Ato o orsodne([email protected]) correspondence for *Author Toronto, Toronto, of Canada. University the 3M2, Medicine, and M5S of Pharmacy, ON Faculty of Biochemistry, Faculty of Dan Department Leslie Sciences, Pharmaceutical of hraooi eSebok,Sebok,Q 1 N,Canada. 5N4, J1H QC Sherbrooke, Sherbrooke, de Pharmacologie bqiyainadihbtdteHC1mdae eyln fthe of recycling HACE1-mediated the b inhibited and ubiquitylation bqiyainadatvto faRbGPs spooe ya by Ve promoted is GTPase a b of activation and Ubiquitylation ARTICLE RESEARCH ß eevd1 pi 03 cetd1 coe 2013 October 12 Accepted 2013; April 12 Received hormones, as such mediators cellular of array 1 vast responses studied physiological a most mediate They and to 2001). largest al., et the Schio Venter 2012; and of (Fredriksson one of form family and represent genome (GPCRs) human receptors G--coupled INTRODUCTION Trafficking Rab, receptor, G-protein-coupled GPCR, WORDS: KEY HECT- the a of HACE1, recycling by the show that promotes b we ligase, studies Here, ubiquitin understood. depletion domain-containing poorly and still are overexpression human vesicle-mediated them proteins connecting of cargo mechanisms of GTPase the targets to and regulation Ras well-recognized step their but are the diseases every of Rab Rab lately. branch almost trafficking. attention largest regulate the great superfamily, form received which has GTPases, transport G-protein-coupled of vesicular regulation trafficking Cargo-mediated of GTPases. that Rab by shown regulated is have receptors others and We ABSTRACT a1a sosre ywsenbo nlss LC-MS/MS analysis. Lys145. undergo to on blot failed ubiquitylated mutant western is Rab11a-K145R Rab11a A by that determined observed experiments as Rab11a, AE,idctn htuiutlto fLs4 sivle in of involved Co-expression is Lys145 Rab11a. of of ubiquitylation activation that indicating HACE1, eetr hog a1adpnetmcaim Interestingly, mechanism. Rab11a-dependent the a through receptor, h bqiyainadatvto faRbGTPase. Rab G- a a of as activation This and such inducing ubiquitylation by protein, trafficking the Rab11a. own cargo its a regulate on with can whereby receptor, protein-coupled regulatory pathway ubiquitylation, demonstrated new novel their a effects a suggests through report functional GTPases other We associated Rab of tested. not of were but mechanism that Rab8a, GTPases and Rab6a Rab of ubiquitylation potentiated lnqeE Sante Clinique la de Sciences des Ste evc eRuaooi,De Rhumatologie, de Service 2 2 rnkLachance ´ronik 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,1113doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. R a1a u o a1aK4R a ciae by activated was Rab11a-K145R, not but Rab11a, AR. ( receptor -adrenergic 2 pai Nadeau ´phanie b RHC1complex AR–HACE1 2 Ri ojnto ihHC1tigrduiutlto of ubiquitylation triggered HACE1 with conjunction in AR ´ ineL e,Sebok,Q 1 N,Canada. 5N4, J1H QC Sherbrooke, Bel, tienne-Le ´ Universite , 1,2 1,2 ´ atmn eMe de partement b aeDegrandmaison Jade , Ste , 2 R,apooyia G-protein-coupled prototypical a AR), ´ paeAngers ´phane eSebok,teCnr eRecherche de Centre the Sherbrooke, de ´ eie Faculte decine, t,20;Hro tal., et Harrow 2005; ¨th, b 2 AR–HACE1-induced b 2 RHC1also AR–HACE1 3 2 n enLcParent Jean-Luc and ´ ntttde Institut , eMe de 1,2 %o the of 4% 3 ,Se Department ´ eieet decine b bsinMarois ´bastien 2 AR– h atta oeta 0 fpecie rg agtGPCRs target drugs prescribed of 2009). 30% Hall, and than Ritter more degradation 2002; then that lysosomal al., fact et can or Pierce The surface and 2009; al., cell endosomes et the (Costanzi into to the internalize recycling and stimulation, undergo GPCRs ligand-responsive agonist of a in Following majority typically membrane form. are plasma signaling-competent GPCRs the C-terminus. to N- intracellular delivered extracellular an an and transmembrane and loops, terminus extracellular seven with three ions topology of loops, molecular intracellular core common peptides, hydrophobic a share a nucleotides, GPCRs All photons. lipids, neurotransmitters, ensonta h PRagoesnI ye1 receptor 1A type II and has angiotensin It GPCR 2012). (Barr al., the et (AT domain that Xiong 2011; been shown al., (Tre-2/Bub2/Cdc16) have been et Marat GEF), TBC1 share 2010; to Rab5 Lambright, known common are (a GAPs Rab Rabex5 a However, GEFs. as Rab as Vsp9 such described and GEF), few, proteins neoplastic Rab39 a (a and Rab6IP1 domain cite as normal To such proteins in date. domain expressed cells) to (differentially GTPases DENN have small GAPs some 2007). these and GEFs for al., few identified very et Rabs, been of (Schwartz number large GAPs) the Despite proteins, (guanine activating hydrolysis GTP nucleotide and (guanine GEFs) GTP factors, to exchange GDP so, of nucleotide exchange do the To promote regulators conformations. distinct (GTP-bound) active and 2011; (GDP-bound) Li, 2012; al., for 1990). et Rutherford, (Kelly foundation from and GTPases resulting Richards Rab essential pathologies are of of an function examples aberrant date and represent and diseases diabetes study bone to neurodegeneration, Cancer, intensive targeting. drug but of effective remains disease area well- to disease, contribute an proteins are dysregulated accessory transport human and how proteins proteins the of Rab coordinate Elucidation in These therapeutically. with underexplored 2011). targets they correlates Novick, recognized which and that a (Hutagalung between has localization GTPase Rab compartments subcellular Each vesicle-mediated 2001). of small McBride, distinct step and Ras-related every (Zerial of almost transport in branch involved largest are the al., GTPases, et forming Seachrist GTPases, 2009; Rab al., al., et et Parent Wikstro 2011; (Hamelin 2002; al., trafficking et GPCR Lachance of 2005; regulators specificity, key as and is GTPases efficacy targets. trafficking pharmaceutical drug new identify actual intracellular the to the also of their but improve comprehension to controlling better essential a events (Hopkins Therefore, disease cellular 2002). of treatment Groom, the and in importance their highlights ieohrGPss a rtissutebtenteinactive the between shuttle proteins Rab GTPases, other Like Rab characterized previously have others and laboratory Our 1A 1,2, )icessteGPbnigo a5.Ti td not study This Rab5a. of GTP-binding the increases R) 1,2 * ,Me me l,20;Zage l,20) oeta 60 than More 2009). al., et Zhang 2008; al., et ¨m lneRobitaille ´lanie 3 aulGe Samuel , ´nier a hlcs three -helices, 1,2 , 111

Journal of Cell Science Agei ta. 04 age l,21;Zage l,2007) al., et Zhang of 2011; expression al., surface et Tang cell 2004; increased al., activity) E3-ligase et deficient (Anglesio with and mutant Myc-HACE1 of (a expression Myc-HACE1-C876S Transient ELISA. by determined ag rti a ietisontrafficking. own its which direct by can GTPases protein Rab cargo for mechanism a regulatory new a uncover 0mntso eyln Fg E.W eete neetdin after interested then opposite were We the 1E). (Fig. did recycling of HACE1-C876S minutes 60 whereas recycling, 5 with time-course 37 treated recycling at by cells minutes 15 studied in further experiments was This recycling. HA-tagged interact of also Seachrist could Immunoprecipitation 2009; receptor the HACE1. al., whether with et assessed we Parent 2000), 2012; al., et al., et (Hammad trafficking bqiyainadatvto fRb1,wihi unregulates turn in which Rab11a, b of activation and ubiquitylation hte AE a bet modulate investigated we to 2000), able al., al., et was et Moore HACE1 Seachrist 2012; 2009; whether al., al., et et Hammad Parent 2010; and 2004; Dong al., 2007; et al., Dong et 2007; (Awwad Wu, proteins Rab various by regulated erpr htc-xrsinof co-expression that report we ciaino -a n aiiae t idn oseii effectors specific to binding its facilitates and K-Ras of activation GEF effect a possessing proteins this recruits the or for whether GEF activity. unclear a necessary as remains acts it seems itself between However, receptor interaction GTPase 2002). direct al., the et a (Seachrist and that underlined receptor also the it activity control but might Rabs, GPCRs that of evidence first the reported only ARTICLE RESEARCH 112 promoted recycling HACE1 that receptor confirmed allow obtained Data to 1E). and (Fig. internalization receptor further 10 in events these specific Because regulate Rab11 2011). and to al., Rab4 et known Rab1, (Tang are that HACE1 GTPases reported with colleagues the associate of recycling and the regulates Tang and with interacts HACE1 RESULTS was ubiquitylation Rab on interactor effect Rab no E3 a Rab but as containing Because 2011) identified Interestingly, repeat reported. al., was However, far. et ankyrin 1) so (Tang ligase and ubiquitylation. protein described domain ubiquitin been (HECT as not HACE1 of has be post-translational such also number one ubiquitylation involving might family, mechanism small GTPases Rab general modifications Rab the a the of of by activation size controlled Considering that the speculate and 2011). could GEFs, al., Rab characterized et (Sasaki ramn ihmnni niie h feto AE on HACE1 1D, Fig. of in effect seen the be the inhibited can of inhibitor, As monensin internalization in recycling 2005). with a increase al., of internalization treatment presence et an the results, (Hamelin in these monensin Because out carried explain were 1C). could assays (Fig. recycling effect receptor no virtually muorcpttdwt h eetr(i.1) h fetof expressed effect transiently The of 1A). trafficking (Fig. on receptor HACE1 co- the HACE1 with endogenous that immunoprecipitated revealed cells HEK293 in expressed bevdi h rsneof presence the in observed AE infcnl erae h paetagonist-induced the apparent that of the established analyses decreased internalization Time-course significantly 1B). required (Fig. not HACE1 is effect enzyme the this of for activity catalytic the that indicating 2 Rrccig bqiyaino te pcfcRb a also was Rabs specific other of Ubiquitylation recycling. AR twsrcnl ecie htmn-bqiyainenhances mono-ubiquitylation that described recently was It m rpaoo o h niae ieprost prevent to periods time indicated the for propranolol M ˚ b ,adte nuae nDE containing DMEM in incubated then and C, b 2 R niaigta AE regulates HACE1 that indicating AR, 2 arnri eetr( receptor -adrenergic b 2 R hra AE-86 had HACE1-C876S whereas AR, b 2 RHC1 oehr u data our Together, AR–HACE1. b 2 Rwt AE nue the induces HACE1 with AR b b 2 m Rb ogl 25%, roughly by AR 2 spoeeo for isoproterenol M Rtafcig Here, trafficking. AR b 2 R rfikn is trafficking AR) b 2 Rwsthen was AR b b 2 2 Rstably AR AR b b b 2 2 2 AR AR AR eiyn h noeosclclzto fHC1with HACE1 of colocalization endogenous the verifying el rnfce ihpDA.C-xrsino a4 or on Rab4a effect of significant any Co-expression have in not pcDNA3. levels did HACE1 with with Rab11a of compared transfected Transfection recycling cells 3A). receptor of (Fig. promoted combinations Rab11a strongly different or with Rab4a cotransfected HACE1, cells in measured sditrhnebyi h rsn eot ssoni i.1Fa– Fig. in and shown HACE1 As report. d, present the in interchangeably used ihHC1oeepeso n salse e oefor role new a obtained established results and the in validated HACE1 overexpression data HACE1 These 2C). with (Fig. control the by inhibited AE-eitdices in increase HACE1-mediated noeosRb1 ihsRAsgiiatyrdcdthe reduced significantly of siRNA depletion the that of with shows recycling 3B Rab11a Fig. system. endogenous our in HACE1 through oprdwt xrsini oto el Fg A.Teewsa was There 2A). (Fig. cells , control in expression with compared oprdwt 5 nenlzto,rsetvl)(i.2B). (60% (Fig. siRNA respectively) siRNA control internalization, HACE1 with 45% with b transfected with transfected cells compared were with cells compared when receptor rfikn fsal expressed stably of trafficking basal 1Fk–s). its in (Fig. seen and progressed that recycling HACE1 to receptor receptor similar as of was the conditions receptor distribution the with with intracellular colocalized colocalization a The predominantly in 1Ff–i). it (Fig. perinuclear and the where towards compartments concentrated region that intracellular HACE1 of into distribution membrane plasma od o ooaiainsuiswr are u sn cells using out carried were studies HA- colocalization expressing stably so, do To rvossuissoe that showed studies Previous regulates HACE1 hog n fteetoGTPases. two these of one through ndtriigwehrHC1regulated 2009; HACE1 al., interested whether et thus were associate we Parent determining 2011), to al., 2004; reported et in (Tang is al., Rab11 HACE1 and Because Rab4 et with 2000). al., (Moore et Rab11 Seachrist and Rab4 infcn ifrne ewe h rfikn rprisof properties trafficking the expressed between transiently differences significant nFg Fthat 1F Fig. in rnfce ihHC1sRA elto fHACE1 of the of Depletion expression surface siRNA. cell the HACE1 reduced significantly with transfected nrclua eilsi E23cls(i.3) Fluorogram 3C). (Fig. cells HEK293 peripheral in in also vesicles but intracellular compartments, mostly colocalized intracellular Rab11a, perinuclear endogenous in with of levels distribution colocalize low reflect at to expressed to showed HA-Rab11a, studies and previously microscopy HACE1 endogenous Confocal shown that 2009). been al., et has (Parent Rab11 it and HACE1 in involved and effectors 2011) al., other et with (Tang HACE1 compete with interact to to Rab4a of ability Interestingly, h eetrrsle nitraiaino the of internalization in of cell stimulation resulted Agonist the conditions receptor pixels). basal of the colocalizing under proximity extracted region, 1Fd, perinuclear the (Fig. the in in cytoplasm, and membrane the throughout found b togyehne yRb1 oepeso whereas co-expression effect Rab11a HACE1-mediated the prevented by on Rab4 of enhanced cotransfection strongly 2 2 3 nraei paetaoitidcditraiaino the of internalization agonist-induced apparent in increase 33% R(upeetr aeilFg 1.Bt ytm eethus were systems Both S1). Fig. material (supplementary AR Rrccigatraoitidcditraiainwas internalization agonist-induced after recycling AR h ucinlipiaino noeosHC1o the on HACE1 endogenous of implication functional The b 2 Rrccig hscudb osbyepandb the by explained possibly be could This recycling. AR ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal b 2 , Rcl ufc xrsinadrecycling. and expression surface cell AR b 5 nclsdpee fHC1cmae with compared HACE1 of depleted cells in 25% b 2 b Rrccigi h rsneo AE was HACE1 of presence the in recycling AR 2 b 2 b Rclclzsitaellrywt endogenous with intracellularly colocalizes AR 2 Rclclz noitaellrcompartments intracellular into colocalize AR RrccigtruhRab11a through recycling AR 2 Rwe xrse ln n niie the inhibited and alone expressed when AR b b 2 2 R oprtv nlssrvae no revealed analyses Comparative AR. Rcmae ihsal expressed stably with compared AR b 2 b Rrccigb 0.W show We 40%. by recycling AR 2 Rrccigi euae by regulated is recycling AR b 2 Rwsasse ncells in assessed was AR b 2 Rrccigwsthus was recycling AR b b b b 2 2 2 2 Rrecycling AR Rrecycling. AR Rrecycling AR Rfo the from AR b b 2 2 AR. AR

Journal of Cell Science EERHARTICLE RESEARCH i.1. Fig. e etpg o legend. for page next See ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal 113

Journal of Cell Science eetratvto Fg A o ae,lns47.B contrast, By 4–7). lanes panel, top b 4A, (Fig. activation receptor ieiaino h ml Taeo t soito with association its or GTPase where 12–15 lanes of small protein the another of dimerization b bottom in arrows see 4A, the (Fig. a Rab11a Indeed, panel). of profile migration bevtos(i.4,) u ale idnssoe htthe 12–15). that showed findings lanes these earlier Our support b 4B,C). immunoprecipitation (Fig. panel, receptor receptor observations to the the with on of up co-immunoprecipitation second normalized experiments Rab11a even independent and multiple activation HACE1 4A, of (Fig. receptor analyses Densitometry by minutes accentuated 120 was which i.1 AE neat with interacts HACE1 1. Fig. ARTICLE RESEARCH 114 of co-immunoprecipitation of on as b co-expression well as Rab11a stimulation or receptor of HACE1 effects the evaluated then We b colocalization between of Colocalization degree high Rab11a. a and is HACE1 there between that revealed analysis u rmlstso el ornfce ihepyvco,Myc- vector, empty with cotransfected cells Rab11a HACE1, of lysates of from carried were out the FLAG-Rab11a reminiscent and of in HA-Rab11a experiments dimerize tagged co-immunoprecipitation differentially condition, could receptor, the Rab11 of this whether presence verify in To a ubiquitylation. Instead, observed partner. Rab11a were another decreased with HACE1 association of , its presence or the dimerization that indicate could spoeeo o o10mntso el xrsigthe expressing interaction cells The 4A. of Fig. the minutes in and 120 HACE1 indicated between proteins to of 0 combinations for isoproterenol F,edgnu AE n a1awsas eetdin detected also was inset). Rab11a 3Cd, (Fig. and compartments perinuclear HACE1 endogenous GFP, fHC1atvtsRb1,wihwudrsl nreduced in result would which Rab11a, co-expression b in activates and presented stimulation HACE1 receptor results that the of suggest with could together 4, This, Fig. 2009). al., et (Parent to in reduction 8–11). returned significant lanes but a panel, caused second the treatment co-expression 4A, HACE1 agonist (Fig. note, minutes of Of 120 minutes at 60 levels basal and 15 after pcfcmncoa rioyi g1cnrlatbd nlstsfo E23clssal xrsigHA- expressing stably cells HEK293 from lysates on antibody control IgG1 isotypic or monoclonal specific peri elw(,,,) cl as 10 bars: Scale (c,h,m,r). yellow in appear n oalwrcpo eyln.Clswr hnfxd emaiie n aee ihrbi oylnlat-AE nioy h hr mg nthe on image third The antibody. anti-HACE1 polyclonal rabbit with labeled and red-labeled permeabilized the fixed, of then image were merged Cells a recycling. represents receptor allow to and h ecnaeo eetritraiainwscluae.()Clswr rae ih5 with treated were Cells (E) calculated. was internalization receptor of percentage the AE-pcfcplcoa nioy h lt hw r ersnaieo he eaaeeprmns B E23clswr ornfce ihFL with cotransfected were cells HEK293 (B) experiments. separate three of representative are shown blots The antibody. polyclonal HACE1-specific spoeeo o 5mntsa 37 at minutes 15 for isoproterenol * 10 containing n cN3 idtp y-AE rMcHC1C7S xrsino elsraercpo a esrdb LS sn LGseii monoclonal FLAG-specific 25 a with pre-treated using were ELISA Cells by (D) measured calculated. was was receptor internalization 5 receptor surface with of cell treated percentage of were Expression Cells Myc-HACE1-C876S. (C) or antibody. Myc-HACE1 wild-type pcDNA3, and a eetdb LS n h ecnaeo eetrrccigwscluae.Rslsaemeans are Results calculated. was Student’s recycling unpaired and receptor paired of using percentage determined was the significance and statistical ELISA by detected was t37 at P 2 2 2 2 2 2 Rpooe AE-eitduiutlto fRab11a of ubiquitylation HACE1-mediated promotes AR 5kaRb1 adadhge ad ahhairby heavier each bands higher and band Rab11a kDa 25 , RGPsrnl rmtdteFLAG-Rab11a/HA-Rab11a the promoted strongly AR-GFP interaction. AR–Rab11a form GDP-bound its in Rab11a with preferentially interacts AR down-modulated was co-immunoprecipitation AR–Rab11a and AR–HACE1 Interestingly, b .5 ** 0.05, ˚ ,adte tmltdwt 5 with stimulated then and C, 2 b RRb1 neato nteasneo tmlto ( stimulation of absence the in interaction AR–Rab11a 2 Rwsc-xrse ihRb1,sgetv of suggestive Rab11a, with co-expressed was AR b P 2 , RGPo ohpoen Fg D.C-xrsinof Co-expression 4D). (Fig. proteins both or AR-GFP m 0.01,*** rpaoo o h niae ieprost rvn ute nenlzto n oalwrcpo eyln.Rcpo elsraeexpression surface cell Receptor recycling. receptor allow to and internalization further prevent to periods time indicated the for propranolol M b , 2 RadHC1epeso oiidthe modified expression HACE1 and AR P b 0kabn perdi ae –1where 8–11 lanes in appeared band kDa 50 , , 2 b RadHC1wr oepesd which co-expressed, were HACE1 and AR .0.()Clclzto nlsso noeosHC1i el tbyepesn HA- expressing stably cells in HACE1 endogenous of analyses Colocalization (F) 0.001. 5ka hsbn a o eetdin detected not was band This kDa. 25 2 RRb1 olwn tmlto with stimulation following AR–Rab11a b b 2 2 Rwsntsgiiatyafce by affected significantly not was AR Radrgltsiscl ufc xrsinadrecycling. and expression surface cell its regulates and AR ˚ ,adte nuae nDE otiig10 containing DMEM in incubated then and C, m spoeeo o 0mntsa 37 at minutes 60 for isoproterenol M m spoeeo o h niae iepros uniiaino ufc eetr a efre yEIAadthe and ELISA by performed was receptors surface of Quantification periods. time indicated the for isoproterenol M m .Clclzn ies(,,,)adfurgasilsrtn HA- illustrating fluorograms and (d,i,n,s) pixels Colocalizing m. b 2 R(,,,)adgenlbldHC1(,,,)sgasweeteaeswt ihdge fcolocalization of degree high a with areas the where signals (b,g,l,q) HACE1 green-labeled and (a,f,k,p) AR , b kDa 8 t 2 5 AR- 0), ˚ ntepeec fmnni.Cl ufc eetr eemaue yEIAand ELISA by measured were receptors surface Cell monensin. of presence the in C t tss(,)o eua w-a NV et(–)floe yBnern post-tests. Bonferroni by followed (D–E) test ANOVA two-way regular or (B,C) -tests m bqiyaino PR a euaetertafcig we the trafficking, ubiquitylating their was regulate HACE1 can whether GPCRs verified of ubiquitylation ytm neetnl,sbtnilptnito fRab11a our of 4E. in Fig. the conditions potentiation both basal when in in revealed not substantial was but detected indicated ubiquitylation 4), Interestingly, was lane 10), panel, constructs system. (lane top Rab4a 4E, (Fig. of of Rab11a of combinations Ubiquitylation the bqii nioya ecie nFg A uaino Lys145 a by of with ubiquitylation analysis 5A. Fig. Rab11a blot in western abolished described by as antibody studied ubiquitin ubiquitylation generated its thus be and was to mutant determined HA-Rab11a-K145R was Lys145 An and ubiquitylated. identified were Rab11a of acids nieNd4 nHC-3uiutnlgs nw obe to known ligase receptor, the HECT-E3-ubiquitin ubiquitylate in to an involved failed Nedd4, HACE1 4G, unlike Fig. in shown niae htHC1wsntregulating not was HACE1 that indicated eiu novd CM/Swspromdon performed was the LC-MS/MS 141 with co-expressed b was that involved, HA-Rab11a Lys the immunoprecipitated identify to and ubiquitylated residue was Rab11a that confirm To recycling and loading GTP of Rab11a regulates Lys145 of Ubiquitylation euae ytercpo n HACE1. and receptor be the would by that proteins regulated Rab11a dimers/oligomers multiple receptor to with simultaneously association or binding the protein by cells. another panel, explained with in Rab11a be dimers top of also form 4D, could can data Rab11a (Fig. our that HACE1 Alternatively, suggest data of These was 7). co-expression which lane 6), the lane by panel, reversed top 4D, (Fig. co-immunoprecipitation b rcs Fg G e ro nbto panel). bottom on arrow this see in specificity 4G, revealing the (Fig. Nedd4, in of process not detected shift but the was HACE1 that of ubiquitylation presence noteworthy Rab11a also reflecting is mobility It in receptor. the of ubiquitylation a1ai el tbyepesn HA- expressing stably endogenous cells of of in depletion ubiquitylation that the Rab11a observed inhibited of we HACE1 activity Furthermore, endogenous ligase 9). effect (lane ubiquitin same Rab11a the E3 produce on the to failed on HACE1-C876S because (lane dependent HACE1 Rab4a was with results This to contrast 14). in 8), (lane co-expressed were in(ae7.Ti a elce ntemgainptenof pattern migration the in condi- reflected same was the This in 7). promoted ubiquitylation greatly (lane whereas was tion 11), Rab11a lane wild-type panel, of top 5A, (Fig. expression rpaoo o h niae ieprost rvn ute internalization further prevent to periods time indicated the for propranolol M 2 2 Ri E23cls he etdscmrsdo the of comprised peptides Three cells. HEK293 in AR with transfected cells HEK293 in proteins, both or HACE1 AR, a1auiutlto a hnasse ntepeec of presence the in assessed then was ubiquitylation Rab11a b AFAE 2 AR m m spoeeo o 5mntsa 37 at minutes 15 for isoproterenol M oesn( eyln niio)o tao vhce o 0minutes 30 for (vehicle) ethanol or inhibitor) recycling (a monensin M KNGLSFIETSALDSTNVEAAFQTILTEIYR ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal b A muorcptto I)wspromduigaHA- a using performed was (IP) Immunoprecipitation (A) 2 Ruiutlto Seo ta. 08.This 2008). al., et (Shenoy ubiquitylation AR 6 b b 2 ...o tlatfv eaaeeprmns The experiments. separate five least at of s.e.m. Rclclzto ihHC1(,,,)aepresented. are (e,j,o,t) HACE1 with colocalization AR 2 Radimnbotn I)wscridotwt an with out carried was (IB) immunoblotting and AR b 2 R el eetetdwt 5 with treated were Cells AR. ˚ ,adte nuae nDMEM in incubated then and C, b 2 b RadHC1co- HACE1 and AR 2 R(i.4) Since 4F). (Fig. AR b b 2 Rtafcigby trafficking AR 2 RadHACE1 and AR b 174 2 m R As AR. AG- M right amino b 2 AR

Journal of Cell Science a1a Rb1-15 r-a1aS5 ihteFLAG- the with HA- -Rab11a-S25N co-expressing or b Cells their -Rab11a-K145R activation. of Rab11a, Rab11a regulation on transferrin the ubiquitylation the in involved as is Rab11. such by an HACE1 recycling whether receptors determined and going other be is proteins receptor to between remains membrane Rab11a also of interaction It of route. recycling ubiquitylation that be in through will whether involved receptors conclude other generally of Rab11a-S25N to with Testing the proteins. necessary as transfected membrane recycling similar the other regulates degree of cells Rab11a-K145R that a the suggests with This to mutant. of Rab11a, compared or recycling pcDNA3 inhibited receptor significantly Fig. transferrin material Rab11a-K145R Rab11a, (supplementary co-expressed S2). Rab11a-S25N of presence of or the recycling Rab11a-K145R in studied receptor we transferrin protein, the membrane another of recycling EERHARTICLE RESEARCH owl-yeH-a1a(i.5,pnl n ,fo o to top from in 5, and 3 panels 5A, (Fig. bottom). HA-Rab11a wild-type to no showed which HA-Rab11-K145R, affects HACE1 of Depletion 2. Fig. eaiepoete nlgu otewell-characterized of the recycling the to for mutant the dominant- analogous confer of Rab11a-S25N to lack dominant-negative properties seems that Lys145 note to on negative promotes interesting Rab11 Rab11a is of why it ubiquitylation after clear minutes However, 15 removal. not only agonist receptor is Rab11a- the time dominant-negative It of recycling the over 5D). HACE1-mediated as (Fig. recycling extent co- mutant receptor similar of S25N a Rab11a-K145R decreased recycling to in also 5C), contrary, HACE1 (Fig. but of not the receptor, effect was early the On effect the this inhibited time. minutes expression However, 15 over 5B). alone (Fig. expressed sustained removal of was agonist co-expression Rab11a after 5B, receptor when the Fig. with of recycling in compared promoted strongly shown Rab11a with As HACE1 5B–D). with together (Fig. or HACE1 alone Rab11a-S25N, or Rab11a-K145R Rab11a, gis AE o 2husadcl ufc eetrepeso A,tepretg frcpo nenlzto fe tmlto ih5 with stimulation after internalization receptor of percentage 5 the with stimulation (A), after expression recycling receptor receptor surface and cell (B), and minutes hours 15 72 for HACE1 against o 0mnts()wsmaue yEIAuigaH-pcfcmncoa nioy fiinyo AE elto ysRAwscnimdb etr blot western by confirmed * was post-tests. siRNA Bonferroni by by depletion HACE1 followed of test Efficiency ANOVA antibody. mean two-way monoclonal are regular Results HA-specific a inset). a (A, using using antibody ELISA HACE1-specific by a measured with analysis was (C) minutes 60 for 2 R ln ri obnto ihHC1Mc eesubjected were HACE1-Myc, with combination in or alone AR, ete atdt sesteefc fHACE1-mediated of effect the assess to wanted then We h ucinlrl fRb1 bqiyainwste assessed then was ubiquitylation Rab11a of role functional The b b 2 Rrccigeprmnsi el htwr co-expressing were that cells in experiments recycling AR 2 R ovrf hte a1aK4Rcudafc the affect could Rab11a-K145R whether verify To AR. b 2 Rtrafficking. AR , 3kabn ncontrast in band kDa 33 E23clssal xrsigHA- expressing stably cells HEK293 m spoeeo o 5mntsa 37 at minutes 15 for isoproterenol M 6 ...o he ofv eaaeeprmns h ttsia infcnewsdetermined was significance statistical The experiments. separate five to three of s.e.m. P , .5 ** 0.05, enfrRb wn otepeec ftreH asa h N- GTPases with together the Rab or at alone of tags HACE1 be with HA Immunoprecipitation co-expressed three were protein. could of that be the presence they can the of weight to that terminus molecular owing higher Rab5 suggesting for of seen band 7B), (Fig. single A Rab11a ubiquitylated. to similar aa odtosadehne yc-xrsinof antibody co-expression in by ubiquitin detected enhanced a was and Rab8a with and conditions Rab6a blotting basal of ubiquitylation western that by showed analyzed and hnasse sfrRb1 ntepeec rasneo HACE1 of absence with or G5 or presence the alone was and in co-expression GTPases Rab11a Rab G4 for these as be of assessed conserved pattern Rab11a- then migration the of to The 7A). vicinity between (Fig. al., the Rab6a boxes shown Rac1-Lys147 in Rab1a, et located and was residues that Lys145 (Castillo-Lluva Lys revealed have it Lys147 analysis Rab8a and this because on Interestingly, HACE1 included 2012). by then was between ubiquitylated and was Rac1 Rab11a made Rab9a, ubiquitylated Rab8a, were Rac1. Rab6a, Rab5a, alignments were Rab4a, Rab2a, sequence Rab1a, GTPases First, Rab investigated. other of Whether co-expression by ubiquitylated also are Rab8a and Rab6a ofatoainit yooi n ebaefatos An fractions. membrane and cytosolic into fractionation to niaeta bqiyaino a1ao y15b AE is GTPase. HACE1 small by results the Lys145 of These on activation 2011). in Rab11a al., involved of in ubiquitylation et seen that is (Lachance form indicate fractions prenylated the membrane only whereas the protein, unprenylated the dominant-negative and of prenylated the forms the in to detected corresponding of were fractions Rab11a cytosolic or of anti-HA forms Two Rab11a-K145R promoted an mutant. Rab11a-S25N of effect co-expression significant using no activation HACE1 had but blot on twofold that than more western shows activation Rab11a by 6 Fig. activated, analyzed antibody. and its GTPase were the in immunoprecipitate samples to Rab11a used was recognizes form GTP-bound specifically that antibody oeua egtfrswr eetdi diint the to higher addition that in the detected showed expressing were proteins extracts forms expected cell of weight of molecular combinations membranes blot indicated western of n HACE1 and P , 0.01. b ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal 2 , Rwr rnfce ihngtv oto CR)o siRNA or (CTRL) control negative with transfected were AR 5kapoen o a2,RbaadRab8a, and Rab6a Rab2a, for proteins kDa 25 ˚ ,adicbto nDE otiig10 containing DMEM in incubation and C, b 2 R(i.7) rlne exposure Prolonged 7B). (Fig. AR m spoeeo for isoproterenol M m propranolol M b 2 Rand AR b 2 b AR 2 115 AR

Journal of Cell Science eodpnl,sgetn httercpo csa cfodin scaffold a as acts receptor the that suggesting panel), second EERHARTICLE RESEARCH 116 affected form and significantly weight not molecular sensitivity was HACE1 higher which in 7B, the by not that Fig. limitation in fact Rab2 was a the for by detected by or ubiquitylation explained assay the be Rab2a of could However, This noticed. 7C). (Fig. HACE1 regulates HACE1 3. Fig. AE ihteRbGPssi umne yteco-expression the the by of augmented co-immunoprecipitation is of panel), GTPases Rab (second the 5A with Fig. HACE1 in worthy as Also note, ubiquitylation. of than other modification translational olwdb ofroips-et AB.* (A,B). post-tests Bonferroni by followed y-AE.Clswr rae ih5 with treated were Cells Myc-HACE1. rvn ute nenlzto n oalwrcpo eyln.Epeso fcl-ufc eetr a eetdb LS n h ecnaeo r of percentage the and ELISA by detected was receptors cell-surface of Expression means recycling. are receptor Results calculated. allow was to recycling and internalization further prevent emaiie n aee ihrbi oylnlat-AE n os niH niois ooaiaino a green-labeled (a) of Colocalization antibodies. anti-HA mouse and anti-HACE1 polyclonal rabbit with labeled and permeabilized a1awt AE r hw ntebto w panels. two bottom the in shown are HACE1 with Rab11a n c lelbldRb1 per ht nd cl as 10 bars: Scale d. in white appears Rab11a blue-labeled (c) and a4,H-a4 y-AE,H-a1a rH-a1a+McHC1 el eetetdwt 5 with treated were Cells Myc-HACE1. + HA-Rab11a or HA-Rab11a, Myc-HACE1, + HA-Rab4a Rab4a, nuae nDE otiig10 containing DMEM in incubated eetrwsdtce yEIAadtepretg frcpo eyln a acltd eut r means are Results calculated. was recycling receptor HA- of expressing percentage stably the cells and ELISA by detected was receptor b 2 Ri h bec faoitsiuain(i.7C, (Fig. stimulation agonist of absence the in AR b 2 Rc-xrsin orsod oapost- a to corresponds co-expression, AR b b 2 2 RrccigtruhRab11a. through recycling AR Rwr rtetdwt eaiecnrl(iTL rsRAaantRb1 o 4husadte ornfce ihpDA or pcDNA3 with cotransfected then and hours 24 for Rab11a against siRNA or (siCTRL) control negative with pretreated were AR m rpaoo o 5mntst rvn ute nenlzto n oalwrcpo eyln.Epeso fcell-surface of Expression recycling. receptor allow to and internalization further prevent to minutes 15 for propranolol M m spoeeo o 5mntsa 37 at minutes 15 for isoproterenol M P , 6 .5 ** 0.05, ...o he eaaeeprmns ttsia nlsswr efre sn h eua w-a NV test ANOVA two-way regular the using performed were analyses Statistical experiments. separate three of s.e.m. P , .1 *** 0.01, A E23clswr rninl ornfce ihFLAG- with cotransfected transiently were cells HEK293 (A) P m , .Furgasilsrtn ooaiainof colocalization illustrating Fluorograms m. .0,**** 0.001, ˚ ,adte nuae nDE otiig10 containing DMEM in incubated then and C, P , .01 C E23clstasetyexpressing transiently cells HEK293 (C) 0.0001. n r paetyacsil,a eosrtdfrRc (Castillo- ubiquitylation. HACE1-mediated Rac1 for Rab11a, for and demonstrated 2012) al., GTPases as in et small the accessible, Lluva the shown on of apparently boxes found is are G5 are and and residues proteins G4 Lys the between corresponding these positioned that helix the illustrates This of on 7D. (Lys145) Fig. structure Rab11a as crystal well as the Lys146), Lys146), and and (Lys144 Lys138 Rab6a (Lys133, Rab Rab8 (Lys147), Rac1 the on sites of, ubiquitylation ubiquitylation and with, interaction GTPases. HACE1 the ial,acmaio ftelclzto fcnimdo potential or confirmed of localization the of comparison a Finally, ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal m spoeeo o 5mntsa 37 at minutes 15 for isoproterenol M 6 ...o iesprt xeiet.()HEK293 (B) experiments. separate five of s.e.m. b 2 RGPwt AE,adclclzto of colocalization and HACE1, with AR-GFP b 2 RadpDA,McHC1 HA- Myc-HACE1, pcDNA3, and AR m fpornllfr6 iue to minutes 60 for propranolol of M b 2 b R b e-aee HACE1 red-labeled (b) AR, 2 RGPwr fixed, were AR-GFP ˚ ,adthen and C, eceptor a -

Journal of Cell Science EERHARTICLE RESEARCH i.4. Fig. e etpg o legend. for page next See ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal 117

Journal of Cell Science i.4. Fig. ARTICLE RESEARCH 118 of regulation the in involved is Lys145 on Rab11a of Ubiquitylation 5. Fig. that of showed regulation the others in and resulting proteins We Rab is with machinery. regulate interact less trafficking GPCRs conversely but receptors Rab trafficking, membrane the receptor whether about membrane well known regulate recently is to transport has vesicular known Rab-mediated trafficking interest. vesicular much attracted of regulation Cargo-specific DISCUSSION niae ntelf.Tebossonaerpeettv ftresprt xeiet.(–)HK9 el eectasetdwt FLAG- with cotransfected were cells HEK293 (B–D) m experiments. Molecular separate antibodies. three polyclonal of specific representative anti-HA are and shown anti-ubiquitin blots anti-FLAG, The with left. out the carried on was indicated (IB) Immunoblotting antibody. monoclonal muoltigwscridotwt niFA,at-bqii,at-y n niH oylnlatbde.Tebossonaerpeettv of representative are shown left. blots the The on antibodies. polyclonal indicated ubiquitylate anti-HA are not and masses anti-Myc does Molecular anti-ubiquitin, HACE1 an anti-FLAG, experiments. (G) anti-HACE1 with experiments. out anti-ubiquitin, carried anti-Rab11a, separate was with three Immunoblotting out of carried representative was are Immunoblotting shown control. blots isotypic The or antibodies. antibodies polyclonal monoclonal anti-Rab11a using out oylnlatbde.Tebossonaerpeettv ftresprt xeiet.()Edgnu a1ai bqiyae yHC1 E23c HEK293 HACE1. by ubiquitylated is anti Rab11a and Endogenous anti-Myc (F) anti-ubiquitin, experiments. anti-FLAG, separate with three out of carried representative HA- was are expressing Immunoblotting shown stably antibodies. blots monoclonal The anti-FLAG antibodies. or polyclonal anti-HA using performed was w-a NV etfloe yBnern ottss * post-tests. Bonferroni by followed test ANOVA two-way oolnlatbd.Imnbotn a are u ihat-LG niGP niMcadat-Aplcoa niois E a1abtntRab4 not but Rab11a anti- (E) an antibodies. using out polyclonal carried anti-HA was and GTPases anti-Myc the anti-GFP, of of anti-FLAG, co-expression Immunoprecipitation with the constructs. out by indicated carried ubiquitylated the was with Immunoblotting cotransfected antibody. were monoclonal cells HEK293 HACE1. of presence obntoso FLAG- of combinations muoltig(B a are u ihat-LG niMcadat-Aplcoa niois h lt r ersnaieo he eaaeexp separate normalize (D) three were percentage. HA-Rab11a of a or representative as Myc-HACE1 are reported immunoprecipitated blots of and The values receptor antibodies. densitometry immunoprecipitated polyclonal The of software. anti-HA amount ImageJ and the with anti-Myc performed anti-FLAG, analyses with Densitometry out (B,C) carried was (IB) Immunoblotting 5 with stimulated niae ieprost rvn ute nenlzto n oalwrcpo eyln.Epeso fcl-ufc eetr a eetdb EL by detected was receptors cell-surface of Expression recycling. receptor means allow are Results to calculated. and was internalization recycling receptor further of prevent percentage to periods time indicated ornfce ihFLAG- with cotransfected AE oepeso n eetrsiuain E23clswr ornfce ihFLAG- with cotransfected were cells HEK293 stimulation. receptor and co-expression HACE1 niae osrcs el eetetdwt 5 with treated were Cells constructs. indicated b 2 RmdltsRb1 ieiainadpooe AE-eitduiutlto fRab11a. of ubiquitylation HACE1-mediated promotes and dimerization Rab11a modulates AR m spoeeo o h niae ie.Imnpeiiain(P ftercpo a are u sn LGseii oolnlantibody. monoclonal FLAG-specific a using out carried was receptor the of (IP) Immunoprecipitation times. indicated the for isoproterenol M b 2 b Rwr rae ihngtv oto sCR)o iN gis AE o 2hus muorcptto fteGPs a carried was GTPase the of Immunoprecipitation hours. 72 for HACE1 against siRNA or (siCTRL) control negative with treated were AR 2 R y-AE,H-a1aadH-a1aK4R muorcptto I)o h Taewspromdwt nanti-HA an with performed was GTPase the of (IP) Immunoprecipitation HA-Rab11a-K145R. and HA-Rab11a Myc-HACE1, AR, b 2 Radteidctdpoen.Imnpeiiaino h eetrwspromduigat-LGmncoa antibody. monoclonal anti-FLAG using performed was receptor the of Immunoprecipitation proteins. indicated the and AR b 2 RadHC1 E23clswr ornfce ihteidctdcntut.Imnpeiiaino h GTPases the of Immunoprecipitation constructs. indicated the with cotransfected were cells HEK293 HACE1. and AR m spoeeo o 5mntsa 37 at minutes 15 for isoproterenol M P , .5 ** 0.05, 6 P , ...o iesprt xeiet.Saitclaaye eepromduigteregular the using performed were analyses Statistical experiments. separate five of s.e.m. .1 *** 0.01, b P b 2 , Repeso rmtsRb1 ieiain hc srvre ythe by reversed is which dimerization, Rab11a promotes expression AR 2 ujc fcretitnersac.Frteimportant the For research. of mechanisms intense new unravelling the current family, is GPCR trafficking pharmacological of own their the direct subject of to elements Wikstro other machinery 2009; with 2002; Rab-associated Hall, interact Smythe, and receptors membrane Ritter 2002; Whether 2009; al., al., 2005; et et al., Parent et Seachrist 2008; Hamelin al., 2011; et al., O’Keeffe et (Esseltine trafficking receptor Rrecycling. AR 0.001. ˚ ,adte nuae nDE otiig10 containing DMEM in incubated then and C, ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal b 2 RadMcHC1 ARb1 rbt rtisand proteins both or HA-Rab11a Myc-HACE1, and AR A E23clswr ornfce ihteindicated the with cotransfected were cells HEK293 (A) A The (A) b 2 RRb1 neato safce by affected is interaction AR–Rab11a b 2 R E23clswere cells HEK293 AR. m rpaoo o the for propranolol M me l,2008). al., et ¨m b 2 he separate three Radthe and AR se are asses S n the and ISA anti-HA d -HA eriments. ais FLAG dto ells

Journal of Cell Science ugse htHC1cue a1aatvto eas we because activation that Rab11a reported caused previously HACE1 that suggested EERHARTICLE RESEARCH lhuhtepyilgclsgiiac fGTPase the form that of indicate GDP-bound data its our in Interestingly, b dimer significance 2004). a al., as et (Pasqualato exists studies Rab11a Crystallography physiological 2008; that understood. al., proposed fully 1998), not et Zheng, is the and (Beck Zhang oligomerization 2001; oligomers al., et and Zhang although dimers 2000; data al., form et Our Inouye Arf 2011). Ras, including al., and GTPases et Several Rho dimerize. (Sasaki can Rab11 Lys147 that on suggest ubiquitylation its by b are mechanisms regulatory and other their intriguing that is indication number few far involved. an and so so be identified been could their that regulation have and GAPs fact Rab The considering and their GEFs importance. Rab pathological regarding when or known physiological partners Surprisingly, is disease. and interacting human trafficking little in vesicular targets of very family as step GTPase well-documented every Rab virtually are the of in members involved 60 are than their targets. more of pharmacological the possible understanding Similarly, novel our identify improve to and to regulation central is trafficking their by Lys145 on Rab11a of Ubiquitylation 6. Fig. Prn ta. 09.Ms pcrmtyadsite-directed by and and of activation Lys145 regulation Rab11a spectrometry the on in in involved ubiquitylated is Mass ubiquitylation is This Rab11 HACE1. 2009). that showed al., mutagenesis et (Parent nrae h omto fRb1 dimers Rab11a of formation the increased co- HACE1 Rab11a. and the activity ligase reduced ubiquitin expression E3 its 15 rH-a1aS5 ncmiainwt FLAG- with combination in HA-Rab11a-S25N or K145R eaaeeprmns estmtyaaye fteqatt fRb1GPta eeimnpeiiae rmtemmrn rcin eeperformed were fractions membrane the from representativ are immunoprecipitated shown were blots that the The Rab11-GTP for antibodies. Immunoprecipita measured of indicated Rab11-GTP. value the quantity activated Densitometry using the blot recognizing software. of western ImageJ specifically by analyses antibody analyzed Densitometry were an experiments. fractions with membrane separate immunoprecipitated and were cytoplasmic the fractions from (M) samples membrane or (C) Cytosolic ttsia infcnewsdtrie sn nupie Student’s unpaired an using determined was significance statistical 2 2 R hc neat ihteGPbudfr fRab11, of form GDP-bound the with interacts which AR, on dependent mechanisms through recycling its regulate to AR ntepeetsuy esoe htHC1itrcswt the with interacts HACE1 that showed we study, present the In b 2 Rrccig hsi knt a activation Ras to akin is This recycling. AR b 2 Ritrcswt ncieRab11a inactive with interacts AR b 2 RRb1 neato.This interaction. AR–Rab11a ncellulo in b 2 RHC1i novdi t activation. its in involved is AR-HACE1 b b 2 2 Rcniinwssta 0% eut r h means the are Results 100%. at set was condition AR Raoeo ihHC1Mc el eesbetdt rcinto 8husatrtransfection. after hours 48 fractionation to subjected were Cells HACE1-Myc. with or alone AR sevidenced as , t ts.* -test. P , 0.05. enee oflyadeswehrRb1 dimerizes. will Rab11a work whether of processes More address these HACE1. fully formation of of of to expression needed the both the be that by by reduced and co-immunoprecipitation be caused oligomers, would or is dimers increased Rab11a receptor tagged that differentially alternatively, edn oteapaac fa of appearance the to leading ihisiaiiyt nac HACE1-mediated enhance to inability its with iesfo h eetr n neato fatv a1awith Rab11a Rab11a regulate of active of dissociation of to interaction in and activation effectors receptors result the and possibly from of dimers could ubiquitylation This with, Rab11a. interaction HACE1 uuesuiswl eelwehrti sseii oti ls of class this to GPCR. specific a is of this the whether co-expression in factor reveal the crucial will example al., a studies of for Future et absence to (Tang the similar described to due was equation, be ubiquitylation could Rab This proteins, on 2011). Rab with effect interact no to but shown been previously has HACE1 te a rtisadt eemn hte te GPCRs other whether the determine like behave to itineraries and trafficking different towards proteins exhibiting specificity this Rab characterize further other to interesting be will a6 n a8,btntohrRbpoen etdi h present of the presence in the tested in proteins ubiquitylated Rab were other study, not but Rab8a, and Rab6a antecuetepsiiiyta h eetrpooe the another promotes and receptor the Rab11a that between possibility interaction the exclude cannot aasgetta the that suggest data a1ai h rsneo h eetr(i.4) hswas This HACE1–Rab11a 4B). The (Fig. HACE1. receptor by of the promoted co- was co-expression interaction of epitope-tagged increased by presence by and inhibited the differentially 4A) in (Fig. Rab11a between lysates cell immunoprecipitation in dimer a Rab11a of appearance the by a4 a o bqiyae by ubiquitylated not was Rab4a ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal E23clswr rnfce ihH-a1a HA-Rab11a- HA-Rab11a, with transfected were cells HEK293 b b , 2 2 6 Rat sasafl opooethe promote to scaffold a as acts AR Rrccig(i.8.Hwvr we However, 8). (Fig. recycling AR 0kabn ftepeitdsz fa of size predicted the of band kDa 50 ...o oridpneteprmns The experiments. independent four of s.e.m. b 2 Rc-xrsin loehr our Altogether, co-expression. AR , 0kabn ncl yae,or lysates, cell in band kDa 50 b 2 RHC1 correlating AR–HACE1, b 2 RadHC1 It HACE1. and AR , 5kaprotein, kDa 25 b 2 Rrecycling. AR e and tes ffour of e b 2 AR. 119 with

Journal of Cell Science niH n niGPHatbde.Tebossonaerpeettv ftresprt xeiet.Arwidctsteapaac fhge mole higher of appearance the indicates Arrow experiments. separate three of upon representative proteins are Rab shown the of blots forms The mass antibodies. anti-GAPDH and anti-HA EERHARTICLE RESEARCH novd pcfct hssest loeiti h fet of effects 120 also the was in Specificity HACE1. exist by also proteins target to of seems ubiquitylation are thus degradation Specificity proteasome-mediated involved. with ubiquitin or incompatible of mono-ubiquitylation chains HACE1-mediated stability that by affected The suggesting not 2012). ubiquitylation, was Rab11a al., of and et degradation Rab8a (Castillo-Lluva Rab6a, proteasomal GTPase causes small HACE1 the by of Lys147 Ubiquitylation to on GTPases. receptor other interesting Rac1 Rab other of of be whether ubiquitylation trafficking modulate and will regulating types Rab11, in It through HACE1 receptors types. of membrane receptor role the other receptor regulation determine the of transferrin in involved recycling the is with of Rab11 of data ubiquitylation that our suggest but receptors membrane FLAG with cotransfected were cells HEK293 (B) yellow. in highlighted are Rab11a and Rac1 of sites ubiquitylation HACE1 and red in b shown are substrates. boxes HACE1 G5 as and Rab8a and of Rab6a alignments of Identification 7. Fig. fatv a1(D T5,Rba(D DX,Rb1 PB1I)adRba(D TF.PtniladcnimdHC1uiutlto ie r shown are sites ubiquitylation HACE1 confirmed and Potential 3TNF). (PDB s Rab8a crystal and from 1OIW) o coordinates (PDB using indicated Rab11a (www.pymol.org) antibody. are 4DKX), PyMOL (PDB masses monoclonal with red. Rab6a prepared Molecular anti-HA in were 3TH5), antibodies. an Images (PDB polyclonal (D) with Rac1 anti-HA experiments. performed active separate and of was three anti-Myc of GTPases anti-ubiquitin, representative the anti-FLAG, are shown with of blots out (IP) carried Immunoprecipitation was GTPases. (IB) Rab Immunoblotting HA-tagged and Myc-HACE1 pcDNA3, 2 Radteidctdcmiain fpDA,McHC1adH-agdRbGPss muoltig(B a are u ihat-LG anti-Myc, anti-FLAG, with out carried was (IB) Immunoblotting GTPases. Rab HA-tagged and Myc-HACE1 pcDNA3, of combinations indicated the and AR oosapiens Homo a1 a1,Rba a4,Rba a6,Rba a9 n a1apoen r hw.Cnevdrsde ewe G4 between residues Conserved shown. are proteins Rab11a and Rab9a Rab8a, Rab6a, Rab5a, Rab4a, Rab2a, Rab1a, Rac1, b A n AE oxrsin C E23clswr ornfce ihFLAG- with cotransfected were cells HEK293 (C) coexpression. HACE1 and 2AR A ceai ersnaino osre oan fsalGPssadsequence and GTPases small of domains conserved of representation Schematic (A) t neato ihGF rGP ilb h ujc ffuture of subject the be will GAPs a or modulates as GEFs Rab11a acts of with HACE1 ubiquitylation interaction Whether whether its or 2010). and Rab11a al., for Swaminathan et GEF 1996; Xu al., ligases et ubiquitin 2006; Rosa E3 Tsygankov, be 2010; HERC1 to and Lambright, shown for Rac1, and were and GTPases, (Barr Rab5, and small for Rab3a c-Cbl other Arf1, Ras, for for for GEFs 5 et Rabex (Baker some instance GAPs However, to response 2013). severely the al., Lys147 and with on interaction Ras its abrogates of Mono-ubiquitylation determined. be results to migrating HACE1. contrast the in with in Rab11a, shifts obtained of induce ubiquitylation to Nedd4 reflecting of pattern, inability the by shown o bqiyaino a1alast t ciainrmisto remains activation its to leads Rab11a of ubiquitylation How ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal b 2 Radteidctdcmiain of combinations indicated the and AR h et The left. the n tructures cular -

Journal of Cell Science bqiyae yHC1a ato ucinlcmlxwith of complex expression functional defined. a be of to remains part is that get as intermediate Rab11a could HACE1 Rab6a an Perhaps by that GTPase. mediate ubiquitylated small to possible this Rab6 conditions of and thus ubiquitylation HACE1 same is the between interaction the It the in in 2011). involved Rab6a al., Rab11 et with post- (Tang mediates interacts of prevent directly lack that HACE1 HACE1 the proteins interacting purified by from 2011). the them that explained of al., be modifications et translational observed could (Tang This HACE1 with ubiquitylation. we interact not However, does Rab6 that 00 n a6-neatn rti ik a6adRab11 and Rab6 2007). links (Kno al., 1 et Rab8 (Miserey-Lenkei protein toward function Rab6a-interacting activity and GEF 2010) its Rabin8 with stimulates interacts Rab11 functionally and transport: are Rab11a intracellular This and in 2011). Rab8a connected al., Rab6a, et because (Lachance interesting GTPases is three these of prenylation EERHARTICLE RESEARCH xeiet.Rb1GF r nw in known are GEFs Rab11 experiments. by activation Rab11a for Model 8. Fig. hspsil htHC1icesscl ufc agtn fthe of targeting is surface It cell 2010). increases al., b HACE1 their et that ankyrin-repeat-containing (Parent GPCRs possible for that of thus export showed et promote known recently can (Mosavi proteins assembly We usually six complex 2004). protein contains domains al., in HACE1 repeat properties activity. scaffolding ligase ankyrin E3 its putative of independent are hsi nlgu oorrcn icvr facmlxbetween complex a of discovery recent Rab11a. our and the to Rab8a analogous Rab6a, is for This specificity shows ubiquitylation hscudb eeatt ua iessascae with associated diseases GTPases. human Rab of to dysregulation and relevant trafficking ligase. GPCR be (2) ubiquitin scaffolding impaired E3 through could an ubiquitylated; and GTPases GTPases This Rab Rab (3) the of be trafficking between and activity own complexes activation; their the can regulate its regulating can GPCRs, by in as GTPases involved such proteins is cargo Rab11a Rab of Rab ubiquitylation of (1) regulation and trafficking GTPases: intracellular of issues three into 02 u ohmnotooshv e enietfe otebest the to identified knowledge. been yet our have of orthologs human no but 2012) a1a(tp2,laigt a1aGPlaigaddmrdsoito se ) n liaeyt iscainfo h eetradatvto fef of ubiquitylatio activation the and in receptor resulting the 1) from (step dissociation HACE1 to recruit ultimately to and scaffold 3), a (step as dissociation acts dimer and and confirmed) GTP-loading be Rab11a regulate to to to remains leading that 2), possibility (step a Rab11a is dimerization Rab11a that 2 Rtruhpoenitrcin ihisakrnrepeats. ankyrin its with interactions protein through AR h atta AE-86 a bet rmt elsurface cell promote to able was HACE1-C876S that fact The nsmay u idnspoiesgiiatnvlinsights novel significant provide findings our summary, In ti lontwrh that noteworthy also is It b 2 RadRbgeranylgeranyltransferase Rab and AR b A eyln se 4). (step recycling 2AR b 2 Rsget httepoenhsfntosthat functions has protein the that suggests AR nvitro in hsi oee nieybecause unlikely however is This . b 2 RtruhHC1mdae ubiquitylation. HACE1-mediated through AR Drosophila b nvitro In 2 AR-HACE1-mediated a htrgltsthe regulates that tde indicate studies Xoge al., et (Xiong de tal., et ¨dler rmCvne h oylnlat-LG oolnlFLAG were monoclonal antibodies anti-FLAG, (9E10) polyclonal The anti-Myc Covance. and from (16B12) anti-HA monoclonal The Reagents METHODS AND MATERIALS 0 vv B ftlbvn eu)a 37 at serum) DMEM bovine in (fetal with FBS maintained supplemented (v/v) (Invitrogen) were 10% Medium) cells Eagle’s Modified HEK293 (Dulbecco’s kidney embryonic Human transfection and culture Cell described as constructs pcDNA3-HA-Rab11a the (The from previously prepared PCR were constructs by HA-Rab11a-K145R HA-Rab11a-S25N, The constructs Plasmid otiig5 CO 5% containing de oke h oa muto N e lt constant. plate per DNA of amount total was the vector keep pcDNA3 to Empty added instructions. manufacturer’s the to the according using performed were confluence 70% n h oylnlat-AE eefo im-lrc.Temouse The Sigma-Aldrich. from were anti-HACE1 polyclonal the and oolnlIgG monoclonal ooTasernwsakn itfo h aoaoyo rRcadLeduc Richard Dr of Human laboratory the Sigma. from (Universite from gift kind purchased Invitrogen. a the were was kit holo-Transferrin and from Sigma. substrate and antibody phosphatase purchased from anti-mouse 546, alkaline purchased goat were were Fluor alkaline-phosphatase-conjugated monensin An reagent Alexa and propranolol antifade to anti-mouse Isoproterenol, goat conjugates Gold 633 anti-Rab11a, Fluor transferrin ProLong monoclonal Alexa human anti-rabbit, rabbit donkey antibodies, The antibody 546 Bioscience. Rab11 Fluor anti-active Alexa NewEast The to anti-Myc-HRP from Abcam. The from Sciences. was conjugated was Life antibody Enzo antibodies from polyclonal were monoclonal (FK2H) was anti- peroxidase Anti-mono- anti-polyubiquitinylated monoclonal Laboratories. Transduction (3F10) mouse BD and The from Science. purchased anti-HA-peroxidase was Applied Rab11a Roche Cruz monoclonal Santa from anti- from purchased the The were (Y-11), beads agarose anti-HA-probe Biotechnology. Protein-G the and antibodies (A-14), GAPDH anti-Myc polyclonal The C rmpDA-y-AE.Tefl-eghPRfamn was fragment PCR PCR full-length with The digested The pcDNA3-Myc-HACE1. Myc-HACE1-C from (SC107534). PCR The OriGene vector. with cDNA pcDNA3 from digested human was the purchased from fragment The PCR template 11426). by (plasmid prepared clone Addgene was from construct purchased Myc-HACE1 was construct Nedd4 nert ftecdn euneo hs osrcswscnimdby confirmed was constructs these of sequencing. sequence dideoxy coding the of Integrity Briefly, ´ ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal eSherbrooke). de Bam b ´ 1 2 iute l,20;Prn ta. 99.TeHA- The 1999). al., et Parent 2004; al., et riault Ritrcswt a1admr qeto akindicates mark (question dimers Rab11a with interacts AR FLAG 2 rnin rnfcino E23clsgont 50– to grown cells HEK293 of transfection Transient . Iand HI M2 nioywsas sda nioyi control. isotypic an as used also was antibody Eco Bam Iadlgtdit h cN3vector. pcDNA3 the into ligated and RI Iand HI 7Smtn a rprdby prepared was mutant 876S ˚ Trans nahmdfe atmosphere humidified a in C Eco Iadlgtdit the into ligated and RI TL1Raet(Mirus) Reagent IT-LT1 M1 nof -specific fectors 121

Journal of Cell Science rnfce ihFLAG- with transfected n hnmitie o nadtoa 8hus h el eete treated then were cells The hours. 0.75 48 with additional an for maintained then and al 0m rsHl H80 .%doyhlt,01 D,1 mM 10 SDS, 0.1% deoxycholate, 0.5% 8.0, pH Na Tris-HCl, mM then were 50 300 cells NaCl, in The harvested hours. and PBS constructs 48 ice-cold for with indicated washed above the described as with maintained transfected were and transiently were cells HEK293 Immunoprecipitation ARTICLE RESEARCH 122 of density a at dishes Petri mm 100 five in 2.0 plated were analysis cells LC-MS/MS HEK293 direct and purification Protein Biolabs) England IgG (New DMP mouse beads to agarose crosslinked experiments, Protein-G spectrometry generated mass for we needed purification post- HA-Rab11a For hours 2010). 72 al., crosslinking the et antibody at Parent DMP 2011; performed using al., western et were (Lachance by oligonucleotide usual experiments as analysis ELISA transfection nM expression the and 10 to Protein blotting according with instructions. (Invitrogen) reagent manufacturer’s 1, HEK293 transfection transfected Control 2000 Ambion. Negative Lipofectamine from were (Silencer purchased siRNA cells the were control number-AM4611) targeting negative catalogue s33240 the and and , s33239 ID human oligonucleotides synthetic The The assays 1999; siRNA al., et Parent Parent 2010; as 2011; al., ELISA al., et et Parent for 2009; Lachance processed al., 2005; then et al., and et (Hamelin hours previously 48 mg/ml 6.5 described then additional 0.1 and an with constructs receptors, for indicated pre-coated maintained the plates cell-surface with 24-well transfected of in (Sigma), poly-L-lysine plated expression were cells of HEK293 we quantification as For receptors processed 2010). cell-surface of al., were et Measurement Parent Cells 2011; al., et (Olympus). (Lachance confocal previously software described scanning 2.0 a Fluoviewer using a with cells microscope inverted 6 HEK293 an to in coupled (FV1000-Olympus) endogenous microscope proteins indicated the transfected detect to or performed was microscopy microscopy Confocal confocal and staining Immunofluorescence nian 0n eppi n 0n hmsai)(im Aldrich). 2010). previously al., (Sigma et described Parent we chymostatin) 2011; as al., nM et out 10 (Lachance carried and then were leupeptin Immunoprecipitations nM nM 9 pepstatin, 10 nM (9 antipain, inhibitors protease with supplemented were buffers ihPSte none nioiswr ltdtiewt lof twice ml washed 1 were 50 with beads in Finally, twice resuspended room 2.5). once eluted PBS, pH washed glycine with were were at for M Beads antibodies buffer (0.1 buffer temperature. blocking unbounded rocker elution room of then at ml (0.1 a PBS rocker 1 buffer with a in on blocking on incubated with minutes and minutes 60 once 8.2) and 60 pH washed added ethanolamine, then for was M were buffer) Beads allowed crosslinking in temperature. was mM ml (15 1 solution crosslinking Subsequently, DMP 8.2). pH fresh triethanolamine, of M (0.2 buffer crosslinking 500 with times three washed 1mM Na eupne n lqoe nts ue.Baswr ahdtiewith twice washed were Beads tubes. test 500 in aliquoted 100 and resuspended Briefly, manufacturer. h nioyue o h sa)o bqiyainlssbfe for NaCl, buffer mM lysis 250 ubiquitylation CaCl 7.5, mM pH or 1 or HEPES, assay) EDTA mM mM the 2 (50 for experiments used ubiquitylation antibody the 80 nuae vrih narce t4 at rocker a on overnight incubated 0olimrinojcieln n mgswr rcse using processed were images and lens objective oil-immersion 60 6 4 m P m 10 fbnigbfe n 30 and buffer binding of l 2 fbnigbfe 01Msdu hsht ufrp .) 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R. Moore, E., Foster, E., E. Millman, L., J. Rosenfeld, V., Iyer, O., H. Awwad, V., C. Fernandez, L., Zhang, N., Melnyk, V., Evdokimova, S., M. Anglesio, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132944/-/DC1 online available material Supplementary material Supplementary la de Fonds the number from [grant award Health salary Que of Senior Recherche Institutes Chercheur Canadian a the and by MOP-184095]; supported was work This manuscript. the Funding and wrote S.A. and V.L., data experiments. the the analyzed performed experiments, S.N. the and conceived J.L.P. S.G. M.R., S.M., J.D., V.L., contributions Author interests. competing no declare authors The interests Competing considered were Data post-tests. * Bonferroni Student’s when by paired significant followed or (GraphPad test 5.0 unpaired ANOVA version the Prism using using Software) performed were analyses Statistical analysis software. 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Chair. Research Lussier uli nye el n ag eldbi.Lstswr hncentrifuged then were Lysates debris. 100,000 cell at large and cells unlysed nuclei, grs ed M-rslne omueIgG mouse to DMP-crosslinked beads agarose n nl,B J. kidney. B. Knoll, normal and versus tumor Wilms’ sporadic J. Genet. in Penninger, Mol. R., Hace1, A. ligase, Brooks-Wilson, protein A., M. Marra, al. S., et Leach, E., P. Grundy, 20) ifrnilepeso fanvlakrncnann 3ubiquitin- E3 containing ankyrin novel a of expression Differential (2004). ora fCl cec 21)17 1–2 doi:10.1242/jcs.132944 111–123 127, (2014) Science Cell of Journal g ˚ .Teeue rcin eenurlzdwt 10 with neutralized were fractions eluted The C. 13 ´bec-Sante o ora 4 at hour 1 for 2061-2074. , 20) niioso hshioiie3kns as eet in defects cause 3- phosphoinositide of Inhibitors (2007). ˚ m n hnpecerdaanwt 20 with again pre-cleared then and C lof10 P m , fPoenGaaoebasprm flst o h 1 for lysate of ml per beads agarose Protein-G of l .5 ** 0.05, FQ)t ...JLP stercpeto h Andre the of recipient the is J.L.P. J.L.P. to (FRQS) ´ 6 6 ocnrto.Clswr hncnrfgdat centrifuged then were Cells concentration. 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Journal of Cell Science arw . rnih . ozlz .M,Tpnr,E,Dehn,M., Diekhans, E., Tapanari, M., J. Gonzalez, A., Frankish, Dupre and J., A. Harrow, Morse, Q., Y. Kuang, M., M. Hammad, The E., Hamelin, A. Malliri, and H. P. Sorensen, M., Daugaard, T., C. Tan, S., Castillo-Lluva, E., Hurt, I., Sinning, K., Wild, J., Bassler, C., Rutz, F., Adolf, Z., Sun, R., Beck, rdiso,R n Schio and R. Fredriksson, S. S. Ferguson, and B. L. Dale, L., J. Esseltine, and H. Xia, C., W. Claycomb, L., M. Lam, H., H., Gu, X., McNeill, Zhang, L., Yang, L., C., Dong, J. Wrana, L., G. Zhang, Wu, and A., C. Dong, A. Sing, K. O., Luu, Jacobson, M., and A. G. Daulat, I. Tikhonova, J., Siegel, S., Costanzi, G. D. Lambright, and F. Barr, EERHARTICLE RESEARCH aet .L,Lbeqe . rii .J n eoi,J L. J. Benovic, and J. M. Orsini, P., Labrecque, L., T. J. B. Kinsella, Parent, and M. H. Reid, Y. B., Z. M. Peng, O’Keeffe, and C. D. Desrosiers, J., T. Cammett, K., L. J. Mosavi, B. Knoll, and W. Dai, E., Alpizar-Foster, E., E. 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(TP receptor A2 thromboxane of endocytosis the constitutive of 5600-5607. by isoform formed beta receptors the of pool intracellular associated cycle. cell the during Y. signaling. cell of regulation in leaders 9 nitni Itp Arcpo rmtsRb T idn n vesicular and binding GTP Rab5 promotes S. receptor S. 1A fusion. Ferguson, type and II angiotensin H. P. Anborgh, vitro. in fibroblasts periodontal in protein Res. Periodontal and J. mRNA procollagenase receptors. endosomes. recycling of dynamics 11480-11488. the in involved interface J. Cherfils, e.Ml elBiol. Cell Mol. Rev. ubiquitination. Rabex-5-mediated by 1377. signaling Ras of cells. al. photoreceptor adult et of Q. maintenance Biol. Y. and PLoS trafficking Lin, rhodopsin L., for required Duraine, G., David, GTPase. Rab11a with interaction direct 2332-2346. a through T. regulated B. is Kinsella, and C. C. receptor. beta2-adrenergic the of degradation Chem. Biol. and J. targeting, lysosomal M. A. 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