The ocular albinism type 1 (OA1) GPCR is ubiquitinatedand its traffic requires endosomal sorting complexresponsible for transport (ESCRT) functionFrancesca Giordanoa,b,c,1, Sabrina Simoesa,b,c, and Graça Raposoa,b,c,2

aInstitut Curie, Centre de Recherche, Paris F-75248, France; bStructure and Membrane Compartments, Centre National de la Recherche Scientifique, UMR144,Paris F-75248, France; and cCell and Tissue Imaging Facility, Infrastructures en Biologie Sante et Agronomie, Paris F-75248, France

Edited by David D. Sabatini, New York University School of Medicine, New York, NY, and approved June 7, 2011 (received for review March 2, 2011)

The function of signaling receptors is tightly controlled by theirintracellular trafficking.Onemajor regulatorymechanismwithin theendo-lysosomal system required for receptor localization and down-regulation is protein modification by ubiquitination and down-stream interactionswith the endosomal sorting complex responsiblefor transport (ESCRT) machinery. Whether and how these mecha-nisms operate to regulate endosomal sorting of mammalian Gprotein-coupled receptors (GPCRs) remains unclear. Here, we ex-plore the involvement of ubiquitin and ESCRTs in the trafficking ofOA1, a pigment cell-specific GPCR, target of mutations in OcularAlbinism type 1, which localizes intracellularly to melanosomes toregulate their biogenesis. Using biochemical and morphologicalmethods in combination with overexpression and inactivationapproaches we show that OA1 is ubiquitinated and that its intracel-lular sorting and down-regulation requires functional ESCRT compo-nents. Depletion or overexpression of subunits of ESCRT-0, -I, and -IIImarkedly inhibits OA1 degradation with concomitant retentionwithin the modified endosomal system. Our data further show thatOA1 ubiquitination is uniquely required for targeting to the intra-lumenal vesicles of multivesicular endosomes, thereby regulatingthe balance between down-regulation and delivery to melano-somes. This studyhighlights the role of ubiquitination and the ESCRTmachinery in the intracellular trafficking of mammalian GPCRs andhas implications for the physiopathology of ocular albinism type 1.

GPR143 | melanosome | multivesicular endosome | Tsg101 | Hrs

Gprotein-coupled receptors (GPCRs) regulate importantphysiological processes through the coordinated action of

their signaling pathways. These pathways are modulated by highlyconserved mechanisms that initially involve receptor endocytosisand subsequent targeting to the lysosome for degradation (down-regulation) and/or recycling to the plasma membrane to restorecellular signaling responsiveness (1). OA1 (GPR143) is a pigmentcell-specific glycoprotein with structural and functional features ofGPCRs (2). Mutations in the OA1 gene underlie ocular albinismtype 1 (3), an X-linked disorder that affects the number and thesize of melanosomes, the lysosome-related organelles (LROs) ofpigment cells devoted to melanin synthesis (4). The retinal pig-ment epithelium (RPE) and skin melanocytes of OA1 patients andcorresponding mouse model display giant melanosomes (“mac-romelanosomes”; refs. 5 and 6). Like other canonical GPCRs,OA1 interacts with arrestins and binds heterotrimeric G proteins(2). However, OA1 displays unique features among GPCRs: Itlocalizes mainly intracellularly, to melanosomes, by virtue ofsorting signals in its cytosolic domain (7). Whereas most GPCRsbind extracellular ligands, the OA1 ligand, presumably the mela-nin precursor L-DOPA (8) present in the lumen of the melano-some, triggers a signaling cascade from the organelle to the cytosol.Although this cascade remains poorly characterized, our recentstudies highlighted that the macromelanosomes result from ab-normal fusion/fission events at early steps of their biogenesis (9).OA1 function in melanogenesis is certainly regulated by a tightbalance between its targeting to melanosomes and its down-regulation. However, the mechanisms that regulate OA1 traffick-ing within the endo-melanosomal system and thereby its localiza-

tion and function remain undefined. Posttranslationalmodificationby ubiquitination controls intracellular trafficking events withinthe endo-lysosomal system (10, 11). Ubiquitin-modified mem-brane proteins delivered from the endocytic or biosyntheticpathways can be recognized by components of the endosomalsorting complex responsible for transport (ESCRT) machineryfor targeting to intraluminal vesicles (ILVs) of multivesicularbodies (MVBs) and for subsequent lysosomal degradation (12).Although ubiquitination and ESCRT function were reported toregulate trafficking of GPCRs in yeast (13–15) and have beenindependently proposed for the down-regulation of selectedmammalian GPCRs (16–18), it remains unclear how and where inthe cell they operate (1). Moreover, the emerging evidences ofubiquitin-independent mechanisms involved in their down-regu-lation reflect the additional complexity of the endocytic sorting ofmammalian GPCRs (19). Using a combination of light and elec-tron microscopy (EM) and biochemical methods, we show thatOA1 down-regulation is dependent on ESCRT function due to itspostranslational modification by ubiquitination. Ubiquitinationcontrols sorting to the intraluminal vesicles of MVBs for appro-priate targeting within the endo-melanosomal network.

Results and DiscussionGPCR OA1 Is Postranslationally Modified by Ubiquitination.A uniquefeature of OA1 as a GPCR is its intracellular localization tolysosomes and melanosomes in melanocytes, and to late endo-somes/lysosomes when transiently expressed in nonmelanocyticcells (7, 9). In endosomes, OA1 is present both at the delimitingand internal membranes, in particular associated with intra-lumenal membrane vesicles (ILVs; ref. 9; Figs. S1 A and B andS2A) suggesting that its distribution within endosomal mem-branes might be a critical step in regulating its function and/ordegradation. To investigate whether OA1 can be modified byubiquitination we have used the approach described for cellsurface associated GPCRs (20). As reported for these GPCRs,ubiquitinated forms of OA1 were not easily detected probablydue to the small amounts of the total cellular complement of thisGPCR that is ubiquitinated at steady state. Thus, we havecoexpressed by transfection Flag-tagged OA1 and HA-taggedubiquitin in the melanocytic cell line MNT1 and in HeLa cells. Ina first step, OA1-Flag was immunoprecipitated from MNT-1 celllysates in stringent conditions (20). Subsequent immunoblotting(IB) with anti-HA antibodies revealed ubiquitinated forms ofOA1, with detectable bands from 68 kDa (monoubiquitin) to 92kDa (four ubiquitins) and a smear at higher molecular weightcorresponding to polyubiquitin chains (Fig. 1Aa). We confirmedthe specificity of these bands by cotransfecting OA1-FLAG with

Author contributions: F.G. and G.R. designed research; F.G. and S.S. performed research;F.G., S.S., and G.R. analyzed data; and F.G. and G.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Department of Cell Biology and Howard Hughes Medical Institute, YaleUniversity School of Medicine, New Haven, CT 06510.

2To whom correspondence should be addressed: E-mail: graposo@curie.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103381108/-/DCSupplemental.

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a control HA-plasmid (Fig. 1Aa). Providing further evidence forubiquitination in an heterologous cell system (Fig. 1Ab), thesame experiments were performed in HeLa cells in which OA1 iscorrectly targeted to endosomes and lysosomes (Figs. S1 A and Band S2A; ref. 7). Ubiquitination occurs generally on lysine (Lys)residues (21). In support of these observations and as shown byimmunofluorescence (IF), OA1-Flag was detected in ubiquitin-HA-positive structures in both HeLa (Fig. S2 A–C) and MNT-1(Fig. S2 G–I) cells. OA1 contains 7 Lys residues with 4 Lys in thethird cytosolic loop (K215, K225, K243, K248) and 3 Lys in thecytosolic tail (K325, K355, K391), all of them potentially in-volved as Ub acceptors. Substitution of all 7 (lysines) Lys (K) toArg (R) (OA1K1-7R) (Fig. S1C) abrogated OA1 ubiquitination intransfected HeLa cells (Fig. 1Bb) and also led to a mild increase

in the unmodified OA1 protein levels (Fig. 1Ba). Overall, ourresults show that OA1 undergoes ubiquitination on Lysin resi-dues both in melanocytic (MNT-1) and nonmelanocytic (HeLa)cells. The ability of OA1 to be ubiquitinated also in HeLa cells,where it is not expressed physiologically, indicates that OA1 canbe basally ubiquitinated, similarly to what was reported for theprotease-activated receptor, PAR1 (22). Several mammalianGPCRs have been shown to be mono- or polyubiquitinated onlysine residues located on their cytosolic side: PAR1 (22), β2AR(23), CXCR4 (16), δ-opioid (24), and k-Opioid receptor (17).Receptor modification by ubiquitin is thought to be required fortheir internalization at the plasma membrane but also to directmembrane cargo, delivered from both endocytic and biosyntheticpathways, to the intraluminal vesicles within late endosomes/

Fig. 1. OA1 is ubiquitinated in MNT-1 cells and HeLa cells. (A)MNT-1 (a) or HeLa cells (b) were transfected with OA1-Flag andeitherubiquitin-HAorempty vectorHA-constructs. Twenty-fourhours after transfection, OA1was immunoprecipitated (IP) withanti-Flag antibody and lysates were immunoblotted (IB) withanti-Flag antibody to detect OA1 and with anti-HA antibody todetect incorporated epitope-tagged ubiquitin. Note the dif-ferent OA1 forms (a doublet of 45–48 kDa and a 60-kDa band).Ubiquitinated forms ofOA1were observed in cells coexpressingOA1-Flag with ubiquitin-HA but not in cells coexpressing OA1-Flag with a control HA-plasmid. Arrows indicate 68- to 92-kDabands. Asterisks indicate a smear at higher molecular weightcorresponding to polyubiquitin chains. (B) HeLa cells weretransfected with Flag-tagged wt (OA1wtFlag) or mutant OA1(OA1K1-7RFlag) constructs and either ubiquitin-HA or emptyvector HA. Immunoprecipitation of OA1 and immunoblottingwith anti-Flag (a) and with anti-HA antibodies (b) were per-formed as in A. Mutation of all lysine in OA1K1-7R abrogatedOA1 ubiquitination. Arrows and asterisk indicate bands anda smear, respectively, corresponding to ubiquitinated forms ofOA1 missing in the ubiquitin-deficient mutant OA1K1-7R.

Fig. 2. The ubiquitin-deficientmutant of OA1 accumulates atthe limiting membrane of en-larged MVBs in HeLa cells. (A andB) Ultrathin cryosections of HeLacells transfectedwith Flag-taggedwild type (OA1wtFlag) or all lysinemutant OA1 (OA1K1-7RFlag) weredouble-immunogold labeled forFlag (PAG 15) to detect OA1 andfor CD63 (PAG 10). WhereasOA1wt is mostly associated withthe ILVs of CD63-positive MVBs(arrows) (A), themutant OA1K1-7R

is present at the limiting mem-brane of enlarged MVBs packedwith ILVs (arrows) (B). (C) Quan-titative evaluation of the labeling(gold particles) for OA1 wild typeor OA1 lysines mutant associatedto the limiting membrane (LM)and ILVs of MVBs shows that80%ofOA1K1-7R is retained at LMcompared with wild-type OA1(10%). (D) A similar quantifica-tion for the all-lysine mutant ofMART-1 (MART-1K1-6R; ref. 29),show that only 30% of MART-1 isretained at the limiting mem-brane of MVBs and 70% is stillsorted to ILVs of MVBs whosemorphology is not affected (E andF). (Scale bars, 150 nm.)

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MVBs (reviewed in ref. 25). In mammalian cells, this process hasbeen well described for the down-regulation of EGF receptor(26). Mammalian GPCRs do not appear to require ubiquitina-tion for efficient endocytosis from plasma membrane but mainlyfor their trafficking after endocytosis (1). Given the exclusiveintracellular localization of OA1, further analysis of its ubiquitin-dependent sorting offers a unique opportunity to dissect thefunction of this posttranslational modification within the endo-cytic system of a mammalian GPCR.

Ubiquitination Is Required for the Targeting of OA1 to ILVs ofMultivesicular Endosomes. To underscore the consequences ofubiquitination inOA1 trafficking, we analyzed by immuno-electronmicroscopy (IEM) the localization in HeLa cells of the two OA1constructs: wild type (OA1wt-Flag) and the all lysine mutant OA1(OA1K1-7R-Flag). In these cells the bulk of OA1 localizes toCD63-positive MVBs where it is mainly associated with the ILVs(Fig. 2A). Strickling, the ubiquitination-defective OA1 mutant,accumulates at the limiting membrane of MVBs (Fig. 2B). Sucheffective sequestration of this GPCR in ILVs of MVBs is sup-ported by quantitative evaluation of the distribution of OA1 wildtype and OA1 Ub mutant on the limiting membrane and ILVs ofMVBs (Fig. 2C). In agreement, OA1-Flag expressed inHeLa cellspartially overlaps with structures positive for K63-linked ubiquitinchains (Fig. S2 D–F) that are known to act as a signal for proteinsorting into the MVB pathway (27). Notably, 40% the MVBsretaining the Ub-defective OA1 mutant were considerably en-larged with heavily packed ILVs compared with those hosting thewild-type OA1. These observations suggest that retention of OA1at the limiting membrane of MVBs is likely to modify the ho-meostasis and/or distribution of proteins involved in the bio-genesis and maturation of these compartments. A similarenlargement of MVBs was also observed in dendritic cells uponexpression of a Ub-mutant of MHC class II molecules (28). Re-inforcing the essential requirement of OA1 ubiquitination inendosomal sorting, the all lysinemutant ofMART-1 (MART-1K1-6R;ref. 29) appeared to be only slightly retained at the limitingmembrane of MVBs (Fig. 2 D–F). Moreover, the expression ofthis mutant did not affect compartment size (Fig. 2 E and F).To further investigate how ubiquitination impacts on the traf-

ficking of OA1 to lysosomes and melanosomes in melanocyticcells, we next analyzed by IEM in MNT1 cells the subcellular lo-calization of the Flag-tagged OA1 wild type (OA1wt) and all lysinemutant OA1 (OA1K1-7R). The mutant OA1K1-7R was retained atthe limitingmembrane ofMVBs also inMNT1 cells (arrows, Fig. 3B–D). These compartments, that were generally not observed incontrol cells (Fig. 3A), were similar to those observed inHeLa cellsupon OA1K1-7R transfection. They were also similar in luminalcontent to those generated when the function of ESCRT compo-nents is impaired in melanocytic cells (see below and ref. 30).Therefore, our results show that ubiquitination of OA1 is uniquelyrequired for its sorting to ILVs of multivesicular endosomes inboth HeLa cells and melanocyticMNT-1 cells. These observationsdiffer from recent findings on the δ-opioid receptor (DOR), forwhich ubiquitination participates to but is dispensable for ILVsorting (24). This is also what we observed for MART-1 (Fig. 2D–F), indicating that the ubiquitination requirements for ILV sortingdiffer depending on the proteins and maybe on their ability tofurther interact with downstream effectors. Interestingly, theamount of mutant, nonubiquitinated form of OA1 associated withmelanosomes is increased (arrowheads, Fig. 3 A and B; quantifi-cation in Fig. 3E). These observations are reminiscent of thosereported for the all-lysine mutant of MART-1 that appeared toaccumulate in melanosomes in the absence of ubiquitination (29).Taken together, our observations indicate that ubiquitination ofOA1 by regulating its down-regulation is likely to control thebalance of receptor within the endo-melanosomal system andensure its intracellular function.

Hrs (ESCRT-0) Is Required for the Endosomal Transport of OA1. Toelucidate the downstream molecular players involved in OA1trafficking and down-regulation we next examined the require-ment for the ESCRT machinery (12). Hrs is a component of the

ESCRT-0 complex directly involved in recognition of ubiquiti-nated membrane cargo on endosomes (31). At steady state, OA1partially overlaps with a small subset of Hrs positive endosomesin MNT1 cells (Fig. S2 J–L) and we have further provided bio-chemical evidence for an interaction between OA1 and Hrs(Fig. 4I). Hrs was detected by IB in lysates immunoprecipitatedwith anti-Flag (OA1) antibody of Hrs-myc and OA1-Flag-transfected MNT-1 cells, but not in lysates of MNT-1 cellscotransfected with Hrs-myc and a Flag empty vector. We nextexamined by IF and IEM the effect of overexpressing Hrs-YFP,one of the Hrs-tagged variants that function as dominant inhib-itors of Hrs-mediated lysosomal sorting of transmembrane pro-teins including receptors (32, 33) and GPCRs (18). IF analysisindicates that overexpression of Hrs-YFP strongly increases lo-calization of endogenous OA1 in the Hrs-positive endosomes(arrows, Fig. 4 A–C) and in the enlarged Hrs-positive clusters ofendosomes that are generated in overexpressing cells (arrows,Fig. 4 D–F). These modified endosomes have been shown to trapubiquitinated cargo destined to ILVs, as demonstrated forMART-1 in melanocytic cells (34) and also for another GPCR,PAR-2 (35). IEM revealed that both endogenous and transfectedOA1 were retained at the limiting membrane of these compart-ments (arrows, Fig. 4G and Inset). In addition, as shown by IB(Fig. 4H), the protein levels of OA1 were significantly increasedwhen coexpressed with Hrs-YFP compared with control cellsexpressing only YFP. Because nonubiquitinated proteins tra-versing early endosomes can also be trapped in the “Hrsosomes”(30), we further inactivated Hrs by RNAi in MNT-1 cells. De-pletion of Hrs results in the accumulation of transiently expressedOA1-Flag in Hrs-depleted cells, as shown by IF (Fig. S3M, arrow;quantification in Fig. S3N). Similar observations were reportedfor a PAR2 construct, another GPCR that undergoes ubiquiti-

Fig. 3. The ubiquitin-deficient OA1 mutant is retained on MVBs and melano-somes inMNT-1 cells. (A and B) Ultrathin cryosections ofMNT-1 cells transfectedwith Flag-taggedwildtype (OA1wtFlag) or all-lysinemutant OA1 (OA1K1-7R Flag)were double-immunogold labeled for Flag (PAG 15) to detect OA1 and Tyrp1(PAG10).OA1K1-7Rwas retainedat the limitingmembraneofMVBs (arrows inB–D) that are generally not observed in wild-type cells (A). Note also the presenceof OA1 at the limiting membrane of mature melanosomes in OA1wt andOA1K1-7R expressing cells (arrowheads, A and B). (Scale bars, 200 nm.) (E)Histogram depicting a quantitative evaluation of OA1 labeling (gold par-ticles) at themelanosomemembrane of OA1wt andOA1K1-7R transfected cells.

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nation and subsequent interaction with Hrs (35). However, de-pletion of Hrs does not result in the accumulation of endogenousOA1 within the cell but rather results in its decreased expressionas analyzed by IB (Fig. S3A). A similar decrease was reported alsofor MART-1 (34), which we use here as a control. IF analysisconfirmed a reduction of both endogenous OA1 and MART-1 inthe majority of siHrs-treated cells (arrows, Fig. S3 F–I) comparedwith control cells (Fig. S3 B–E). These observations suggest thatthe absence of functional Hrs impacts on their normal traffickingwithout fully impairing their degradation, as reported for theEGFR (36). In addition, we found by quantitative real-time PCRthat the reduction of the endogenous OA1 andMART-1 proteinswas certainly related to a down-regulation of their transcripts insiHrs-treated cells (Fig. S3 J–L). This observation indicates thatHrs depletion, could also consequently impact on the transcrip-tion of these melanosomal proteins. Taken together, our resultsindicate that the trafficking of OA1 requires its ubiquitination and

functional Hrs. Such requirements slightly differ from what waspreviously reported for the DOR, the down-regulation of whichappeared dependent on Hrs despite lack of ubiquitination (18).

OA1 Sorting and Degradation Requires ESCRT-I Function. One of thedownstream effectors of Hrs in the regulation of endosomal pro-tein sorting is ESCRT-I. Tsg101 is a “core” component of thiscomplex and can bind both ubiquitinated cargo and Hrs (37).Knockdown of Tsg101 has been shown to significantly inhibit ly-sosomal sorting and degradation of various membrane receptorsincluding EGFR (38) and the GPCRGABA(B)-receptor (39). Toinvestigate the involvement of ESCRT-I in OA1 sorting, we de-pleted Tsg101 by RNAi from MNT-1 cells. Efficient knockdown(>90%) of Tsg101 protein was confirmed by IB (Fig. 5A). Re-vealing a role for ESCRT-I in OA1 trafficking, depletion of Tsg101interfered with OA1 degradation resulting in a significant increasein OA1 levels relative to control cells (Fig. 5A). As a control and inagreement with previous observations (30), MART-1 levels alsoincreased upon Tsg101 down-regulation (Fig. 5A). IF analysisshowed that in Tsg101-depleted cells endogenous OA1 accumu-lates in enlarged vesicular structures throughout the cytoplasmthat were mostly overlapping with those positive for MART-1(Fig. 5 B–I). IEM revealed characteristic aberrant membranousstructures (class E compartments, ref. 38) depicting both OA1

Fig. 4. OA1 interacts with Hrs and Hrs overexpression traps OA1 at thelimiting membrane of endosomes. (A–F) Immunofluorescence analysis ofMNT-1 cells transfected with fluorescent Hrs-YFP (A and D) and labeled forendogenous OA1 (B and E). C and F represent merged images from the twoleft panels. Insets are 2.5× magnifications of boxed regions. Arrows indicatearea of colocalization. (Scale bar, 10 μm.) (G) Ultrathin cryosections of MNT-1cells transfected with Hrs-YFP alone (Inset) or in combination with OA1-Flag(G) were double immunogold labeled with anti-GFP (Hrs) and anti-OA1(endogenous OA1) (Inset) or GFP (Hrs) and anti-Flag (transfected OA1) (G),respectively. Both endogenous and transfected OA1 (PAG 15) are retained atthe limiting membrane of the Hrs-positive (PAG 10) endosomes. Note theinterconnected network of large endosomal vacuoles densely packed withsmall ILVs. (Scale bars, 200 nm.) (H) Total cell lysates of MNT-1 cotransfectedwith either Hrs-YFP or YFP and OA1-flag were analyzed by immunoblottingwith the indicated antibodies. The arrow indicates the accumulation of OA1upon coexpression with Hrs-YFP, but not with YFP alone. (I) MNT-1 cells weretransfected with either OA1-Flag or Flag empty vector and Hrs-myc. Lysateswere immunoprecipitated with monoclonal anti-Flag antibody, fractionatedby SDS/PAGE, and immunoblotted with anti-myc (Hrs) or Flag (OA1). Thearrow indicates a specific band detected by anti-myc antibody in OA1-immunoprecipitates but not in immunoprecipitates of empty-Flag vector.

Fig. 5. Knockdown of Tsg101 (ESCRT-I) affects OA1 sorting and degradationin MNT-1 cells. MNT-1 cells were treated with control siRNA (siCtrl) or siRNAsspecific for Tsg101 (siTsg101), for Tsg101 and MART-1 (siTsg101/MART-1) andfor MART-1 (siMART-1). (A) Whole cell lysates were analyzed by immuno-blotting for OA1, Tsg101, MART-1, or tubulin as a control. Note the effectivedepletion of Tsg101 and the enrichment of OA1 and MART-1 in siTsg101-treated cells. The amount of OA1 is significantly increased also in siTsg101/MART-1-treated cells. Arrows point to the 60-kDa form of OA1. (B–I) Cellstreated with siRNAs targeting Tsg101 (siTsg101) (F–I) or Ctrl siRNAs (siCtrl) (B–E) were analyzed by IFMusing antibodies toOA1 (B and F) andMART-1 (C andG). Overlays are shown inD andH. Boxed regions correspond to 5×magnifiedareas in E and I. (Scale bars, 10 μm.) (J and K) Ultrathin cryosections of Tsg101-depletedMNT-1 cells were immunogold labeled for OA1 (PAG 15) andMART-1 (PAG 10). OA1 localizes at the limitingmembrane of MART-1 positive class Emembranous structures (J) and aberrant MVBs (J Inset) and is also associatedwith autophagosomal-like compartments (K). (Scale bars, 200 nm.)

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and MART-1 at their limiting membranes (Fig. 5J and Inset).OA1 was also found at the membrane of MART-1 containingautophagosome-like compartments generated in siTsg101 cells(30, 40) (arrows, Fig. 5K). We recently reported that MART-1interacts with OA1 and acts as an escort for this GPCR at earlysteps of its biosynthetic pathway (9). Therefore, the accumulationof OA1 in Tsg101-depleted cells could be merely a consequenceof the accumulation of MART-1 itself. To test whether the effectof Tsg101 on OA1 was mediated by MART-1, we concomitantlyinactivated Tsg101 and MART-1 in MNT-1 cells. WB analysisrevealed an accumulation of OA1 also in Tsg101/MART1-de-pleted cells (Fig. 5A), in agreement with a specific effect of Tsg101on OA1 protein sorting and degradation independent of its as-sociation with MART-1. These results also strengthen a role forMART-1 in the maintenance of OA1 stability that precedes theinvolvement of Tsg101 in OA1 sorting (9). Consistent with a blockin the trafficking that precedes OA1 delivery to lysosomes anddegradation, colocalization of endogenous OA1 with the lyso-somal protein LAMP1 was also reduced (Fig. S4 A-H). Overall,these observations indicate that OA1 degradation and sortingwithin the endo-lysosomal system requires both ESCRT-0 andESCRT-I function alike other ubiquitinated cargoes (38) butunlike theDOR, for which degradation was dependent onHrs butnot Tsg101 (18). Upon Tsg101 inactivation, OA1 was alsodetected at the limiting membrane of intracellular vesicles, similarto those in which Tyrp1 traffics for delivery to melanosomes (Fig.S5B; ref. 30) explaining the reduced delivery to melanosomes(Fig. S5D). Whereas ESCRT-0, -I, and -II are primarily involvedin recognition and sequestration of ubiquitinated cargoes inendosomemembranes and in membrane bud formation, ESCRT-III subunits are essentially required for ILV formation and scis-sion (41).

OA1 Sorting and Degradation Requires ESCRT-III Function. To in-vestigate the possible implication of ESCRT-III as downstreameffectors of OA1 down-regulation, we depleted Vps24 by RNAiin MNT1 cells. We then analyzed OA1 protein expression insiVps24-treated cells by IB. Similarly to Tsg101 depletion, Vps24depletion led to an accumulation of OA1, as well as MART-1,compared with control cells (Fig. 6I). IF analysis showed thatdepletion of Vps24 affects OA1 distribution in a manner similarto MART-1, resulting in a dispersed localization of both proteinsthroughout the cell (Fig. 6 A–H). IEM showed that the OA1 andMART-1 positive structures correspond to small endosomes(Fig. 6J). OA1 was also retained at the limiting membrane ofsmall MVBs (arrows, Fig. 6K), which is typically generated whenhVps24 is depleted in HeLa cells (42). Together these findingsindicate that OA1 degradation and sorting to ILVs of MVBs isalso affected by Vps24 depletion. Similarly to what was observedfor the all lysine mutant of OA1 (Fig. 3 B and E) and MART-1(29) depletion of Vps24 did not affected OA1 localization tomelanosomes (Fig. S5 C and D). Moreover, and differently fromcontrol cells in which OA1 is also detected in small ILVs insidemelanosomes (Fig. S5A Inset) and (9), in Vps24-depleted cellsOA1 was detected almost exclusively at their limiting membrane(Fig. S5C). These observations suggest that OA1 ubiquitinationand ESCRT-III components could be involved in an inwardbudding step also at the melanosomal membrane. Reinforcingthe involvement of the ESCRT machinery in OA1 intracellulartrafficking, overexpression of a dominant negative version of theAAA ATPase Vps4 (Vps4E228Q), known to block the traffick-ing of ubiquitinated cargoes (43), also increased levels of en-dogenous OA1 relative to those observed in control cells by IB(Fig. S5E). Together, our results indicate that the degradationand sorting of OA1 within endo-lysosomal membranes requiresnot only ESCRT-0 and -I, but also requires ESCRT-III function.

Conclusion. Taken together, our data uniquely highlight how theintracellular trafficking of a GPCR is regulated through ubiq-uitination and the ESCRT machinery. We show that the productof the Ocular Albinism type 1 gene, the GPCR OA1, is ubiq-uitinated, and that this ubiquitination is essential for its targetingto the intraluminal vesicles of MVBs in nonmelanocytic and

melanocytic cells. Our results extend former studies by providingfurther evidence for the direct involvement of ubiquitination andthe ESCRT machinery in the sequestration of a mammalianGPCR in ILVs of MVBs, a step essential in the regulation of thetrafficking of several signaling receptors. In melanocytic cells,ubiquitination of OA1 is likely to control the balance betweendown-regulation and delivery to melanosomes, where this GPCRfunctions to maintain melanosome identity and composition (9).Our study has also important implications in the physiopathologyof ocular albinism type 1, an X-linked genetic disorder caused bymutations in the OA1 gene (44). Notably, two “gain of lysines”mutations (T232K, E233K) were reported in OA1 patients (45).In view of our data, it is tempting to speculate that these mu-tations could possibly generate additional acceptor sites of ubi-quitin and consequently affect OA1 degradation. Importantly,an impairment of OA1 degradation might also affect OA1 sig-naling within the endomembrane system (2), which may be tightlyrelated to the balance between down-regulation and localizationto the melanosome. The OA1 downstream effectors are still ob-scure, but it is surely a challenge for the future to investigate howits intracellular trafficking and regulatory mechanisms impact inOA1 signaling and function.

Materials and MethodsCell Culture, Transfection, and siRNA Depletion. MNT-1 and HeLa cells werecultured and transfected with plasmids and oligonucleotides as described (9,46, 47) using Lipofectamine 2000 or Oligofectamine (Invitrogen). Cells wereanalyzed 24–48 h (plasmids) or 72 h (oligos) after transfection.

Antibodies. Polyclonal anti-human OA1, raised against the C terminus of thehuman OA1 (9), MART-1 7c10 (ABCAM), monoclonal anti Flag M2, polyclonalanti Flag, and polyclonal anti HA were from SIGMA-Aldrich. Sources of otherantibodies are listed in SI Materials and Methods.

Fig. 6. Knockdown of Vps24 (ESCRT-III) affects OA1 sorting and degradationin MNT-1 cells. MNT-1 cells were treated with control siRNA (siCtrl) or siRNAsspecific for Vps24 (siVps24). (A–H) Cells were analyzed by IFM using anti-OA1(A and E) and anti-MART-1 (B and F) antibodies. Overlay is shown in D, andboxed regions are 2.5×magnifications. (D andH) Brightfield (BF) images of thesame cells. (Insets) Overlays of BF and OA1-MART-1 fluorescent signals. (Scalebars, 10 μm.) (I) Whole cell lysates were analyzed by immunoblotting for OA1,MART-1, Vps24, or tubulin, as indicated. Note the accumulation of OA1 andMART-1 in Vps24-depleted cells. Arrows point to the different maturationforms of OA1. (J and K) IEM analysis was performed in siVps24-treated cellswith the indicated antibodies. In Vps24-depleted cells, OA1 (PAG 10) is foundat the limitingmembrane ofMART-1 (PAG 15)-positive “coated” endosomes(arrows, J) and multivesicular endosomes (arrows, K). (Scale bars, 200 nm.)

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Plasmids and siRNAs. The OA1-Flag plasmid was reported elsewhere (9). Flag-tagged plasmid encoding OA1 with single K to R substitutions, OA1K1-6R, wasobtained using the PCR-based QuikChange Lightning multi site-directedmutagenesis kit (Stratagene). Source of other plasmids and siRNAs are listedin SI Materials and Methods.

Quantitative Real-Time PCR. Total RNA was extracted from siRNA and siCtrltransfectedMNT-1 cells using RNeasyMini kit (Quiagen). The same amount ofcDNA was synthesized using SuperScript II (Invitrogen) and random primers.Real-time PCR was carried out with the GeneAmp 7000 Sequence DetectionSystem (Applied Biosystems; ref. 9) and normalized using the ribosomal geneS26. Primers used for quantitative PCR of OA1, MART-1, and the referencegene S26 were as described.(9).

Biochemistry. SDS/PAGE and immunoblotting were carried out by standardmethods and as described (9). Immunoprecipitations from MNT-1 or HeLacells were performed using Protein G-agarose beads (Invitrogen; ref. 9). Forubiquitin detection, MNT-1 cells (108 cells) or HeLa cells (106 cells) expressingOA1-Flag and HA-ubiquitin or HA-empty vector were lysed for 30 min on icein 1% Triton X-100 (50 mM Tris·HCl/150 mM NaCl/10 mM EDTA, pH 7.2/0.1%SDS) supplemented with complete protease inhibitors mixture (Roche Di-agnostic), 20 mM N-ethylmaleimide (NEM) and in stringent conditions (20).OA1-Flag was immunoprecipitated as detailed in SI Materials and Methods.

Immunofluorescence (IF) and Immuno Electron Microscopy (IEM). HeLa andMNT-1 cells cultured on coverslips were fixed with 4% paraformaldehyde insodium phosphate buffer (PBS) and immunofluorescence was carried out asdescribed (9). For immuno electron microscopy, cells were fixed with a mix-ture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer andprocessed for ultracryomicrotomy and immunogold labeling (48). Ultrathincryosections were single- or double-immunogold labeled with antibodiesand protein A coupled to 10 or 15-nm gold, as indicated. Sections wereobserved under a CM120 electron microscope (FEI, Eindoven), equipped witha KeenView camera (Soft Imaging System; SIS, Germany). For quantificationof OA1 labeling, gold particles were counted in randomly selected in-tracellular compartments in each of two separate experiments. Data arepresented as mean ± SD.

ACKNOWLEDGMENTS. We are grateful to G. Strouss, G. Hassink, S. Urbé,D. Rimoldi, R. Tsapis, H. Stenmark, E. Santonico, and V. Marigo for helpfulsuggestions and for generous gifts of reagents. We thank our colleaguesG. van Niel, C. Delevoye, M. Romao, D. Tenza, and I. Hurbain for discussionsduring the course of this work. We also thank V. Fraisier and L. Sengmanivongfor assistance with deconvolution processing and W. Faigle for help with massspectrometry. This work was supported by Institut Curie, Centre National de laRecherche Scientifique, and the Association pour la Recherche contre le Can-cer. F.G. was a fellow from the Fondation pour la Recherche Médicale.

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