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RESEARCH ARTICLE

Ubiquitylation and activation of a Rab GTPase is promoted by ab2AR–HACE1 complex

Veronik Lachance1,2, Jade Degrandmaison1,2, Sebastien Marois1,2, Melanie Robitaille3, Samuel Genier1,2,Stephanie Nadeau1,2, Stephane Angers3 and Jean-Luc Parent1,2,*

ABSTRACT

We and others have shown that trafficking of G-protein-coupled

receptors is regulated by Rab GTPases. Cargo-mediated regulation

of vesicular transport has received great attention lately. Rab

GTPases, which form the largest branch of the Ras GTPase

superfamily, regulate almost every step of vesicle-mediated

trafficking. Rab GTPases are well-recognized targets of human

diseases but their regulation and the mechanisms connecting them

to cargo proteins are still poorly understood. Here, we show by

overexpression and depletion studies that HACE1, a HECT-

domain-containing ubiquitin ligase, promotes the recycling of the

b2-adrenergic receptor (b2AR), a prototypical G-protein-coupled

receptor, through a Rab11a-dependent mechanism. Interestingly,

the b2AR in conjunction with HACE1 triggered ubiquitylation of

Rab11a, as observed by western blot analysis. LC-MS/MS

experiments determined that Rab11a is ubiquitylated on Lys145.

A Rab11a-K145R mutant failed to undergo b2AR–HACE1-induced

ubiquitylation and inhibited the HACE1-mediated recycling of the

b2AR. Rab11a, but not Rab11a-K145R, was activated by b2AR–

HACE1, indicating that ubiquitylation of Lys145 is involved in

activation of Rab11a. Co-expression of b2AR–HACE1 also

potentiated ubiquitylation of Rab6a and Rab8a, but not of other

Rab GTPases that were tested. We report a novel regulatory

mechanism of Rab GTPases through their ubiquitylation, with

associated functional effects demonstrated on Rab11a. This

suggests a new pathway whereby a cargo protein, such as a G-

protein-coupled receptor, can regulate its own trafficking by inducing

the ubiquitylation and activation of a Rab GTPase.

KEY WORDS: GPCR, G-protein-coupled receptor, Rab, Trafficking

INTRODUCTIONG-protein-coupled receptors (GPCRs) represent ,4% of the

human genome and form one of the largest and most studied

family of proteins (Fredriksson and Schioth, 2005; Harrow et al.,

2012; Venter et al., 2001). They mediate physiological responses

to a vast array of cellular mediators such as hormones,

neurotransmitters, lipids, nucleotides, peptides, ions and

photons. All GPCRs share a common molecular topology with

a hydrophobic core of seven transmembrane a-helices, three

intracellular loops, three extracellular loops, an extracellular N-

terminus and an intracellular C-terminus. GPCRs are typically

delivered to the plasma membrane in a ligand-responsive and

signaling-competent form. Following agonist stimulation, the

majority of GPCRs internalize into endosomes and can then

undergo recycling to the cell surface or lysosomal degradation

(Costanzi et al., 2009; Pierce et al., 2002; Ritter and Hall, 2009).

The fact that more than 30% of prescribed drugs target GPCRs

highlights their importance in the treatment of disease (Hopkins

and Groom, 2002). Therefore, a better comprehension of the

cellular events controlling their intracellular trafficking is

essential to improve the actual drug efficacy and specificity,

but also to identify new pharmaceutical targets.

Our laboratory and others have previously characterized Rab

GTPases as key regulators of GPCR trafficking (Hamelin et al.,

2005; Lachance et al., 2011; Parent et al., 2009; Seachrist et al.,

2002; Wikstrom et al., 2008; Zhang et al., 2009). More than 60

Rab GTPases, forming the largest branch of Ras-related small

GTPases, are involved in almost every step of vesicle-mediated

transport (Zerial and McBride, 2001). Each Rab GTPase has a

distinct subcellular localization that correlates with the

compartments between which they coordinate transport

(Hutagalung and Novick, 2011). These proteins are well-

recognized targets in human disease, but to date are

underexplored therapeutically. Elucidation of how dysregulated

Rab proteins and accessory proteins contribute to disease remains

an area of intensive study and an essential foundation for

effective drug targeting. Cancer, neurodegeneration, diabetes and

bone diseases represent examples of pathologies resulting from

aberrant function of Rab GTPases (Kelly et al., 2012; Li, 2011;

Richards and Rutherford, 1990).

Like other GTPases, Rab proteins shuttle between the inactive

(GDP-bound) and active (GTP-bound) conformations. To do so,

distinct regulators promote the exchange of GDP to GTP (guanine

nucleotide exchange factors, GEFs) and GTP hydrolysis (guanine

nucleotide activating proteins, GAPs) (Schwartz et al., 2007).

Despite the large number of Rabs, very few GEFs and GAPs have

been identified for these small GTPases to date. To cite a few,

some DENN (differentially expressed in normal and neoplastic

cells) domain proteins such as Rab6IP1 (a Rab39 GEF), and Vsp9

domain proteins such as Rabex5 (a Rab5 GEF), have been

described as Rab GEFs. However, Rab GAPs are known to share

a common TBC1 (Tre-2/Bub2/Cdc16) domain (Barr and

Lambright, 2010; Marat et al., 2011; Xiong et al., 2012). It has

been shown that the GPCR angiotensin II type 1A receptor

(AT1AR) increases the GTP-binding of Rab5a. This study not

1Service de Rhumatologie, Departement de Medecine, Faculte de Medecine etdes Sciences de la Sante, Universite de Sherbrooke, the Centre de RechercheClinique Etienne-Le Bel, Sherbrooke, QC J1H 5N4, Canada. 2Institut dePharmacologie de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. 3Departmentof Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, and theDepartment of Biochemistry, Faculty of Medicine, University of Toronto, Toronto,ON M5S 3M2, Canada.

*Author for correspondence (jean-luc.parent@usherbrooke.ca)

Received 12 April 2013; Accepted 12 October 2013

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 111–123 doi:10.1242/jcs.132944

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only reported the first evidence that GPCRs might control activityof Rabs, but it also underlined that a direct interaction between

the receptor and the GTPase seems necessary for this effect(Seachrist et al., 2002). However, it remains unclear whether thereceptor itself acts as a GEF or recruits proteins possessing a GEFactivity.

It was recently described that mono-ubiquitylation enhancesactivation of K-Ras and facilitates its binding to specific effectors(Sasaki et al., 2011). Considering the small number of

characterized Rab GEFs, and the size of the Rab family, onecould speculate that activation of Rab GTPases might also becontrolled by a general mechanism involving post-translational

modifications such as ubiquitylation. However, Rabubiquitylation has not been described so far. Interestingly,HACE1 (HECT domain and ankyrin repeat containing E3

ubiquitin protein ligase 1) was identified as a Rab interactor(Tang et al., 2011) but no effect on Rab ubiquitylation wasreported. Because b2-adrenergic receptor (b2AR) trafficking isregulated by various Rab proteins (Awwad et al., 2007; Dong and

Wu, 2007; Dong et al., 2010; Hammad et al., 2012; Moore et al.,2004; Parent et al., 2009; Seachrist et al., 2000), we investigatedwhether HACE1 was able to modulate b2AR trafficking. Here,

we report that co-expression of b2AR with HACE1 induces theubiquitylation and activation of Rab11a, which in turn regulatesb2AR recycling. Ubiquitylation of other specific Rabs was also

observed in the presence of b2AR–HACE1. Together, our datauncover a new regulatory mechanism for Rab GTPases by whicha cargo protein can direct its own trafficking.

RESULTSHACE1 interacts with and regulates the recycling of the b2ARTang and colleagues reported that Rab1, Rab4 and Rab11

associate with HACE1 (Tang et al., 2011). Because theseGTPases are known to regulate specific events in b2ARtrafficking (Hammad et al., 2012; Parent et al., 2009; Seachrist

et al., 2000), we assessed whether the receptor could also interactwith HACE1. Immunoprecipitation of HA-tagged b2AR stablyexpressed in HEK293 cells revealed that endogenous HACE1 co-

immunoprecipitated with the receptor (Fig. 1A). The effect ofHACE1 on trafficking of transiently expressed b2AR was thendetermined by ELISA. Transient expression of Myc-HACE1 andMyc-HACE1-C876S (a mutant with deficient E3-ligase activity)

(Anglesio et al., 2004; Tang et al., 2011; Zhang et al., 2007)increased cell surface expression of b2AR by roughly 25%,indicating that the catalytic activity of the enzyme is not required

for this effect (Fig. 1B). Time-course analyses established thatHACE1 significantly decreased the apparent agonist-inducedinternalization of the b2AR, whereas HACE1-C876S had

virtually no effect (Fig. 1C). Because an increase in thereceptor recycling could explain these results, internalizationassays were carried out in the presence of a recycling inhibitor,

monensin (Hamelin et al., 2005). As can be seen in Fig. 1D,treatment with monensin inhibited the effect of HACE1 oninternalization of b2AR, indicating that HACE1 regulates b2ARrecycling. This was further studied by recycling time-course

experiments in cells treated with 5 mM isoproterenol for15 minutes at 37 C, and then incubated in DMEM containing10 mM propranolol for the indicated time periods to prevent

further receptor internalization and to allow receptor recycling(Fig. 1E). Data obtained confirmed that HACE1 promoted b2ARrecycling, whereas HACE1-C876S did the opposite after

60 minutes of recycling (Fig. 1E). We were then interested in

verifying the endogenous colocalization of HACE1 with b2AR.To do so, colocalization studies were carried out using cells

stably expressing HA-b2AR. Comparative analyses revealed nosignificant differences between the trafficking properties oftransiently expressed b2AR compared with stably expressedb2AR (supplementary material Fig. S1). Both systems were thus

used interchangeably in the present report. As shown in Fig. 1Fa–d, HACE1 and b2AR colocalize into intracellular compartmentsfound throughout the cytoplasm, in the proximity of the cell

membrane and in the perinuclear region, under basal conditions(Fig. 1Fd, extracted colocalizing pixels). Agonist stimulation ofthe receptor resulted in internalization of the b2AR from the

plasma membrane into intracellular compartments and in adistribution of HACE1 that concentrated towards the perinuclearregion where it predominantly colocalized with the receptor

(Fig. 1Ff–i). The intracellular distribution of HACE1 and itscolocalization with the receptor was similar to that seen in basalconditions as receptor recycling progressed (Fig. 1Fk–s).

The functional implication of endogenous HACE1 on the

trafficking of stably expressed b2AR was assessed in cellstransfected with HACE1 siRNA. Depletion of HACE1significantly reduced the cell surface expression of the b2AR

compared with expression in control cells (Fig. 2A). There was a,33% increase in apparent agonist-induced internalization of thereceptor when cells were transfected with HACE1 siRNA

compared with cells transfected with control siRNA (60%compared with 45% internalization, respectively) (Fig. 2B).b2AR recycling after agonist-induced internalization was

inhibited by ,25% in cells depleted of HACE1 compared withthe control (Fig. 2C). These data validated the results obtainedwith HACE1 overexpression and established a new role forHACE1 in b2AR cell surface expression and recycling.

HACE1 regulates b2AR recycling through Rab11aPrevious studies showed that b2AR recycling is regulated by

Rab4 and Rab11 (Moore et al., 2004; Parent et al., 2009;Seachrist et al., 2000). Because HACE1 is reported to associatewith Rab4 and Rab11 (Tang et al., 2011), we were thus interested

in determining whether HACE1 regulated b2AR recyclingthrough one of these two GTPases. b2AR recycling was thusmeasured in cells cotransfected with different combinations ofHACE1, Rab4a or Rab11a (Fig. 3A). Transfection of HACE1

strongly promoted receptor recycling compared with levels incells transfected with pcDNA3. Co-expression of Rab4a orRab11a did not have any significant effect on b2AR recycling.

Interestingly, b2AR recycling in the presence of HACE1 wasstrongly enhanced by Rab11a co-expression whereascotransfection of Rab4 prevented the HACE1-mediated effect

on b2AR recycling. This could be possibly explained by theability of Rab4a to interact with HACE1 (Tang et al., 2011) andto compete with other effectors involved in b2AR recycling

through HACE1 in our system. Fig. 3B shows that depletion ofendogenous Rab11a with siRNA significantly reduced therecycling of the b2AR when expressed alone and inhibited theHACE1-mediated increase in b2AR recycling by 40%. We show

in Fig. 1F that b2AR colocalizes intracellularly with endogenousHACE1 and it has been shown previously to colocalize withRab11 (Parent et al., 2009). Confocal microscopy studies showed

that endogenous HACE1 and HA-Rab11a, expressed at low levelsto reflect distribution of endogenous Rab11a, colocalized mostlyin perinuclear intracellular compartments, but also in peripheral

intracellular vesicles in HEK293 cells (Fig. 3C). Fluorogram

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Fig. 1. See next page for legend.

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analysis revealed that there is a high degree of colocalizationbetween HACE1 and Rab11a. Colocalization between b2AR-GFP, endogenous HACE1 and Rab11a was also detected in

perinuclear compartments (Fig. 3Cd, inset).

b2AR promotes HACE1-mediated ubiquitylation of Rab11aWe then evaluated the effects of receptor stimulation as well as ofHACE1 or Rab11a co-expression on co-immunoprecipitation ofb2AR–HACE1 and b2AR–Rab11a following stimulation with

isoproterenol for 0 to 120 minutes of cells expressing thecombinations of proteins indicated in Fig. 4A. The interactionbetween HACE1 and the b2AR was not significantly affected byreceptor activation (Fig. 4A, top panel, lanes 4–7). By contrast,

b2AR–Rab11a co-immunoprecipitation was down-modulatedafter 15 and 60 minutes of agonist treatment but returned tobasal levels at 120 minutes (Fig. 4A, second panel, lanes 8–11).

Of note, HACE1 co-expression caused a significant reduction inthe b2AR–Rab11a interaction in the absence of stimulation (t50),which was accentuated by receptor activation even up to

120 minutes (Fig. 4A, second panel, lanes 12–15).Densitometry analyses of multiple independent experiments ofHACE1 and Rab11a co-immunoprecipitation with the receptor

normalized on the receptor immunoprecipitation support theseobservations (Fig. 4B,C). Our earlier findings showed that theb2AR interacts preferentially with Rab11a in its GDP-bound form(Parent et al., 2009). This, together with the results presented in

Fig. 4, could suggest that receptor stimulation and co-expressionof HACE1 activates Rab11a, which would result in reducedb2AR–Rab11a interaction.

Interestingly, b2AR and HACE1 expression modified themigration profile of Rab11a (Fig. 4A, see arrows in bottompanel). Indeed, a ,50 kDa band appeared in lanes 8–11 where

the b2AR was co-expressed with Rab11a, suggestive ofdimerization of the small GTPase or its association withanother protein of ,25 kDa. This band was not detected in

lanes 12–15 where b2AR and HACE1 were co-expressed, whichcould indicate that the presence of HACE1 decreased Rab11adimerization or its association with another partner. Instead, a,25 kDa Rab11a band and higher bands each heavier by ,8 kDa

were observed in this condition, reminiscent of Rab11aubiquitylation. To verify whether Rab11 could dimerize in thepresence of the receptor, co-immunoprecipitation experiments of

differentially tagged HA-Rab11a and FLAG-Rab11a were carriedout from lysates of cells cotransfected with empty vector, Myc-HACE1, b2AR-GFP or both proteins (Fig. 4D). Co-expression of

b2AR-GFP strongly promoted the FLAG-Rab11a/HA-Rab11a

co-immunoprecipitation (Fig. 4D, top panel, lane 6), which wasreversed by the co-expression of HACE1 (Fig. 4D, top panel,lane 7). These data suggest that Rab11a can form dimers in cells.

Alternatively, our data could also be explained by the associationof Rab11a with another protein or with receptor dimers/oligomersbinding simultaneously to multiple Rab11a proteins that would be

regulated by the receptor and HACE1.Rab11a ubiquitylation was then assessed in the presence of

b2AR, HACE1 or both proteins, in HEK293 cells transfected with

the combinations of constructs indicated in Fig. 4E.Ubiquitylation of Rab11a (Fig. 4E, top panel, lane 4), but notof Rab4a (lane 10), was detected in basal conditions in oursystem. Interestingly, substantial potentiation of Rab11a

ubiquitylation was revealed when both the b2AR and HACE1were co-expressed (lane 8), in contrast to results with Rab4a (lane14). This was dependent on the E3 ubiquitin ligase activity of

HACE1 because HACE1-C876S failed to produce the same effecton Rab11a (lane 9). Furthermore, we observed that depletion ofendogenous HACE1 inhibited the ubiquitylation of endogenous

Rab11a in cells stably expressing HA-b2AR (Fig. 4F). Sinceubiquitylation of GPCRs can regulate their trafficking, weverified whether HACE1 was ubiquitylating the b2AR. As

shown in Fig. 4G, HACE1 failed to ubiquitylate the receptor,unlike Nedd4, an HECT-E3-ubiquitin ligase known to beinvolved in b2AR ubiquitylation (Shenoy et al., 2008). Thisindicated that HACE1 was not regulating b2AR trafficking by

ubiquitylation of the receptor. It is also noteworthy that the shiftin mobility reflecting Rab11a ubiquitylation was detected in thepresence of HACE1 but not of Nedd4, revealing specificity in this

process (Fig. 4G, see arrow on bottom panel).

Ubiquitylation of Lys145 regulates Rab11a GTP loading and recyclingof b2ARTo confirm that Rab11a was ubiquitylated and to identify the Lysresidue involved, LC-MS/MS was performed on

immunoprecipitated HA-Rab11a that was co-expressed with theb2AR in HEK293 cells. Three peptides comprised of the141AFAEKNGLSFIETSALDSTNVEAAFQTILTEIYR174 aminoacids of Rab11a were identified and Lys145 was determined to be

ubiquitylated. An HA-Rab11a-K145R mutant was thus generatedand its ubiquitylation studied by western blot analysis with aubiquitin antibody as described in Fig. 5A. Mutation of Lys145

abolished Rab11a ubiquitylation by b2AR and HACE1 co-expression (Fig. 5A, top panel, lane 11), whereas ubiquitylationof wild-type Rab11a was greatly promoted in the same condi-

tion (lane 7). This was reflected in the migration pattern of

Fig. 1. HACE1 interacts with b2AR and regulates its cell surface expression and recycling. (A) Immunoprecipitation (IP) was performed using a HA-specific monoclonal or isotypic IgG1 control antibody on lysates from HEK293 cells stably expressing HA-b2AR and immunoblotting (IB) was carried out with anHACE1-specific polyclonal antibody. The blots shown are representative of three separate experiments. (B) HEK293 cells were cotransfected with FLAG-b2ARand pcDNA3, wild-type Myc-HACE1 or Myc-HACE1-C876S. Expression of cell surface receptor was measured by ELISA using a FLAG-specific monoclonalantibody. (C) Cells were treated with 5 mM isoproterenol for the indicated time periods. Quantification of surface receptors was performed by ELISA and thepercentage of receptor internalization was calculated. (D) Cells were pre-treated with 25 mM monensin (a recycling inhibitor) or ethanol (vehicle) for 30 minutesat 37˚C, and then stimulated with 5 mM isoproterenol for 60 minutes at 37˚C in the presence of monensin. Cell surface receptors were measured by ELISA andthe percentage of receptor internalization was calculated. (E) Cells were treated with 5 mM isoproterenol for 15 minutes at 37˚C, and then incubated in DMEMcontaining 10 mM propranolol for the indicated time periods to prevent further internalization and to allow receptor recycling. Receptor cell surface expressionwas detected by ELISA and the percentage of receptor recycling was calculated. Results are means 6 s.e.m. of at least five separate experiments. Thestatistical significance was determined using paired and unpaired Student’s t-tests (B,C) or regular two-way ANOVA test (D–E) followed by Bonferroni post-tests.*P,0.05, **P,0.01,***P,0.001. (F) Colocalization analyses of endogenous HACE1 in cells stably expressing HA-b2AR. Cells were treated with 5 mMisoproterenol for 15 minutes at 37˚C, and then incubated in DMEM containing 10 mM propranolol for the indicated time periods to prevent further internalizationand to allow receptor recycling. Cells were then fixed, permeabilized and labeled with rabbit polyclonal anti-HACE1 antibody. The third image on the rightrepresents a merged image of the red-labeled b2AR (a,f,k,p) and green-labeled HACE1 (b,g,l,q) signals where the areas with a high degree of colocalizationappear in yellow (c,h,m,r). Scale bars: 10 mm. Colocalizing pixels (d,i,n,s) and fluorograms illustrating HA-b2AR colocalization with HACE1 (e,j,o,t) are presented.

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HA-Rab11-K145R, which showed no ,33 kDa band in contrastto wild-type HA-Rab11a (Fig. 5A, panels 3 and 5, from top to

bottom).The functional role of Rab11a ubiquitylation was then assessed

in b2AR recycling experiments in cells that were co-expressingRab11a, Rab11a-K145R or Rab11a-S25N, alone or together with

HACE1 (Fig. 5B–D). As shown in Fig. 5B, co-expression ofHACE1 with Rab11a strongly promoted recycling of the receptorcompared with when Rab11a was expressed alone 15 minutes

after agonist removal (Fig. 5B). However, this effect was notsustained over time. On the contrary, Rab11a-K145R co-expression inhibited the early effect of HACE1 in recycling of

the receptor, but also decreased receptor recycling over time(Fig. 5C), to a similar extent as the dominant-negative Rab11a-S25N mutant (Fig. 5D). It is not clear why Rab11a promotes

HACE1-mediated recycling of the receptor only 15 minutes afteragonist removal. However, it is interesting to note that lack ofubiquitylation of Rab11 on Lys145 seems to confer dominant-negative properties analogous to the well-characterized

dominant-negative Rab11a-S25N mutant for the recycling ofthe b2AR. To verify whether Rab11a-K145R could affect therecycling of another membrane protein, we studied recycling of

the transferrin receptor in the presence of co-expressed Rab11a,Rab11a-K145R or Rab11a-S25N (supplementary material Fig.S2). Rab11a-K145R significantly inhibited recycling of the

transferrin receptor compared with cells transfected withpcDNA3 or Rab11a, to a degree similar as the Rab11a-S25Nmutant. This suggests that Rab11a-K145R regulates the recyclingof other membrane proteins. Testing of other receptors will be

necessary to conclude whether ubiquitylation of Rab11a isgenerally involved in recycling of membrane proteins goingthrough that route. It also remains to be determined whether an

interaction between other receptors such as the transferrinreceptor and HACE1 is involved in the regulation of theirrecycling by Rab11.

We then wanted to assess the effect of HACE1-mediatedubiquitylation on Rab11a activation. Cells co-expressing HA-Rab11a, -Rab11a-K145R or -Rab11a-S25N with the FLAG-

b2AR, alone or in combination with HACE1-Myc, were subjected

to fractionation into cytosolic and membrane fractions. Anantibody that specifically recognizes Rab11a in its activated,

GTP-bound form was used to immunoprecipitate the GTPase andsamples were analyzed by western blot using an anti-HAantibody. Fig. 6 shows that HACE1 co-expression promotedRab11a activation more than twofold but had no significant effect

on activation of Rab11a-K145R or of the dominant-negativeRab11a-S25N mutant. Two forms of Rab11a were detected in thecytosolic fractions corresponding to prenylated and unprenylated

forms of the protein, whereas only the prenylated form is seen inthe membrane fractions (Lachance et al., 2011). These resultsindicate that ubiquitylation of Rab11a on Lys145 by HACE1 is

involved in activation of the small GTPase.

Rab6a and Rab8a are also ubiquitylated by co-expression of b2ARand HACE1Whether other Rab GTPases were ubiquitylated was theninvestigated. First, sequence alignments were made betweenRab1a, Rab2a, Rab4a, Rab5a, Rab6a, Rab8a, Rab9a, Rab11a and

Rac1. Rac1 was included because it was shown to beubiquitylated by HACE1 on Lys147 (Castillo-Lluva et al.,2012). Interestingly, this analysis revealed that Rab1a, Rab6a

and Rab8a have Lys residues located in the vicinity of Rab11a-Lys145 and Rac1-Lys147 between the conserved G4 and G5boxes (Fig. 7A). The migration pattern of these Rab GTPases was

then assessed as for Rab11a in the presence or absence of HACE1co-expression alone or with b2AR (Fig. 7B). Prolonged exposureof western blot membranes of cell extracts expressing theindicated combinations of proteins showed that higher

molecular weight forms were detected in addition to theexpected ,25 kDa proteins for Rab2a, Rab6a and Rab8a,similar to Rab11a (Fig. 7B), suggesting that they could be

ubiquitylated. A single band of higher molecular weight can beseen for Rab5 owing to the presence of three HA tags at the N-terminus of the protein. Immunoprecipitation of Rab GTPases

that were co-expressed with HACE1 alone or together with b2ARand analyzed by western blotting with a ubiquitin antibodyshowed that ubiquitylation of Rab6a and Rab8a was detected in

basal conditions and enhanced by co-expression of b2AR and

Fig. 2. Depletion of HACE1 affects b2AR trafficking. HEK293 cells stably expressing HA-b2AR were transfected with negative control (CTRL) or siRNAagainst HACE1 for 72 hours and cell surface receptor expression (A), the percentage of receptor internalization after stimulation with 5 mM isoproterenol for15 minutes (B), and receptor recycling after stimulation with 5 mM isoproterenol for 15 minutes at 37˚C, and incubation in DMEM containing 10 mM propranololfor 60 minutes (C) was measured by ELISA using a HA-specific monoclonal antibody. Efficiency of HACE1 depletion by siRNA was confirmed by western blotanalysis with a HACE1-specific antibody (A, inset). Results are mean 6 s.e.m. of three to five separate experiments. The statistical significance was determinedusing a regular two-way ANOVA test followed by Bonferroni post-tests. *P,0.05, **P,0.01.

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enceHACE1 (Fig. 7C). However, Rab2a ubiquitylation was not

noticed. This could be explained by a limitation in sensitivity

of the assay or by the fact that the higher molecular weight formdetected for Rab2 in Fig. 7B, which was not significantly affectedby HACE1 and b2AR co-expression, corresponds to a post-

translational modification other than ubiquitylation. Also worthyof note, as in Fig. 5A (second panel), co-immunoprecipitation ofHACE1 with the Rab GTPases is augmented by the co-expression

of the b2AR in the absence of agonist stimulation (Fig. 7C,second panel), suggesting that the receptor acts as a scaffold in

the HACE1 interaction with, and ubiquitylation of, the RabGTPases.

Finally, a comparison of the localization of confirmed or potentialubiquitylation sites on Rac1 (Lys147), Rab6a (Lys144 and Lys146),Rab8 (Lys133, Lys138 and Lys146), as well as Rab11a (Lys145) on

the crystal structure of the corresponding proteins is shown inFig. 7D. This illustrates that these Lys residues are found on the a-helix positioned between the G4 and G5 boxes of the small GTPases

and are apparently accessible, as demonstrated for Rac1 (Castillo-Lluva et al., 2012) and Rab11a, for HACE1-mediated ubiquitylation.

Fig. 3. HACE1 regulates b2AR recycling through Rab11a. (A) HEK293 cells were transiently cotransfected with FLAG-b2AR and pcDNA3, Myc-HACE1, HA-Rab4a, HA-Rab4a + Myc-HACE1, HA-Rab11a, or HA-Rab11a + Myc-HACE1. Cells were treated with 5 mM isoproterenol for 15 minutes at 37˚C, and thenincubated in DMEM containing 10 mM propranolol for 15 minutes to prevent further internalization and to allow receptor recycling. Expression of cell-surfacereceptor was detected by ELISA and the percentage of receptor recycling was calculated. Results are means 6 s.e.m. of five separate experiments. (B) HEK293cells stably expressing HA-b2AR were pretreated with negative control (siCTRL) or siRNA against Rab11a for 24 hours and then cotransfected with pcDNA3 orMyc-HACE1. Cells were treated with 5 mM isoproterenol for 15 minutes at 37˚C, and then incubated in DMEM containing 10 mM of propranolol for 60 minutes toprevent further internalization and to allow receptor recycling. Expression of cell-surface receptors was detected by ELISA and the percentage of receptorrecycling was calculated. Results are means 6 s.e.m. of three separate experiments. Statistical analyses were performed using the regular two-way ANOVA testfollowed by Bonferroni post-tests (A,B). *P,0.05, **P,0.01, ***P,0.001, ****P,0.0001. (C) HEK293 cells transiently expressing b2AR-GFP were fixed,permeabilized and labeled with rabbit polyclonal anti-HACE1 and mouse anti-HA antibodies. Colocalization of (a) green-labeled b2AR, (b) red-labeled HACE1and (c) blue-labeled Rab11a appears white in d. Scale bars: 10 mm. Fluorograms illustrating colocalization of b2AR-GFP with HACE1, and colocalization ofRab11a with HACE1 are shown in the bottom two panels.

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Fig. 4. See next page for legend.

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DISCUSSIONCargo-specific regulation of vesicular trafficking has recentlyattracted much interest. Rab-mediated vesicular transport is wellknown to regulate membrane receptor trafficking, but less isknown about whether membrane receptors conversely regulate

the Rab trafficking machinery. We and others showed thatGPCRs interact with Rab proteins resulting in the regulation of

receptor trafficking (Esseltine et al., 2011; Hamelin et al., 2005;

O’Keeffe et al., 2008; Parent et al., 2009; Ritter and Hall, 2009;Seachrist et al., 2002; Smythe, 2002; Wikstrom et al., 2008).Whether membrane receptors interact with other elements of theRab-associated machinery to direct their own trafficking is the

subject of current intense research. For the importantpharmacological GPCR family, unravelling new mechanisms of

Fig. 4. b2AR modulates Rab11a dimerization and promotes HACE1-mediated ubiquitylation of Rab11a. (A) The b2AR–Rab11a interaction is affected byHACE1 co-expression and receptor stimulation. HEK293 cells were cotransfected with FLAG-b2AR and Myc-HACE1, HA-Rab11a or both proteins andstimulated with 5 mM isoproterenol for the indicated times. Immunoprecipitation (IP) of the receptor was carried out using a FLAG-specific monoclonal antibody.Immunoblotting (IB) was carried out with anti-FLAG, anti-Myc and anti-HA polyclonal antibodies. The blots are representative of three separate experiments.(B,C) Densitometry analyses performed with ImageJ software. The densitometry values of immunoprecipitated Myc-HACE1 or HA-Rab11a were normalized tothe amount of immunoprecipitated receptor and reported as a percentage. (D) b2AR expression promotes Rab11a dimerization, which is reversed by thepresence of HACE1. HEK293 cells were cotransfected with the indicated constructs. Immunoprecipitation of the GTPases was carried out using an anti-FLAGmonoclonal antibody. Immunoblotting was carried out with anti-FLAG, anti-GFP, anti-Myc and anti-HA polyclonal antibodies. (E) Rab11a but not Rab4a isubiquitylated by the co-expression of b2AR and HACE1. HEK293 cells were cotransfected with the indicated constructs. Immunoprecipitation of the GTPaseswas performed using anti-HA or anti-FLAG monoclonal antibodies. Immunoblotting was carried out with anti-FLAG, anti-ubiquitin, anti-Myc and anti-HApolyclonal antibodies. The blots shown are representative of three separate experiments. (F) Endogenous Rab11a is ubiquitylated by HACE1. HEK293 cellsstably expressing HA-b2AR were treated with negative control (siCTRL) or siRNA against HACE1 for 72 hours. Immunoprecipitation of the GTPase was carriedout using anti-Rab11a monoclonal antibodies or isotypic control. Immunoblotting was carried out with anti-Rab11a, anti-ubiquitin, anti-HACE1 and anti-HApolyclonal antibodies. The blots shown are representative of three separate experiments. (G) HACE1 does not ubiquitylate b2AR. HEK293 cells werecotransfected with FLAG-b2AR and the indicated proteins. Immunoprecipitation of the receptor was performed using anti-FLAG monoclonal antibody.Immunoblotting was carried out with anti-FLAG, anti-ubiquitin, anti-Myc and anti-HA polyclonal antibodies. The blots shown are representative of three separateexperiments. Molecular masses are indicated on the left.

Fig. 5. Ubiquitylation of Rab11a on Lys145 is involved in the regulation of b2AR recycling. (A) HEK293 cells were cotransfected with the indicatedcombinations of FLAG-b2AR, Myc-HACE1, HA-Rab11a and HA-Rab11a-K145R. Immunoprecipitation (IP) of the GTPase was performed with an anti-HAmonoclonal antibody. Immunoblotting (IB) was carried out with anti-FLAG, anti-ubiquitin and anti-HA specific polyclonal antibodies. Molecular masses areindicated on the left. The blots shown are representative of three separate experiments. (B–D) HEK293 cells were cotransfected with FLAG-b2AR and theindicated constructs. Cells were treated with 5 mM isoproterenol for 15 minutes at 37˚C, and then incubated in DMEM containing 10 mM propranolol for theindicated time periods to prevent further internalization and to allow receptor recycling. Expression of cell-surface receptors was detected by ELISA and thepercentage of receptor recycling was calculated. Results are means 6 s.e.m. of five separate experiments. Statistical analyses were performed using the regulartwo-way ANOVA test followed by Bonferroni post-tests. *P,0.05, **P,0.01, ***P,0.001.

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their trafficking is central to improve our understanding of their

regulation and to identify novel possible pharmacological targets.Similarly, the more than 60 members of the Rab GTPase familyare involved in virtually every step of vesicular trafficking and

are well-documented as targets in human disease. Surprisingly,very little is known regarding their regulation and theirinteracting partners when considering their number andphysiological or pathological importance. The fact that so few

Rab GEFs and Rab GAPs have been identified so far is intriguingand could be an indication that other regulatory mechanisms areinvolved.

In the present study, we showed that HACE1 interacts with theb2AR to regulate its recycling through mechanisms dependent onits E3 ubiquitin ligase activity and Rab11a. HACE1 co-

expression reduced the b2AR–Rab11a interaction. Thissuggested that HACE1 caused Rab11a activation because wepreviously reported that b2AR interacts with inactive Rab11a

(Parent et al., 2009). Mass spectrometry and site-directedmutagenesis showed that Rab11 is ubiquitylated on Lys145 byHACE1. This ubiquitylation is involved in Rab11a activation andin the regulation of b2AR recycling. This is akin to Ras activation

by its ubiquitylation on Lys147 (Sasaki et al., 2011). Our datasuggest that Rab11 can dimerize. Several GTPases including Ras,Rho and Arf form dimers and oligomers (Beck et al., 2008;

Inouye et al., 2000; Zhang et al., 2001; Zhang and Zheng, 1998),although the physiological significance of GTPaseoligomerization is not fully understood. Crystallography studies

proposed that Rab11a exists as a dimer in its GDP-bound form(Pasqualato et al., 2004). Interestingly, our data indicate that theb2AR, which interacts with the GDP-bound form of Rab11,

increased the formation of Rab11a dimers in cellulo, as evidenced

by the appearance of a ,50 kDa band of the predicted size of a

Rab11a dimer in cell lysates (Fig. 4A) and by increased co-immunoprecipitation between differentially epitope-taggedRab11a in the presence of the receptor (Fig. 4B). This was

inhibited by co-expression of HACE1. The HACE1–Rab11ainteraction was promoted by b2AR co-expression. Altogether, ourdata suggest that the b2AR acts as a scaffold to promote theHACE1 interaction with, ubiquitylation of and activation of

Rab11a. This could possibly result in dissociation of Rab11adimers from the receptors and interaction of active Rab11a witheffectors to regulate b2AR recycling (Fig. 8). However, we

cannot exclude the possibility that the receptor promotes theinteraction between Rab11a and another ,25 kDa protein,leading to the appearance of a ,50 kDa band in cell lysates, or

alternatively, that increased co-immunoprecipitation ofdifferentially tagged Rab11a is caused by the formation ofreceptor dimers or oligomers, and that both of these processes

would be reduced by the expression of HACE1. More work willbe needed to fully address whether Rab11a dimerizes.

Rab4a was not ubiquitylated by b2AR–HACE1, correlatingwith its inability to enhance HACE1-mediated b2AR recycling.

Rab6a and Rab8a, but not other Rab proteins tested in the presentstudy, were ubiquitylated in the presence of b2AR and HACE1. Itwill be interesting to further characterize this specificity towards

other Rab proteins and to determine whether other GPCRsexhibiting different trafficking itineraries behave like the b2AR.HACE1 has previously been shown to interact with Rab proteins,

but no effect on Rab ubiquitylation was described (Tang et al.,2011). This could be due to the absence of a crucial factor in theequation, similar to for example the co-expression of a GPCR.

Future studies will reveal whether this is specific to this class of

Fig. 6. Ubiquitylation of Rab11a on Lys145 by b2AR-HACE1 is involved in its activation. HEK293 cells were transfected with HA-Rab11a, HA-Rab11a-K145R or HA-Rab11a-S25N in combination with FLAG-b2AR alone or with HACE1-Myc. Cells were subjected to fractionation 48 hours after transfection.Cytosolic (C) or membrane (M) fractions were immunoprecipitated with an antibody specifically recognizing activated Rab11-GTP. Immunoprecipitates andsamples from the cytoplasmic and membrane fractions were analyzed by western blot using the indicated antibodies. The blots shown are representative of fourseparate experiments. Densitometry analyses of the quantity of Rab11-GTP that were immunoprecipitated from the membrane fractions were performed withImageJ software. Densitometry value measured for the b2AR condition was set at 100%. Results are the means 6 s.e.m. of four independent experiments. Thestatistical significance was determined using an unpaired Student’s t-test. *P,0.05.

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membrane receptors but our data with the transferrin receptorsuggest that ubiquitylation of Rab11 is involved in the regulation

of recycling of other receptor types. It will be interesting todetermine the role of HACE1 in regulating trafficking of othermembrane receptors through Rab11, and whether other receptor

types modulate ubiquitylation of Rab GTPases. Ubiquitylation ofRac1 on Lys147 by HACE1 causes proteasomal degradation ofthe small GTPase (Castillo-Lluva et al., 2012). The stability ofRab6a, Rab8a and Rab11a was not affected by HACE1-mediated

ubiquitylation, suggesting that mono-ubiquitylation or ubiquitinchains incompatible with proteasome-mediated degradation areinvolved. Specificity thus seems to also exist in the effects of

ubiquitylation of target proteins by HACE1. Specificity was also

shown by the inability of Nedd4 to induce shifts in the migratingpattern, reflecting ubiquitylation of Rab11a, in contrast to results

obtained with HACE1.How ubiquitylation of Rab11a leads to its activation remains to

be determined. Mono-ubiquitylation of Ras on Lys147 severely

abrogates its interaction with and the response to GAPs (Baker etal., 2013). However, some GEFs for other small GTPases, forinstance Rabex 5 for Ras, c-Cbl for Rap1 and Rac1, and HERC1for Arf1, Rab3a and Rab5, were shown to be E3 ubiquitin ligases

(Barr and Lambright, 2010; Rosa et al., 1996; Swaminathan andTsygankov, 2006; Xu et al., 2010). Whether HACE1 acts as aGEF for Rab11a or whether ubiquitylation of Rab11a modulates

its interaction with GEFs or GAPs will be the subject of future

Fig. 7. Identification of Rab6a and Rab8a as HACE1 substrates. (A) Schematic representation of conserved domains of small GTPases and sequencealignments of Homo sapiens Rac1, Rab1a, Rab2a, Rab4a, Rab5a, Rab6a, Rab8a, Rab9a and Rab11a proteins are shown. Conserved residues between G4and G5 boxes are shown in red and HACE1 ubiquitylation sites of Rac1 and Rab11a are highlighted in yellow. (B) HEK293 cells were cotransfected with FLAG-b2AR and the indicated combinations of pcDNA3, Myc-HACE1 and HA-tagged Rab GTPases. Immunoblotting (IB) was carried out with anti-FLAG, anti-Myc,anti-HA and anti-GAPDH antibodies. The blots shown are representative of three separate experiments. Arrow indicates the appearance of higher molecularmass forms of the Rab proteins upon b2AR and HACE1 coexpression. (C) HEK293 cells were cotransfected with FLAG-b2AR and the indicated combinations ofpcDNA3, Myc-HACE1 and HA-tagged Rab GTPases. Immunoprecipitation (IP) of the GTPases was performed with an anti-HA monoclonal antibody.Immunoblotting (IB) was carried out with anti-FLAG, anti-ubiquitin, anti-Myc and anti-HA polyclonal antibodies. Molecular masses are indicated on the left. Theblots shown are representative of three separate experiments. (D) Images were prepared with PyMOL (www.pymol.org) using coordinates from crystal structuresof active Rac1 (PDB 3TH5), Rab6a (PDB 4DKX), Rab11a (PDB 1OIW) and Rab8a (PDB 3TNF). Potential and confirmed HACE1 ubiquitylation sites are shownin red.

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experiments. Rab11 GEFs are known in Drosophila (Xiong et al.,2012) but no human orthologs have yet been identified to the bestof our knowledge.

It is also noteworthy that b2AR-HACE1-mediatedubiquitylation shows specificity for Rab6a, Rab8a and Rab11a.This is analogous to our recent discovery of a complex betweenthe b2AR and Rab geranylgeranyltransferase a that regulates the

prenylation of these three GTPases (Lachance et al., 2011). Thisis interesting because Rab6a, Rab8a and Rab11a are functionallyconnected in intracellular transport: Rab11 interacts with Rabin8

and stimulates its GEF activity toward Rab8 (Knodler et al.,2010) and Rab6a-interacting protein 1 links Rab6 and Rab11function (Miserey-Lenkei et al., 2007). In vitro studies indicate

that Rab6 does not interact with HACE1 (Tang et al., 2011).However, we observed that HACE1 mediates Rab6aubiquitylation. This could be explained by the lack of post-translational modifications of the purified proteins that prevent

them from interacting in vitro. This is however unlikely becauseHACE1 directly interacts with Rab11 in the same conditions(Tang et al., 2011). It is thus possible that an intermediate is

involved in the interaction between HACE1 and Rab6 to mediatethe ubiquitylation of this small GTPase. Perhaps Rab6a could getubiquitylated by HACE1 as part of a functional complex with

Rab11a that remains to be defined.The fact that HACE1-C876S was able to promote cell surface

expression of b2AR suggests that the protein has functions that

are independent of its E3 ligase activity. HACE1 contains sixputative ankyrin repeat domains usually known for theirscaffolding properties in protein complex assembly (Mosavi etal., 2004). We recently showed that ankyrin-repeat-containing

proteins can promote export of GPCRs (Parent et al., 2010). It isthus possible that HACE1 increases cell surface targeting of theb2AR through protein interactions with its ankyrin repeats.

In summary, our findings provide significant novel insightsinto three issues of intracellular trafficking and regulation of RabGTPases: (1) Rab GTPases can be ubiquitylated; (2)

ubiquitylation of Rab11a is involved in its activation; and (3)cargo proteins such as GPCRs, can regulate their own traffickingby regulating the activity of Rab GTPases through scaffoldingcomplexes between the Rab GTPases and an E3 ubiquitin ligase.

This could be relevant to human diseases associated withimpaired GPCR trafficking and dysregulation of Rab GTPases.

MATERIALS AND METHODSReagentsThe monoclonal anti-HA (16B12) and anti-Myc (9E10) antibodies were

from Covance. The polyclonal anti-FLAG, monoclonal FLAGM1-specific

and the polyclonal anti-HACE1 were from Sigma-Aldrich. The mouse

monoclonal IgG1 FLAGM2 antibody was also used as an isotypic control.

The polyclonal anti-Myc (A-14), the anti-HA-probe (Y-11), the anti-

GAPDH antibodies and Protein-G agarose beads were from Santa Cruz

Biotechnology. The monoclonal anti-HA-peroxidase (3F10) was

purchased from Roche Applied Science. The mouse monoclonal anti-

Rab11a was purchased from BD Transduction Laboratories. Anti-mono-

and anti-polyubiquitinylated monoclonal antibodies conjugated to

peroxidase (FK2H) were from Enzo Life Sciences. The anti-Myc-HRP

polyclonal antibody was from Abcam. The anti-active Rab11 antibody

was from NewEast Bioscience. The rabbit monoclonal anti-Rab11a,

Alexa Fluor 546 donkey anti-rabbit, Alexa Fluor 633 goat anti-mouse

antibodies, human transferrin conjugates to Alexa Fluor 546, and

ProLong Gold antifade reagent were purchased from Invitrogen.

Isoproterenol, propranolol and monensin were purchased from Sigma.

An alkaline-phosphatase-conjugated goat anti-mouse antibody and the

alkaline phosphatase substrate kit were purchased from Sigma. Human

holo-Transferrin was a kind gift from the laboratory of Dr Richard Leduc

(Universite de Sherbrooke).

Plasmid constructsThe HA-Rab11a-S25N, HA-Rab11a-K145R constructs were prepared

by PCR from the pcDNA3-HA-Rab11a constructs as described

previously (Theriault et al., 2004; Parent et al., 1999). The HA-

Nedd4 construct was purchased from Addgene (plasmid 11426). The

Myc-HACE1 construct was prepared by PCR from the human cDNA

clone template purchased from OriGene (SC107534). The PCR

fragment was digested with BamHI and EcoRI and ligated into the

pcDNA3 vector. The Myc-HACE1-C876S mutant was prepared by

PCR from pcDNA3-Myc-HACE1. The full-length PCR fragment was

digested with BamHI and EcoRI and ligated into the pcDNA3 vector.

Integrity of the coding sequence of these constructs was confirmed by

dideoxy sequencing.

Cell culture and transfectionHuman embryonic kidney HEK293 cells were maintained in DMEM

(Dulbecco’s Modified Eagle’s Medium) (Invitrogen) supplemented with

10% (v/v) FBS (fetal bovine serum) at 37 C in a humidified atmosphere

containing 5% CO2. Transient transfection of HEK293 cells grown to 50–

70% confluence were performed using the TransIT-LT1 Reagent (Mirus)

according to the manufacturer’s instructions. Empty pcDNA3 vector was

added to keep the total amount of DNA per plate constant.

Fig. 8. Model for Rab11a activation by b2AR through HACE1-mediated ubiquitylation. Briefly, b2AR interacts with Rab11a dimers (question mark indicatesthat Rab11a dimerization is a possibility that remains to be confirmed) and acts as a scaffold to recruit HACE1 (step 1) resulting in the ubiquitylation ofRab11a (step 2), leading to Rab11a GTP-loading and dimer dissociation (step 3), and ultimately to dissociation from the receptor and activation of effectorsto regulate b2AR recycling (step 4).

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ImmunoprecipitationHEK293 cells were transiently transfected with the indicated constructs

and were maintained as described above for 48 hours. The cells were then

washed with ice-cold PBS and harvested in 300 ml of lysis buffer (150 mM

NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% deoxycholate, 0.1% SDS, 10 mM

Na4P2O7, 1% IGEPAL, and 5 mM EDTA or 1 mM CaCl2 depending on

the antibody used for the assay) or ubiquitylation lysis buffer for

ubiquitylation experiments (50 mM HEPES, pH 7.5, 250 mM NaCl,

2 mM EDTA or 1 mM CaCl2, 0.5% IGEPAL, 1 mM PMSF, 1 mM NaF,

1 mM Na3VO4, 10% glycerol and 10 mM N-ethylmaleimide). Both

buffers were supplemented with protease inhibitors (9 nM pepstatin, 9 nM

antipain, 10 nM leupeptin and 10 nM chymostatin) (Sigma Aldrich).

Immunoprecipitations were then carried out as we described previously

(Lachance et al., 2011; Parent et al., 2010).

Immunofluorescence staining and confocal microscopyConfocal microscopy was performed to detect the indicated endogenous

or transfected proteins in HEK293 cells using a scanning confocal

microscope (FV1000-Olympus) coupled to an inverted microscope with a

660 oil-immersion objective lens and images were processed using

Fluoviewer 2.0 software (Olympus). Cells were processed as we

described previously (Lachance et al., 2011; Parent et al., 2010).

Measurement of cell-surface receptorsFor quantification of expression of cell-surface receptors, 6.56105

HEK293 cells were plated in 24-well plates pre-coated with 0.1 mg/ml

poly-L-lysine (Sigma), transfected with the indicated constructs and then

maintained for an additional 48 hours and then processed for ELISA as

described previously (Hamelin et al., 2005; Lachance et al., 2011; Parent

et al., 2009; Parent et al., 2010; Parent et al., 1999; Theriault et al., 2004).

siRNA assaysThe synthetic oligonucleotides ID s33239 and s33240 targeting the

human HACE1 gene, s16703 and s16704 targeting the human RAB11A

gene, and the negative control siRNA (Silencer Negative Control 1,

catalogue number-AM4611) were purchased from Ambion. HEK293

cells were transfected with 10 nM oligonucleotide using the

Lipofectamine 2000 transfection reagent (Invitrogen) according to the

manufacturer’s instructions. Protein expression analysis by western

blotting and ELISA experiments were performed at 72 hours post-

transfection as usual (Lachance et al., 2011; Parent et al., 2010).

DMP antibody crosslinkingFor HA-Rab11a purification needed for mass spectrometry experiments,

we generated Protein-G agarose beads DMP (New England Biolabs)

crosslinked to mouse IgG1 anti-HA antibody as described by the

manufacturer. Briefly, 100 ml of Protein-G agarose beads were

resuspended and aliquoted in test tubes. Beads were washed twice with

500 ml of binding buffer (0.1 M sodium phosphate buffer pH 8.0). Then

80 ml of binding buffer and 30 mg of anti-HA antibody were added and

incubated overnight on a rocker at 4 C. The following day, beads were

washed three times with 500 ml of binding buffer and twice with 1 ml of

crosslinking buffer (0.2 M triethanolamine, pH 8.2). Subsequently, 1 ml

of fresh DMP solution (15 mM in crosslinking buffer) was added and

crosslinking was allowed for 60 minutes on a rocker at room

temperature. Beads were then washed once with blocking buffer (0.1

M ethanolamine, pH 8.2) and incubated in 1 ml of blocking buffer for

60 minutes on a rocker at room temperature. Beads were washed once

with PBS then unbounded antibodies were eluted twice with 1 ml of

elution buffer (0.1 M glycine pH 2.5). Finally, beads were washed twice

with PBS, resuspended in 50 ml PBS, and stored at 4 C until use.

Protein purification and direct LC-MS/MS analysisHEK293 cells were plated in five 100 mm Petri dishes at a density of

2.06106 cells per dish. The following day, the cells were transiently

transfected with FLAG-b2AR, HA-Rab11a with or without Myc-HACE1

and then maintained for an additional 48 hours. The cells were then treated

with 0.75 mM of epoxomicin or 0.42% DMSO for 4 hours. Subsequently,

the cells were washed twice with ice-cold PBS and harvested in 1 ml per

dish of ubiquitylation lysis buffer supplemented with protease inhibitors.

After 60 minutes of incubation in lysis buffer at 4 C, the lysates were

centrifuged for 30 minutes at 13,500 g at 4 C. Lysates were pooled and

pre-cleared with 20 ml of Protein-G agarose beads per ml of lysate for 1 h

on a rocker at 4 C and then pre-cleared again with 20 ml of Protein-G

agarose beads per ml overnight. The following day, 100 ml of Protein-G

agarose beads DMP-crosslinked to mouse IgG1 anti-HA antibody was

added to pre-cleared lysates and incubated overnight on a rocker at 4 C.

Beads were then washed three times with lysis buffer, and three times with

50 mM ammonium bicarbonate, pH 7.8, to remove any residual detergent.

Finally, the antibody complex was eluted four times with 100 ml of elution

buffer (0.1 M glycine, pH 2.5), pre-heated to 37 C, for 10 minutes on a

rocker at 30 C. The eluted fractions were neutralized with 10 ml of Tris-

HCl, pH 8.0. The resulting peptide mixture was analyzed by liquid

chromatography-tandem MS (LC-MS/MS) using a LTQ-XL Linear Ion

Trap Mass spectrometer (Thermo Scientific) as described previously

(Daulat et al., 2012).

Subcellular fractionationHEK293 cells were grown overnight in 100 mm Petri dishes before being

transfected. Forty-eight hours after transfection, cells were suspended in

1 ml of hypotonic buffer (0.16 PBS) supplemented with protease

inhibitors (9 mM pepstatin, 9 mM antipain, 10 mM leupeptin and

10 mM chymostatin) and incubated on ice for 10 minutes before being

broken with 20–30 strokes of a Dounce homogenizer depending on cell

confluency. 100 ml of 106 PBS was then added to the hypotonic cell

solution to obtain a 16 concentration. Cells were then centrifuged at

3000 rpm for 20 minutes at 4 C. Supernatant was conserved and was

centrifugated again at 10,000 rpm for 10 minutes at 4 C to remove

nuclei, unlysed cells and large cell debris. Lysates were then centrifuged

at 100,000 g for 1 hour at 4 C to give supernatant and pellet fractions.

The fractions were subjected to immunoprecipitation and western blot

analysis using specific antibodies.

Densitometry analysesThe densitometry analyses were performed with ImageJ software.

Statistical analysisStatistical analyses were performed using Prism version 5.0 (GraphPad

Software) using the unpaired or paired Student’s t-test and Two-way

ANOVA test followed by Bonferroni post-tests. Data were considered

significant when *P,0.05, **P,0.01, ***P,0.001, ****P,0.0001.

Competing interestsThe authors declare no competing interests.

Author contributionsV.L., J.D., S.M., M.R., S.G. and S.N. performed the experiments. V.L., S.A. andJ.L.P. conceived the experiments, analyzed the data and wrote the manuscript.

FundingThis work was supported by the Canadian Institutes of Health [grant numberMOP-184095]; and a Chercheur Senior salary award from the Fonds de laRecherche Quebec-Sante (FRQS) to J.L.P. J.L.P. is the recipient of the Andre-Lussier Research Chair. V.L. received a studentship from the FRQS during part ofthis work.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.132944/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2014) 127, 111–123 doi:10.1242/jcs.132944

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