothe cellfromthebloodstreamandnotinitsutilizationwithinthecell(8,9).oncethis wasestablished,thecauseofthisfaultyglucosemovementbecamethenextand ongoingfocus,withtheroleofglut4translocationbeingacentraltheme(10,11). Asreviewedabove,GLUT4transportersaretheinsulin responsivechannels throughwhichglucosetraversestheplasmamembraneinmycocytesand adipocytes.defectsinthepresenceofglut4attheplasmamembraneare correlatedwithinsulinresistance.inmousemodels,decreasedpresenceofglut4 attheplasmamembranethroughpartialglut4knockdownresultsininsulin resistance(12,13,14,15).inaddition,inhumanswithinsulinresistanceortype2 diabetes,thereisdecreasedglut4attheplasmamembraneinresponsetoinsulin (16).ThusitcanbesaidthatthetranslocationofGLUT4totheplasmamembranein

9 8 responsetoinsulinisnotonlydefectiveininsulinresistanceandtype2diabetes, butalsoamajorcontributortothepathophysiology.however,multiple mechanismslikelycontributetothesedefects.theseincludefaultyinsulinsignal transduction(17,18),down regulationofglut4,andmislocalizationofglut4 awayfromthegsvcompartment(19,20,21). GLUT4LocalizationtoGSVs TounderstandtheconceptofGLUT4mislocalization,whatisknownabout theformationofgsvsshouldbereviewed.eventheveryexistenceofgsvstook timetodemonstrate.inthenon insulinstimulatedstate,glut4ismainlylocalized tointracellularmembranecompartments(22,23).infibroblasts(preadipocytes), exogenouslyexpressedglut4isthoughttocyclebetweenendosomesandthe trans Golginetwork(TGN)(24,25).However,inmatureadipocytes,GLUT4is largelyexcludedfromendosomes.thishasbeenshowninstudiesthatusedhorseradishperoxidase conjugatedtransferrintoablatecompartmentsoftherecycling pathway(24).infibroblasts,thisresultsinthedestructionofthemajorityofglut4. However,whenthisisdoneinmatureadipocytes,over50%ofGLUT4remains unaffected,suggestingthatitissequesteredinspecializedcompartments,namely GSVs. TheexistenceofGSVsisalsosupportedbythefindingthatvesiclesenriched inglut4,irapandvamp2,butlackingcommonendosomalproteinssuchas transferrinandcellubrevin,havebeenisolatedandappeartobeinsulin responsive (26).Ithasbeenfurthershownthatthesesmall,specializedvesiclesarenotfoundin fibroblasts,andthatthedevelopmentofthesevesiclescorrelateswithadipocyte

10 9 maturationandinsulinresponsiveness.theproductionofgsvsinmaturing adipocytesislinkedtotheexpressionofthecargoadaptorproteinsortilin(27,28, 29).Importantly,thisworkfurtherdemonstratedthatexogenousexpressionof sortilinin3t3 L1preadipocytesresultedinGSVformationandthatsortilin knockdownin3t3 L1adipocytespreventedtheformationofGSVs.Asadipocytes mature,glut4canbeseentoco localizewithtgnmarkerssuchassyntaxin6and 16.ThissuggeststhatatleastsomestepsofGSVformationtrafficthroughthis regionofthetgn.thistheoryisfurthersupportedbythefindingthatirap,an integralproteiningsvs,hassugarmodificationsconsistentwithitspassagethrough thetgn(21). AdditionaldatasupportingtheexistenceofGSVscomesfromtheobservation thatthehalf lifeofglut4issignificantlyshorterinmatureadipocytesthanitisin fibroblasts.thissuggeststheglut4hasbeensequesteredfromthegeneral recyclingpathwaywhereinitcanbereadilydegradedbytargetingtolysosomes. Interestingly,followingstimulationwithinsulin,therateofGLUT4degradation increases,suggestingthatinsulinmobilizesglut4fromthissequestration compartment.finalevidenceforthegsvcompartmentcomesfromthefindingthat thereappearstobeadosedependentresponseofglut4mobilizationfollowing insulinstimulation(30).inthisstudy,lowinsulindosesresultedinonly10 20%of intracellularglut4cyclingthroughtheplasmamembrane,whilehighinsulindoses resultedin~70%ofglut4cyclingthoughtheplasmamembrane.thisgraded responsewouldfitwiththepresenceofasequesteredglut4pool(gsvs)thatcan beaccessedtodifferentextentsinthepresenceofinsulin.takenalltogether,the

11 10 evidencepointstotheexistenceofgsvswhoseformationlikelyinvolvesstepsata sub domainofthetgn. DirectevidenceforthemislocalizationofGSVsintheinsulinresistantstate, type2diabetesandgestationaldiabetescomesfromcellfractionationstudies(19, 20,21).Instudiesexaminingtissuefromhealthysubjects,GLUT4isfoundinthe lightmembrane(iecellsurface)compartmentinadipocytesandmycocytes. However,ininsulinresistantanddiabeticstates,itisfoundchieflyinthedense membrane(ieintracellular)compartment.onestudyalsoshowedthatirap s(one ofthetwoproteinknowntoassociatewithgsvs)distributionwassimilarlyaltered. Thesestudiesusedbiopsiesfromfastingindividuals,suggestingthatGLUT4isnot beingcompartmentalizedproperlywithinunstimulatedadiposeandmusclecells. ThesereportsraisethepossibilitythatunlessGLUT4isselectivelytraffickedtothe GSVcompartment,itwillnotbeaccessibletoinsulinsignaling.Additionalstudiesin diabeticpatientshavealsohighlightedtheimportanceofpropergsvformation.for example,obesepatientswithincreasedtnf alphaexpressionshoweddownregulationofsortilin(31).assortilinisnecessaryandsufficientforgsvformation intissueculturemodels,thislinkageofsortilinexpressiontoaninsulinresistant state,againemphasizestheimportanceofproperglut4localizationinthe pathophysiologyoftype2diabetes. ARoleforTUGinGLUT4Trafficking DespitetheapparentimportanceofGSVformationandGLUT4localization, exactlyhowglut4aretargetedtoandkeptingsvs,aswellashowinsulin mobilizesthesevesiclestothecellsurface,isnotwellunderstood.thisareaisthe

12 11 mainfocusofworkintheboganlaboratory.inparticular,workonaproteincalled TUG(tether,containingaUBXdomain,forGLUT4)hasshownpromiseinclarifying thisissue.datasupportthenotionthatincultured3t3 L1adipocytes,TUGbinds directlytoglut4andsequestersitspecificallyingsvs,thusexcludingitfromthe plasmamembrane(32,33,34,35).overexpressionoftugappearstoenhance targetingofglut4togsvs,andincreasestheinsulin responsivepoolofglut4 proteins.conversely,adecreaseintugabundance(usingrnai)orinhibitingits action(usingatruncated,dominantnegativeform,termedubxc terminalorubx Cter)preventstheaccumulationofGLUT4inGSVs.TruncationoftheC terminalof TUG(anywherefromaminoacid270on)preventsTUG sabilitytosequestergsvs awayfromtheplasmamembrane.importantly,disruptionoftugaction(using RNAiorthedominantnegativeform)resultsintargetingofGLUT4tothecell surface,andmimicsinsulinactiontoalargeextent.thisfitswiththeideathattug functionallytethersglut4toanintracellularanchoringsitewithinunstimulated cells.inthisproposedmodelthetuginassociationwithglut4ingsvsbindstoan anchoringsiteandtherebylocalizesthegsvsproperly,inaconfigurationfrom whichtheycanbemobilized,withincells.however,itshouldbenotedthatthe associationoftugwithgsvsandtheanchoringsiteisnotnecessarilyastaticone. Forexample,GSVsmaybeallowedtomovewithintheTGNsub domainwhile remainingsequesteredfromthegeneralrecyclingpathway.themolecularnature ofthisanchoringsiteisnotfullyunderstood.nonetheless,dataareconsistentwith theideathatinsulinstimulatesdissociationofatug GLUT4proteincomplexto releaseglut4tothecellsurface,andtoenhanceglucoseuptake.

13 12 PresentdatafromtheBoganlaboratorysupportamodelinwhichfollowing insulin stimulation,tugisproteolyticallyprocessedandgivesrisetoanew ubiquitin likemodifier,termedtugul(fortugubiquitin like).tuguljoinsa familyofabout11knownubiquitin likemodifiers(ubls),includingubiquitinitself (36,37,38).Thesepeptidesaregenerallysynthesizedaspartoflarger,precursor proteins,whicharecleavedinasite specificmannertoliberatethematureubl(as thenterminalcleavageproduct).theublcanthenbeattachedcovalentlytotarget proteinsorlipids.inthecaseoftug,cleavageisaregulatedeventthatoccurs rapidlyafterinsulinstimulationin3t3 L1adipocytes.TUGcleavageseparatesanN terminalregion(tugul),whichbindsglut4,fromcterminalregions,whichbind theanchoringproteins.preliminarydatasuggestthattugulmoveswithglut4to theplasmamembrane,andthec terminalproductremainsbehindandisdegraded bytheproteasome.thismodelpredictsthatsubsequentlyendocytosedglut4 wouldrequireintact,newly synthesizedtugproteinforretentioningsvsof unstimulatedcells.mosttugresidesinthecytosol,anditmayberecruitedto membranescontainingendocytosedglut4fromthislargereservoir. Inthesustainedpresenceofinsulin,endocytosedGLUT4maynotcycle throughgsvs,andthereneednotbeongoingtugprocessinganddegradation. Thus,TUGisanessentialcompartmentofaretentionmechanismthatislikely engagedonlyintheabsenceofinsulin. Ubc9andGLUT4Trafficking Anotherproteinthatlikelyplaysacrucialroleinthetargetingandstoringof GLUT4inGSVsisUbc9,aSUMOconjugatingenzyme(39,40).Ubc9bindstothe

14 13 GLUT4Cterminus,andcontrolstheaccumulationofGLUT4inGSVsinL6myoblasts and3t3 L1adipocytes.Similartoubiquitinconjugatingenzymes,Ubc9catalyzes thecovalentattachmentofaubl,sumo,totargetproteins.sumois12kd,andits attachmenttotargetproteinsmodulatestheiractivitiesorintracellularlocations (41,42,43).TheUbc9datasuggestthatSUMOmodification( SUMOylation )may functioninglut4trafficking.ofnote,presentdatafromtheboganlaboratory showsthecterminalremnantoftugisobservedinboth42kdaand54kdaforms. Theexpectedsizeofthecleavageproductis42kDa,andthe54kDaformmaybea modifiedformofthe42kdacleavageproduct.onepossibility,whichwasexamined duringthecourseofthiswork,wasthatthetugc terminalproductissumoylated, andthatthismodificationofthetugc terminalcleavageproductmaybeinvolved inremovingitfromtheanchoringsite.thiswouldvacatetheanchoringsitesothat itisavailableforsubsequentcyclesofglut4retentionandrelease. Usually,SUMOmodificationsareaddedtoalysineresidueamidsta consensussite:aliphaticresidue lysine anyaminoacid glutamicacid(44).several residuesmeetingthesecriteriacanbefoundwithintug.however,aswillbe discussed,multipleexperimentswerenotconsistentwiththisideaoftug SUMOylation.ThisraisedthepossibilityoftheputativeTUGmodifierbeing somethingotherthansumo. InadditiontoSUMOylation,othercommonpost translationalprotein modificationsincludeacetylation,methylation,ubiquitination,andadpribosylation.ofnote,theseothermodificationsalsofrequentlyoccuratlysines.in addition,previousworkhasshownthatsomelysinesarealternativelyacetylated

15 14 andsumoylated,whichmadeacetylationthelogicalmodificationtoconsidernext (45,46,47,48).Further,theUniProtKBonlinedatabaseshowedthatoneofTUG scterminalresidueswasasiteofacetylation(refq9bze9).arecentpapershowed thatlysinemodificationoftheenzymeornithinecarbamoyltransferasedecreased theaffinityofthisenzymeforoneofitssubstrates.inaddition,theacetylationof thisenzymewasdependentontheextracellularglucoseandaminoacidavailability (49).ThisresultedinthehypothesisthatTUGacetylationmayalteritsaffinityfora GSVanchoringprotein,allowingGLUT4tobereleasedtotheplasmamembrane.In addition,thisacetylationcouldbedependentoneitherextracellularglucose concentration,ortheinsulinsignalingcascade.theideathatacetylationcanbedue totheconcentrationofmetabolicproductsisalsosupportedbyworkshowingthat deacetylationcausesbreakdownofnadtoproducenicotinamideandadp ribose. Buildupofnicotinamideshiftsthehomeostasistofavoracetylationandbuildupof NADfavorsdeacetylation.Thus,ifTUG sacetylationstatealtersitsaffinityforthe anchorandthisinturnaffectsthelocalizationofglut4,theconcentrationof metabolicproducts suchasnad couldhavearole. GCC185asaPutativeAnchor AfinalquestiontoconsideriswhereGSVsareanchoredwithinthecell.The membranedensityworkdoneindiabetichumansstronglysuggestedthatproper GLUT4localizationplayedakeyroleininsulin responsiveness.tugisknownto bindglut4withinitsn terminusandthatdeletionofthec terminusoftugresults initsinabilitytoretainglut4intracellularly.thissuggeststhatthec terminal bindstoananchoringsiteforgsvs.againthisterm anchor isusedinthe

16 15 functionalsenseandisnotmeanttoimplyastaticinteraction.giventhatgsvsseem toatleasttrafficthroughthetgn,itispossiblethataproteinassociatedwiththe TGNistheanchor(50).OnecandidateforthisanchoristheknowntetherGolgin Coiled Coilprotein185(GCC185).GolginproteinsareintegraltotheGolgicomplex andfrequentlyactasanchorsforvesiclesmovingtoandfromthisorganelle. ThreereasonshighlightGCC185asaputativeanchor.1.Thisproteinhas beenshowntolocalizetothetgnandtobeanecessarytetherfortransportvesicles comingfromendosomes(51).thismodelwasbestworkedoutinregardstothe mannose 6 phosphatereceptor(m6pr),areceptorforlysosomalenzymesthat trafficsbetweenlateendosomes/lysosomesandthetgn.afterreleasingitscargoto lysosomes,m6prrecyclesbackthroughthetgn,usinggcc185asitsanchor. Withoutthisinteraction,M6PR saremislocalizedandinsteadfoundatrandom locationswithininthecell.asimilarmodelcouldbeimaginedforglut4vesicles, whichalsomustrecyclebackfromtheplasmamembrane.alsoofnote,whenglut4 colocalizeswithsyntaxin6/16atthetgn,thissamesubdomainisalsolabeledwith M6PR(12,22).ThisfurtheremphasizestheideathatGCC185maybeananchorfor boththem6prandforglut4.2.gcc185hasbeenshowntointeractwithrabs(52, 53,54).RabsaresmallGTPasesthatareactivewhenGTP boundandinactivewhen GDP bound.theyhavediverseroles(54,55).thoserelevanttothetopicathand includevesiclebudding,tethering,andfusion(57);vesiclemotilitywhenmolecular motorsareinvolved(58);andcontrolofsignalingpathways.rabsareknowntobe involvedinthesestepsinglut4trafficking,andsincegcc185hasbeenshownto associatewithmultiplerabs,itispossiblethatitisduringitsassociationwith

17 16 GCC185thatGSVsareformedandthenlatermovedfromtheirintracellularsiteto theplasmamembrane(56,59).thisbringsupreason3:gcc185anchorsnoncentrosomalmicrotubules.glut4usesbothactinandmicrotubulestoarriveatthe plasmamembrane(60).inparticular,insulinloadsglut4ontokif5bmicrotubule motorstomobilizeitfromtheperinuclearregion,nearthegolgiapparatus,andto targetittothecellperiphery(61).itispossiblethatgsvsareloadedfromtheirsite inassociationwithgcc185ontothesemicrotubulestotrafficouttotheplasma membraneinresponsetoinsulin. Summary ClarifyinghowGLUT4istargetedandstoredinitsspecificvesiclesiskeyto understandingthecascadeofeventsafterinsulinsignaling.datasuggesttug associatesspecificallywithglut4thatisstoredingsvs,andthatthesegsvsmay befunctionallytetheredatthetgntoawaitinsulinsignaling.identificationofthe anchoringproteinthattugusestoholdgsvsinplaceaswellasbetter characterizationofhowgsvsaremobilizedtotheplasmamembranewillclarify howinsulinactstocontrolglut4targetingandglucoseuptake.

18 17 SpecificAims: Thelong rangegoaloftheprojectproposedhereistobetterunderstandgsv formation,localization,andinsulin inducedtranslocation.asoutlinedabove,itwas hypothesizedthattugactsasafunctionaltetherbetweenglut4withingsvsand ananchoringproteinatthetgn(seefigure1).itwashypothesizedthatthistgn anchorproteinisgcc185.additionally,itwashypothesizedthattugmodification participatesinabiochemicalmechanismforthereleaseofgsvstotheplasma membrane.thismodificationmayoccuratalysineresidueinthec terminaloftug andwasconsideredtobeeithersumoylationoracetylation.totestthese hypotheses,hereitisproposed: 1.TocharacterizeinteractionsamongGLUT4,GCC185,andTUG,andtolearnif theseproteinsbindeachothersimultaneouslyasacomplexinhek293and unstimulated3t3 L1adipocytes. 2.TolearnifTUGisSUMOylatedoracetylated,todefinethesite,andtostudythe functionalroleofthismodification. Accomplishmentoftheseaimswillclarifybiochemicalmechanismsthat mediatetheactionofinsulintoenhanceglucoseuptake.thesemechanismshave highrelevanceforthepathogenesisofinsulinresistanceanddiabetes.

19 18

20 19 Methods: Aim1a.TotestwhetherGCC185bindstoTUGaswellastoGLUT4,initial experimentsemployedcoimmunoprecipitation(co IP)fromlysatesoftransiently transfected293cells.transfectionwascarriedoutbytreatingplatesof293cells grownindmemand10%bovinegrowthserumwithamixtureoflipofectamine, DNA,andDMEM.Ingeneral,onemicrogramofDNA,15microlitersof Lipofectamine,and250microlitersofDMEMwereusedforeachtransfectedplate. Thesenumbersweretitratedupordownwiththegoalofhavingequallevelsof proteinexpressionofthetransfecteddnaineachplate(thiswasdeterminedby comparingtheprelysateproteinlevels).cellswereallowedtogrowfor48 72hours post treatmentandwerethenlysedoniceusingice coldtnet(50 mm Tris, ph 8.0, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100).Thisallowedfortheinvestigationof non covalentinteractions. AplasmidencodingGCC185wasacquiredfromthePfefferlabatStanford Universityandwasamyc taggedconstruct.tugwasusedeitherintheuntagged formandpulleddownwiththespecificantibodymadeintheboganlab,calledl1c, orintheflag taggedform.glut4wasusedeitherintheuntaggedformandblotted forwiththespecificantibodyyu126,orwasusedinthev5taggedform.these proteinsweretransfectedinallpossiblecombinationstoprobeforpossible interactions.co IP swerecarriedoutusingaffinitymatrixbeadstothevarious proteintags(ieflag,v5,myc)orusingthespecificantibodytotheproteinfollowed byproteingbeads.westernblotsweremadewiththepre lysatesandeluates. Thesesampleswererunouton4 12%polyacrylamidegelsandovernightdrygel

21 20 transfersweresetupatalowvoltage(2 4Volts)toallowforcompletetransferof GCC185,alargeproteinof185kDa.MembranesweredevelopedusingPiercePICO ECLreagent.Imageswereacquiredonfilm. Aim1b.TomaptheinteractionbetweenGCC185andTUG,aseriesof truncationsofbothproteinswereusedtocarryoutcoimmunoprecipitationsin293 cells.againaffinitymatrixbeadsfortheflagormyctagwereused.thetug truncationsalreadyexistedinthelabinthefollowingforms:tugubx(aminoacids ),flag TUGdelta18(aminoacids1 532),andflag L1N1(TUGproteinwith aminoacids ).TwooftheGCC185truncationconstructswereobtained fromthepfefferlabandincludeddeltac110(aminoacids1 1574)anddeltaCC123 (aminoacids ).AdditionaltruncationconstructswerecreatedusingPCR followedbytopocloning,andwerebasedonfragmentsthatwouldfoldintocoiledcoilsbasedonthepaperbyhayesetal(53).thefragmentsincludedcc123(amino acids1 861),C110(aminoacids )andC343(aminoacids ).All ofthegcc185constructsweretaggedwithmyc. Aim1c.ToassesswhetherTUGmodificationisnecessaryforinteractionwith theputativeanchorproteingcc185.thelysinesitemostlikelytobemodifiedwas locatedandmutatedusingpcrtechniques.thisconstructwastermedtugk549r, anditmutatedthepenultimatelysinetoanarginine.itwasusedin coimmunoprecipitationswiththemyc GCC185constructtoassessforincreasedor decreasedinteraction. Aim2a.ToassesswhetherTUGisatargetofUbc9 mediatedsumoylation. AnoutlineofexperimentstoassessforproteinSUMOylationcanbefoundinan

22 21 overviewbyok Kyong,Park SargeandSarge(62).Inthefirstexperiment performed,sumo 1,andSUMO 2weretaggedwiththeHAtagandthentransiently transfectedinto293cellsalongwithtug.thehatagwasthenimmunoprecipitated andtugwasblottedfor.transfectedcellswerelysedinboiling.1%sdstoassess forcovalentmodificationswithsumo.inthesecondexperiment,tugwas immunoprecipitatedusingthel1cantibodyandproteingbeads,andsumowas blottedfor.inthethirdexperiment,purificationswerecarriedoutusingaffinity matrixbeadscontainingasumo interactionmotifandthentugwasblottedfor.in thefourthexperiment,affinitymatrixbeadscontainingboundsumowereusedto pulldownproteinstowhichsumonormallybound.beadscontainingbound ubiquitin,whichisnotbelievedtobindtug,wereusedasanegativecontrol.again, TUGwasblottedfor.TheTUGK549Rconstructwasagainusedinexperiments alongsidewildtypetugtoassessforanydifference. Aim2b.ToexaminewhetherSUMOmodifiesTUGin3T3 L1adipocytes,cells stablyexpressingaglut4reporterproteinwereused.thisreportercontainsseven mycepitopetags,aswellasgfpfusedinframeatthecarboxyterminus.ithasbeen validatedextensively,andisexpressedataboutfivefoldtheabundanceof endogenousglut4.themyctagsfacilitateimmunoprecipitationofglut4and associatedproteins,usinganimmobilizedmonoclonalantibody.inaddition,stable 3T3 L1cellshavealreadybeengeneratedandcharacterizedthatcontainboththe GLUT4reporterandoverexpressedTUG,ortheGLUT4reporterandadominant negative(ubx Cter)TUGfragment.Thesethreecelllineswerethenstablyinfected withtheha SUMO1andHA SUMO2vector.Infectionswereachievedbyfirst

23 22 transientlytransfecting293cellswiththedesireddnaandthepclvectorsystem (63).Themediafromthese293plateswasthencollectedandaddedtothe3T3L1 plates.successfuluptakewasconfirmedusingfacsandcellswerethensortedto get100%infectionrates.thesecellswerethendifferentiatedintomature adipocytes,treatedintheabsenceofinsulinorinthepresenceofinsulinfor2,5,or 10minutesandthenimmunoprecipitatedusingtheHAtag.Eluateswerethen immunoblottedtoassessforthepresenceofassociatedtug,andtodetermineif insulinaffectedthisassociation.toconcentratetheinteractionofthedesired proteins,thisexperimentwasalsodoneaftermakingmembranesandcytosol.in thisexperiment,threeplatesofthesametypewerecombined,andlysateswere spunintheultracentrifugefor30minutesat100,000rpm.theseparated membranesandcytosolwerethenimmunoprecipitatedusingthehatagandtug wasblottedfor. Aim2c.ToexaminewhetheracetylationisthemodificationofTUG,293cells weretransientlytransfectedwithtugortugk549r.after36hours,thesecells weretreatedwithtrichostatina(tsa)ataconcentrationof0.5micromolarfor16 21hoursandwithnicotinamide(NAM)ataconcentrationof5milliMolarforthelast 6hours.BothTSAandNAMareknowntoinhibitdeacetylation.Cellswerethen lysedinboiling0.1%sds,triton X100wasaddedto1%,andTUGwas immunoprecipitatedusingthel1cantibody.eluateswerethenblottedusinga commercialantibodytoacetylatedlysine.

24 23 Results 1a:DeterminationofaninteractionbetweenGCC185andTUG.Intransfected 293cells,immunoprecipitationofthemyctagonGCC185resultedinthe simultaneousprecipitationoftuginthecelllinesinwhichbothtugandgcc185 weretransfected.inthelaneswithonlymyc GCC185oronlyTUG,thiseffectwas notseen(seefigure2).theinteractionoftugandgcc185wasdemonstratedeven inlane6offigure2,wherethetransfectionofthegcc185constructoccurredata lowerefficiency.theconverseofthisexperimentwasalsodoneandshowedthat whentheflagtagontugwasimmunoprecipitated,therewas coimmunoprecipitationofgcc185.again,thiseffectwasonlyseeninthecelllines inwhichbothflag TUGandmyc GCC185wereexpressed(seeFigure3).Bothof theseexperimentswererepeatedonthreeseparateoccasions. Figure 2. Immunoprecipitation of myc-gcc185 and Western Blot of TUG. In this experiment, 293 cells were transfected with combinations of TUG, TUG K549R mutant, GFP-Golgin-160, and myc-gcc185. Golgin-160, another large Golgi-resident protein was used to compare it to GCC185. The myc tag on GCC185 was immunoprecipitated and equal amounts of the various proteins were blotted for. TUG and TUG K549R were blotted for with anti-l1c, GCC185 with anti-myc, and GFP-golgin 160 with anti-gfp.

25 24 Figure3.Immunoprecipitationofflag TUGandWesternBlotofmycGCC185. In the reciprocal experiment to the one in Figure 2, 293 cells were transfected with combinations of flag-tug, flag-tug delta18, and myc-gcc185. The flag tag was immunoprecipitated and equal amounts of the various proteins were blotted for. TUG and flag-tug delta18 were blotted for with anti-flag and GCC185 with anti-myc. ExperimentswerealsodoneinwhichV5 GLUT4,myc GCC185andflag TUG wereexpressedandthemyctagwasimmunoprecipitated.thiswasdonewiththe goalofshowingthattugwasacentralproteinbindingbothglut4andgcc185,so thatpullingdowngcc185wouldbringdowntugandtheassociatedglut4in complex.unfortunately,thev5 GLUT4constructdidnotexpressinalloftheplates inwhichitwastransfected,sotheseresultscouldnotbeinterpreted(datanot shown). 1b.MappingtheinteractionbetweenTUGandGCC185.Inthisexperiment, flag TUG,flag L1N1(TUGproteinwithaminoacids ),myc deltac110 (GCC185proteinwithaminoacids1 1574),andmyc deltacc123(gcc185protein withaminoacids )weretransfectedinto293cells.Themyctagwas immunoprecipitated,andtheflagtagwasblottedfor.therewasaspecificbandin thelaneswithflag TUGandmyc deltacc123(seefigure4).therewasnobandin

26 25 thelanewithflag TUGdelta18andmyc GCC185(seeFigure3).Theabsenceofa bandinthelanewithflag TUGdelta18andmyc GCC185wasseenintwoseparate experiments.unfortunately,thecellstransfectedwithmyc deltac110diedon multipleoccasions,soitwasnotpossibletonarrowdownthesiteongcc185any further. 1c.DeterminingwhetherTUGmodificationisnecessaryforinteractionwith GCC185.Fortheinitialassessmentofthis,293cellsweretransfectedwith combinationsoftug,tugk549r whosepenultimatelysinehasbeenaltered,with thepurposeofmakingitunmodifiable andmyc GCC185.Whenthemyctagwas immunoprecipitated,andtheeluatewasblottedfortug,itwasseenthatgcc185 andtugcamedowntogether,butthattugk549randgcc185didnotcomedown togetherorcamedownsignificantlyless(seefigure2).thetugk549rmutantwas transfectedatanefficiencythatwasabout50%lessthanthatofthetugconstruct, howeverthedifferenceintheamountoftugk549randtugintheeluatelanesis greaterthan50%.thisresultwasseenontwoseparateoccasions.

27 26 2a.AssessingTUGforSUMO modificationin293cells.inmultipleseparate experiments,293cellsweretransfectedwithdifferentcombinationsofha SUMO1, HA SUMO2,andTUG.InsomeexperimentstheHAtagwasimmunoprecipitatedand TUGwasblottedfor,andinotherexperimentsTUGwasimmunoprecipitatedand thehatagwasblottedfor.innoneoftheseexperimentsdidthetwoproteinscome downtogether.whenaffinitymatrixbeadscontainingsumo interactingmotifs wereused,notugcamedown.whenaffinitymatrixbeadscontainingboundsumo wereused,theydidnotbindtotug,asnotugcamedownintheeluate.finally, whentugandthemutanttugk549rweretransfectedtoseeifsumointeracted withoneandnottheother,nodifferencecouldbedetected. 2b.AssessingTUGforSUMO modificationin3t3l1adipocytes.sixsetsof 3T3L1adipocyteswereanalyzed:thefirstexpressedexcessGLUT4andHA SUMO1, thesecondexpressedexcessglut4,tug,andha SUMO1,thethirdexpressed excessglut4,ubx CterminalTUGfragment,andHA SUMO1,thefourthexpressed excessglut4andha SUMO2,thefifthexpressedGLUT4,TUG,andHA SUMO2, andthesixthexpressedexcessglut4,ubx CterminalTUGfragment,andHA SUMO 2.Aninsulintimecourse(0,2,5,and10minutes)wassetup,theHAtagwas immunoprecipitated,andtugwasblottedforintheeluates.innoneoftheplates wastugseentocomedown.inasimilarexperiment,membranesandcytosolwere separatedinthefirst3celltypesandhawasagainimmunoprecipitated,thistime withouttheinsulintimecourse.whentugwasblottedforintheeluates,itwasnot seentocomedowninanyofthelanes.

28 27 2c.AssessingTUGforacetylationin293cells.293cellsweretransfectedwith eithertugortugk549randthentreatedwitheithernadandtsa(toprevent deacetylation)orwithnothing.tugwasthenimmunoprecipitatedinthecell lysates.intheprelysates,theplatestreatedwithnadandtsahadsignificantly moreproteinontheacetylatedlysineblotascomparedtotheuntreatedplates.in theeluatesblottedforacetylatedlysine,therewasabandinthetuglanetreated withnadandtsa,butthisbandranat51kdaasopposedtothenormallevelfor TUG,whichis60kDa.Thus,itwasunclearwhatthisbandrepresented.Ofnote, anothermemberofthelaboratory(charisseorme)conductedanexperimentin whichsheimmunoprecipitatedtheflag tagsonbothflag taggedfulllengthtugand flag taggedtugdelta18.shethenblottedtheeluateswithanacetylated lysine antibody(whichdetectsacetylated lysineresidues).theimmunoprecipitated proteinwasdetectedwhenflag TUGwasused,butnotwhenflag TUGdelta18was used(figure5).thissuggeststhattugisacetylatedanditscterminal18amino acidsarerequiredforthisacetylation.

29 28

30 29 Discussion Attheoutsetofthisproject,thegoalwastogainabetterunderstandingof howgsvsformandwheretheylocalizewithinadipocytesandmyocytesinthe absenceofinsulin.coimmunoprecipitationdatafrom293cellssuggestthatgcc185 andtugdoinfactinteract.thisissupportedbythefactthattugcanbepulled downwhengcc185isimmunoprecipitated,withthereciprocalfindingalsobeing demonstrated.thisdataislimitedbythefactthatitwasacquiredfrom293cells,a celllinethatisnotinsulinresponsive.however,tugwasshowntosequester GLUT4awayfromthecellsurfaceintransfected293cells(32).Thus,althoughitisa promisingresult,itneedstobeexpandeduponwithexperimentsininsulinresponsivecelllines,suchasthe3t3l1adipocytes.insuchexperiments,inaddition toseeinganinteractionbetweentugandgcc185,onemightalsoexpecttoseea lossofthisinteractionafterstimulationofthecellswithinsulin.thiswouldfitthe hypothesizedmodelinwhichtugfunctionallytethersgsvstoanintracellular anchor,fromwhichgsvsarereleasedfollowinginsulinstimulation. InregardstomappingtheinteractionbetweenTUGandGCC185,the preliminarydatasuggestthatthecterminusoftugisnecessaryforthisinteraction, sincethetugdelta18constructdoesnotappeartointeractwithgcc185.these datafitwithprevioustugdata,whichshowedthatthenterminusoftug interactedwithglut4andthatthecterminusoftugwasnecessaryforitto sequestergsvsawayfromtheplasmamembrane.thefindingthatwhentuglacked itscterminusitwasnolongerabletosequestergsvs,isconsistentwiththisnew

31 30 findingthatthecterminusoftugisnecessarytobindtogcc185,theputative anchor. Furthermore,thefactthatafragmentofTUG scterminus,theubx Cter, appearstoactasadominantnegativealsofitsthismodel.presumably,thisubx Cterfragmentcanbindtotheanchoringsite,andinsodoingoccupiestheplaces wherefulllengthtugwouldnormallybind.sincetheubx Cterfragmentlacksthe GLUT4bindingsite,thiswouldpreventTUGfrombeingabletotetherGSVs.This makesitimportanttotestthehypothesisthattheubx CterfragmentandGCC185 bindtooneanother.italsoraisestheneedforfunctionaldatainaninsulinresponsivecellline.forexample,inthelabthereisa740++3t3l1adipocytecell linethathaspermanentlyknocked downtug.ifthetugdelta18constructwere addedbacktothis,itshouldbeunabletorescuepropergsvlocalization,sinceit wouldpresumablylacktheanchor bindingsite. MappingoftheTUGbindingsiteonGCC185willalsobeimportant.The preliminarydatasuggestthattugbindstothec terminusofgcc185.further narrowingofthisinteractionwasnotcompletedasplatestransfectedwiththeother fragmentsdied. Itwouldalsobecrucialtodemonstratetheco localizationofgcc185and GLUT4inaninsulinresponsivecell linesuchas3t3 L1adipocytes.Thiswas attemptedusingelectroporationfollowedbycellstainingbutthecellsdidnot survive.itseemslikelythattheharshconditionsimposedonthecellsfromthe combinationofthesetwomethodsresultedinthisendpoint.assuch,acherry GCC185constructwascreatedtobeusedalongsidethealreadyexistingGFP GLUT4

32 31 construct.byusingthesetwoconstructs,electroporationwouldstillbenecessary, butthestainingstepcouldbeskipped,sincebothconstructswouldfluoresce.in addition,someofthesecellswouldhavetobetreatedwithinsulintoshowthatthis resultsinlossofco localization.alternatively,stablecelllinescouldbemade expressingcherry GCC185andGFP GLUT4,ortransientlystablecellscouldbe createdwiththetwoconstructsusinganadenovirusvector. ThedatashowingthattheTUGK549Rmutantsignificantlylessensthe interactionbetweentugandgcc185ispromising.thismutationwasmadebased onthefactthatthissiteseemedalikelysiteofmodificationandwasseeninthe UniProtKBdatabasetobeanacetylatedresidue.Thismutationwouldpreventsuch acetylationfromtakingplacebyeliminatingthelysine.itisthereforeinteresting thatthismutantcannotinteractwithgcc185,andsuggeststhattugacetylationis necessaryforitsassociationwithgcc185.toassessthetruesignificanceofthis finding,experimentswouldhavetobeset upinaninsulinresponsivecellline.for example,ifthe740++celllinewereused,thek549rmutantcouldbeaddedback anditwouldbeexpectedtobeunabletolocalizegsvstotheirappropriate intracellularlocation,sinceitwouldnotbindtogcc185,thepresumedanchoring site.amutantoftheubx CterwasalsomadewiththisK549R,andthiscouldbe testedin3t3l1adipocytesaswell.thepredictedoutcomeherewouldbethatthe mutantubx Cterwouldnolongerbeabletoactasadominantnegativeconstruct, sinceitpresumablycouldnolongerinteractwithgcc185. Attemptstoverifythatacetylationoccursatthissiteyieldednon specific results.hence,theseexperimentsshouldfirstbere attemptedusingapositive

33 32 controlsuchasp 53,whichisknowntobeacetylated.Itisalsopossibletoraise antibodiesagainstspecificacetylatedlysinecomplexes,thoughthisisanexpensive andtime consumingendeavor.itwouldalsobepossibletolookattheroleof acetylationin3t3l1cellseitherbyusingdeacetylaseinhibitors,whichmight strengthentheinteractionbetweengcc185andtugandhindergsvtranslocation, orbypromotingdeacetylases,whichmightpreventtheinteractionbetweengcc185 andtug. Aspartofourinitialhypothesis,weproposedthatthe54kDafragmentsof TUGrepresentedamodifiedformofprocessedTUGthatalloweditsremovalfrom theanchoringsite.wehadhopedtocharacterizethismodification.giventhevariety ofexperimentsdoneinboth293and3t3l1adipocytes,alongwiththerepetitionof saidexperiments,itseemsthatsumoisnotthemodifieroftug.thismaybe consistentwiththeworkfromliuetal.,inwhichitwasshownthatthecatalytically inactiveformofubc9wasjustaseffectiveatallowinggsvmovementtothecell surfaceastheactiveformofubc9(40).sincethecatalyticroleofubc9isto facilitatesumoylation,theobservationthataninactiveubc9stillallowsforgsv translocationsuggeststhatsumoylationdoesnotplayaroleintheuntetheringof GSVs.Yet,becausethisworkusedanadenovirustoexpressUbc9transiently,and giventhatthe54kdfragmentisproducedaftertugcleavage,itisformallypossible thatdisruptingsumoylationmightnotaffecttheinitialcycleofgsvrelease. Nonetheless,itseemsmostlikelythatTUGSUMOylationisnotinvolvedinGSV translocation.

34 33 Thenatureofthemodificationcausingtheshiftfroma42kDatoa54kDa productremainsunknown.onepossibilityisthatthismodificationisadpribosylation.deacetylationandadp ribosylationhavebeenshowntooccurin concertandarebothstimulatedbynad(64).ifacetylationoftugisneededforitto associatewithgcc185,thentug sdeacetylationandsubsequentadp ribosylation mayallowittodissociate.further,theadp ribosylationcouldaccountforthe54 kdabandoftugseenfollowinginsulinstimulation.thishypothesisdeserves furtherinvestigation. Itshouldbestatedthatinexperimentsmeanttorepresentaninsulinresponsivecellmodel,3T3L1adipocyteswereusedtobeconsistentwithother GLUT4literatureinwhichthisisgenerallythemodelcellsystem.Thisisinpart becausemyocytecelllineshaveproveddifficulttomanipulate.itispresumedthat thetugandtheanchorwouldplayasimilarroleinmycocytes,sincethesecellsare similarlyresponsivetoinsulin.however,thisissomethingthatwouldeventually havetobeverifiedinamyocytecellline.additionally,gsvmovementtotheplasma membraneinmycocytescanalsobestimulatedbymusclecontraction,anditisnot clearwhetherthisusesthesamesignaltransductionpathwayasinsulintorelease GSVs. Althoughtheseresultsneedtobedevelopedfurther,theyareexciting. AdditionalcharacterizationoftheTUG/GCC185interactionmayfurthersupportthe ideathatgcc185isindeedtheanchortowhichthegsv TUGcomplexbinds.This wouldthenprovideaclearersenseoftheproteinsinvolvedintheinsulinresponsivecomplex.aspreviouslyreviewed,theformationandlocationofgsvs

35 34 playakeyroleinglucosehomeostasis,makingthisvaluableinformation.ifgcc185 infactplaysthisrole,itwouldexplainwhyglut4andirapcolocalizewithtgn markersatvariouspointsinadipocytematuration.thefactthatgcc185is associatedwithmicrotubuleswouldalsoprovideatransportmechanismwhereby GSVscouldmovetotheplasmamembrane.Inaddition,theassociationofGCC185 withrabs,wouldputat handthecatalyststoallowthistranslocation. ItisalsoinstructivetoconsiderthefactthatTUGisexpressedinalmost everycelltype.thisraisesthepossibilitythattugplaysasimilarfunctional tetheringroleinmultiplecelltypes.ifthatisthecase,theinteractionoftugand GCC185maybeconserved,withtheTUG associatedvesiclebeingthevariable.for thisreason,othercelltypeswithregulatedsecretionshouldbeinvestigatedtoseeif asimilarset upexists.suchcelltypesaremultipleandwouldincludegastric parietal(inwhichtheh+/k+pumpistranslocatedtothecellsurface),renaldistal collectingtubules(inwhichaquaporinchannelsaretranslocated),andneurons(in whichamp Aglutamatereceptorsaretranslocated)forastart.Giventhefinelycontrolledstepsthatmustgointoformingthesesecretedvesiclesandthen stimulatingtheirrelease,itwouldmakesenseiftheunderlyingproteinswere conserved.

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