The ocular type 1 (OA1) GPCR is ubiquitinated and its traffic requires endosomal sorting complex responsible for transport (ESCRT) function

Francesca 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 their tion and function remain undefined. Posttranslational modification intracellular trafficking. One major regulatory mechanism within the by ubiquitination controls intracellular trafficking events within fi endo-lysosomal system required for receptor localization and down- the endo-lysosomal system (10, 11). Ubiquitin-modi ed mem- regulation is modification by ubiquitination and down- brane delivered from the endocytic or biosynthetic stream interactions with the endosomal sorting complex responsible pathways can be recognized by components of the endosomal sorting complex responsible for transport (ESCRT) machinery for transport (ESCRT) machinery. Whether and how these mecha- for targeting to intraluminal vesicles (ILVs) of multivesicular nisms operate to regulate endosomal sorting of mammalian G bodies (MVBs) and for subsequent lysosomal degradation (12). protein-coupled receptors (GPCRs) remains unclear. Here, we ex- Although ubiquitination and ESCRT function were reported to plore the involvement of ubiquitin and ESCRTs in the trafficking of regulate trafficking of GPCRs in yeast (13–15) and have been OA1, a pigment -specific GPCR, target of mutations in Ocular independently proposed for the down-regulation of selected Albinism type 1, which localizes intracellularly to melanosomes to mammalian GPCRs (16–18), it remains unclear how and where in regulate their biogenesis. Using biochemical and morphological the cell they operate (1). Moreover, the emerging evidences of methods in combination with overexpression and inactivation ubiquitin-independent mechanisms involved in their down-regu- approaches we show that OA1 is ubiquitinated and that its intracel- lation reflect the additional complexity of the endocytic sorting of lular sorting and down-regulation requires functional ESCRT compo- mammalian GPCRs (19). Using a combination of light and elec- nents. Depletion or overexpression of subunits of ESCRT-0, -I, and -III tron microscopy (EM) and biochemical methods, we show that OA1 down-regulation is dependent on ESCRT function due to its markedly inhibits OA1 degradation with concomitant retention fi fi postranslational modi cation by ubiquitination. Ubiquitination within the modi ed endosomal system. Our data further show that controls sorting to the intraluminal vesicles of MVBs for appro- OA1 ubiquitination is uniquely required for targeting to the intra- priate targeting within the endo-melanosomal network. lumenal vesicles of multivesicular , thereby regulating the balance between down-regulation and delivery to melano- Results and Discussion somes. This study highlights the role of ubiquitination and the ESCRT GPCR OA1 Is Postranslationally Modified by Ubiquitination. A unique machinery in the intracellular trafficking of mammalian GPCRs and feature of OA1 as a GPCR is its intracellular localization to has implications for the physiopathology of . and melanosomes in , and to late endo- somes/lysosomes when transiently expressed in nonmelanocytic GPR143 | melanosome | multivesicular | Tsg101 | Hrs cells (7, 9). In endosomes, OA1 is present both at the delimiting and internal membranes, in particular associated with intra- lumenal membrane vesicles (ILVs; ref. 9; Figs. S1 A and B and protein-coupled receptors (GPCRs) regulate important A physiological processes through the coordinated action of S2 ) suggesting that its distribution within endosomal mem- G branes might be a critical step in regulating its function and/or their signaling pathways. These pathways are modulated by highly fi conserved mechanisms that initially involve receptor endocytosis degradation. To investigate whether OA1 can be modi ed by and subsequent targeting to the for degradation (down- ubiquitination we have used the approach described for cell regulation) and/or recycling to the plasma membrane to restore surface associated GPCRs (20). As reported for these GPCRs, cellular signaling responsiveness (1). OA1 (GPR143) is a pigment ubiquitinated forms of OA1 were not easily detected probably due to the small amounts of the total cellular complement of this cell-specific glycoprotein with structural and functional features of GPCR that is ubiquitinated at steady state. Thus, we have GPCRs (2). Mutations in the OA1 underlie ocular albinism coexpressed by transfection Flag-tagged OA1 and HA-tagged type 1 (3), an X-linked disorder that affects the number and the ubiquitin in the melanocytic cell line MNT1 and in HeLa cells. In size of melanosomes, the lysosome-related (LROs) of a first step, OA1-Flag was immunoprecipitated from MNT-1 cell pigment cells devoted to synthesis (4). The retinal pig- lysates in stringent conditions (20). Subsequent immunoblotting ment epithelium (RPE) and skin melanocytes of OA1 patients and (IB) with anti-HA antibodies revealed ubiquitinated forms of corresponding mouse model display giant melanosomes (“mac- ” OA1, with detectable bands from 68 kDa (monoubiquitin) to 92 romelanosomes ; refs. 5 and 6). Like other canonical GPCRs, kDa (four ubiquitins) and a smear at higher molecular weight OA1 interacts with arrestins and binds heterotrimeric G proteins corresponding to polyubiquitin chains (Fig. 1Aa). We confirmed (2). However, OA1 displays unique features among GPCRs: It the specificity of these bands by cotransfecting OA1-FLAG with localizes mainly intracellularly, to melanosomes, by virtue of sorting signals in its cytosolic domain (7). Whereas most GPCRs bind extracellular ligands, the OA1 ligand, presumably the mela- Author contributions: F.G. and G.R. designed research; F.G. and S.S. performed research; nin precursor L-DOPA (8) present in the lumen of the melano- F.G., S.S., and G.R. analyzed data; and F.G. and G.R. wrote the paper. some, triggers a signaling cascade from the to the . Although this cascade remains poorly characterized, our recent The authors declare no conflict of interest. studies highlighted that the macromelanosomes result from ab- This article is a PNAS Direct Submission. normal fusion/fission events at early steps of their biogenesis (9). 1Present address: Department of Cell Biology and Howard Hughes Medical Institute, Yale OA1 function in melanogenesis is certainly regulated by a tight University School of Medicine, New Haven, CT 06510. balance between its targeting to melanosomes and its down- 2To whom correspondence should be addressed: E-mail: [email protected]. regulation. However, the mechanisms that regulate OA1 traffick- This article contains supporting information online at www.pnas.org/lookup/suppl/doi: ing within the endo-melanosomal system and thereby its localiza- 10.1073/pnas.1103381108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103381108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. OA1 is ubiquitinated in MNT-1 cells and HeLa cells. (A) MNT-1 (a)orHeLacells(b) were transfected with OA1-Flag and either ubiquitin-HA or empty vector HA-constructs. Twenty-four hours after transfection, OA1 was immunoprecipitated (IP) with anti-Flag antibody and lysates were immunoblotted (IB) with anti-Flag antibody to detect OA1 and with anti-HA antibody to detect incorporated epitope-tagged ubiquitin. Note the dif- ferent OA1 forms (a doublet of 45–48 kDa and a 60-kDa band). Ubiquitinated forms of OA1 were observed in cells coexpressing OA1-Flag with ubiquitin-HA but not in cells coexpressing OA1- Flag with a control HA-plasmid. Arrows indicate 68- to 92-kDa bands. Asterisks indicate a smear at higher molecular weight corresponding to polyubiquitin chains. (B)HeLacellswere transfected with Flag-tagged wt (OA1wtFlag)ormutantOA1 (OA1K1-7RFlag) constructs and either ubiquitin-HA or empty vector HA. Immunoprecipitation of OA1 and immunoblotting with anti-Flag (a) and with anti-HA antibodies (b)wereper- formed as in A. Mutation of all lysine in OA1K1-7R abrogated OA1 ubiquitination. Arrows and asterisk indicate bands and a smear, respectively, corresponding to ubiquitinated forms of OA1 missing in the ubiquitin-deficient mutant OA1K1-7R.

a control HA-plasmid (Fig. 1Aa). Providing further evidence for in the unmodified OA1 protein levels (Fig. 1Ba). Overall, our ubiquitination in an heterologous cell system (Fig. 1Ab), the results show that OA1 undergoes ubiquitination on Lysin resi- same experiments were performed in HeLa cells in which OA1 is dues both in melanocytic (MNT-1) and nonmelanocytic (HeLa) correctly targeted to endosomes and lysosomes (Figs. S1 A and B cells. The ability of OA1 to be ubiquitinated also in HeLa cells, and S2A; ref. 7). Ubiquitination occurs generally on lysine (Lys) where it is not expressed physiologically, indicates that OA1 can residues (21). In support of these observations and as shown by be basally ubiquitinated, similarly to what was reported for the immunofluorescence (IF), OA1-Flag was detected in ubiquitin- protease-activated receptor, PAR1 (22). Several mammalian HA-positive structures in both HeLa (Fig. S2 A–C) and MNT-1 GPCRs have been shown to be mono- or polyubiquitinated on (Fig. S2 G–I) cells. OA1 contains 7 Lys residues with 4 Lys in the lysine residues located on their cytosolic side: PAR1 (22), β2AR third cytosolic loop (K215, K225, K243, K248) and 3 Lys in the (23), CXCR4 (16), δ-opioid (24), and k- (17). cytosolic tail (K325, K355, K391), all of them potentially in- Receptor modification by ubiquitin is thought to be required for volved as Ub acceptors. Substitution of all 7 (lysines) Lys (K) to their internalization at the plasma membrane but also to direct Arg (R) (OA1K1-7R)(Fig. S1C) abrogated OA1 ubiquitination in membrane cargo, delivered from both endocytic and biosynthetic transfected HeLa cells (Fig. 1Bb) and also led to a mild increase pathways, to the intraluminal vesicles within late endosomes/

Fig. 2. The ubiquitin-deficient mutant of OA1 accumulates at the limiting membrane of en- larged MVBs in HeLa cells. (A and B) Ultrathin cryosections of HeLa cells transfected with Flag-tagged wild type (OA1wtFlag) or all lysine mutant OA1 (OA1K1-7RFlag) were double-immunogold labeled for Flag (PAG 15) to detect OA1 and for CD63 (PAG 10). Whereas OA1wt is mostly associated with the ILVs of CD63-positive MVBs (arrows) (A), the mutant OA1K1-7R is present at the limiting mem- brane of enlarged MVBs packed with ILVs (arrows) (B). (C) Quan- titative evaluation of the labeling (gold particles) for OA1 wild type or OA1 lysines mutant associated to the limiting membrane (LM) and ILVs of MVBs shows that 80% of OA1K1-7R is retained at LM compared with wild-type OA1 (10%). (D) A similar quantifica- tion for the all-lysine mutant of MART-1 (MART-1K1-6R; ref. 29), show that only 30% of MART-1 is retained at the limiting mem- brane of MVBs and 70% is still sorted to ILVs of MVBs whose morphology is not affected (E and F). (Scale bars, 150 nm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103381108 Giordano et al. Downloaded by guest on September 30, 2021 MVBs (reviewed in ref. 25). In mammalian cells, this process has ESCRT-0 complex directly involved in recognition of ubiquiti- been well described for the down-regulation of EGF receptor nated membrane cargo on endosomes (31). At steady state, OA1 (26). Mammalian GPCRs do not appear to require ubiquitina- partially overlaps with a small subset of Hrs positive endosomes tion for efficient endocytosis from plasma membrane but mainly in MNT1 cells (Fig. S2 J–L) and we have further provided bio- for their trafficking after endocytosis (1). Given the exclusive chemical evidence for an interaction between OA1 and Hrs intracellular localization of OA1, further analysis of its ubiquitin- (Fig. 4I). Hrs was detected by IB in lysates immunoprecipitated dependent sorting offers a unique opportunity to dissect the with anti-Flag (OA1) antibody of Hrs-myc and OA1-Flag- function of this posttranslational modification within the endo- transfected MNT-1 cells, but not in lysates of MNT-1 cells cytic system of a mammalian GPCR. cotransfected with Hrs-myc and a Flag empty vector. We next examined by IF and IEM the effect of overexpressing Hrs-YFP, Ubiquitination Is Required for the Targeting of OA1 to ILVs of one of the Hrs-tagged variants that function as dominant inhib- Multivesicular Endosomes. To underscore the consequences of itors of Hrs-mediated lysosomal sorting of transmembrane pro- ubiquitination in OA1 trafficking, we analyzed by immuno-electron teins including receptors (32, 33) and GPCRs (18). IF analysis microscopy (IEM) the localization in HeLa cells of the two OA1 indicates that overexpression of Hrs-YFP strongly increases lo- constructs: wild type (OA1wt-Flag) and the all lysine mutant OA1 calization of endogenous OA1 in the Hrs-positive endosomes (OA1K1-7R-Flag). In these cells the bulk of OA1 localizes to (arrows, Fig. 4 A–C) and in the enlarged Hrs-positive clusters of CD63-positive MVBs where it is mainly associated with the ILVs endosomes that are generated in overexpressing cells (arrows, (Fig. 2A). Strickling, the ubiquitination-defective OA1 mutant, Fig. 4 D–F). These modified endosomes have been shown to trap accumulates at the limiting membrane of MVBs (Fig. 2B). Such ubiquitinated cargo destined to ILVs, as demonstrated for effective sequestration of this GPCR in ILVs of MVBs is sup- MART-1 in melanocytic cells (34) and also for another GPCR, ported by quantitative evaluation of the distribution of OA1 wild PAR-2 (35). IEM revealed that both endogenous and transfected type and OA1 Ub mutant on the limiting membrane and ILVs of OA1 were retained at the limiting membrane of these compart- MVBs (Fig. 2C). In agreement, OA1-Flag expressed in HeLa cells ments (arrows, Fig. 4G and Inset). In addition, as shown by IB partially overlaps with structures positive for K63-linked ubiquitin (Fig. 4H), the protein levels of OA1 were significantly increased chains (Fig. S2 D–F) that are known to act as a signal for protein when coexpressed with Hrs-YFP compared with control cells sorting into the MVB pathway (27). Notably, 40% the MVBs expressing only YFP. Because nonubiquitinated proteins tra- retaining the Ub-defective OA1 mutant were considerably en- versing early endosomes can also be trapped in the “Hrsosomes” larged with heavily packed ILVs compared with those hosting the (30), we further inactivated Hrs by RNAi in MNT-1 cells. De- wild-type OA1. These observations suggest that retention of OA1 pletion of Hrs results in the accumulation of transiently expressed at the limiting membrane of MVBs is likely to modify the ho- OA1-Flag in Hrs-depleted cells, as shown by IF (Fig. S3M, arrow; CELL BIOLOGY meostasis and/or distribution of proteins involved in the bio- quantification in Fig. S3N). Similar observations were reported genesis and maturation of these compartments. A similar for a PAR2 construct, another GPCR that undergoes ubiquiti- enlargement of MVBs was also observed in dendritic cells upon expression of a Ub-mutant of MHC class II molecules (28). Re- inforcing the essential requirement of OA1 ubiquitination in endosomal sorting, the all lysine mutant of MART-1 (MART-1K1-6R; ref. 29) appeared to be only slightly retained at the limiting membrane of MVBs (Fig. 2 D–F). Moreover, the expression of this 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 melanocytic cells, we next analyzed by IEM in MNT1 cells the subcellular lo- calization of the Flag-tagged OA1 wild type (OA1wt) and all lysine mutant OA1 (OA1K1-7R). The mutant OA1K1-7R was retained at the limiting membrane of MVBs also in MNT1 cells (arrows, Fig. 3 B–D). These compartments, that were generally not observed in control cells (Fig. 3A), were similar to those observed in HeLa cells upon OA1K1-7R transfection. They were also similar in luminal content 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 uniquely required for its sorting to ILVs of multivesicular endosomes in both HeLa cells and melanocytic MNT-1 cells. These observations differ from recent findings on the δ-opioid receptor (DOR), for which ubiquitination participates to but is dispensable for ILV sorting (24). This is also what we observed for MART-1 (Fig. 2 D– F), indicating that the ubiquitination requirements for ILV sorting differ depending on the proteins and maybe on their ability to further interact with downstream effectors. Interestingly, the amount of mutant, nonubiquitinated form of OA1 associated with melanosomes is increased (arrowheads, Fig. 3 A and B; quantifi- cation in Fig. 3E). These observations are reminiscent of those reported for the all-lysine mutant of MART-1 that appeared to accumulate in melanosomes in the absence of ubiquitination (29). Fig. 3. The ubiquitin-deficient OA1 mutant is retained on MVBs and melano- Taken together, our observations indicate that ubiquitination of somes in MNT-1 cells. (A and B) Ultrathin cryosections of MNT-1 cells transfected OA1 by regulating its down-regulation is likely to control the with Flag-tagged wildtype (OA1wtFlag) or all-lysine mutant OA1 (OA1K1-7R Flag) balance of receptor within the endo-melanosomal system and were double-immunogold labeled for Flag (PAG 15) to detect OA1 and Tyrp1 ensure its intracellular function. (PAG 10). OA1K1-7R was retained at the limiting membrane of MVBs (arrows in B– D) that are generally not observed in wild-type cells (A). Note also the presence Hrs (ESCRT-0) Is Required for the Endosomal Transport of OA1. To of OA1 at the limiting membrane of mature melanosomes in OA1wt and elucidate the downstream molecular players involved in OA1 OA1K1-7R expressing cells (arrowheads, A and B). (Scale bars, 200 nm.) (E) trafficking and down-regulation we next examined the require- Histogram depicting a quantitative evaluation of OA1 labeling (gold par- ment for the ESCRT machinery (12). Hrs is a component of the ticles) at the melanosome membrane of OA1wt and OA1K1-7R transfected cells.

Giordano et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 nation and subsequent interaction with Hrs (35). However, de- functional Hrs. Such requirements slightly differ from what was pletion of Hrs does not result in the accumulation of endogenous previously reported for the DOR, the down-regulation of which OA1 within the cell but rather results in its decreased expression appeared dependent on Hrs despite lack of ubiquitination (18). as analyzed by IB (Fig. S3A). A similar decrease was reported also for MART-1 (34), which we use here as a control. IF analysis OA1 Sorting and Degradation Requires ESCRT-I Function. One of the confirmed a reduction of both endogenous OA1 and MART-1 in downstream effectors of Hrs in the regulation of endosomal pro- the majority of siHrs-treated cells (arrows, Fig. S3 F–I) compared tein sorting is ESCRT-I. Tsg101 is a “core” component of this with control cells (Fig. S3 B–E). These observations suggest that complex and can bind both ubiquitinated cargo and Hrs (37). fi the absence of functional Hrs impacts on their normal traf cking Knockdown of Tsg101 has been shown to significantly inhibit ly- without fully impairing their degradation, as reported for the sosomal sorting and degradation of various membrane receptors EGFR (36). In addition, we found by quantitative real-time PCR including EGFR (38) and the GPCR GABA(B)-receptor (39). To that the reduction of the endogenous OA1 and MART-1 proteins investigate the involvement of ESCRT-I in OA1 sorting, we de- was certainly related to a down-regulation of their transcripts in fi J–L pleted Tsg101 by RNAi from MNT-1 cells. Ef cient knockdown siHrs-treated cells (Fig. S3 ). This observation indicates that (>90%) of Tsg101 protein was confirmed by IB (Fig. 5A). Re- Hrs depletion, could also consequently impact on the transcrip- vealing a role for ESCRT-I in OA1 trafficking, depletion of Tsg101 tion of these melanosomal proteins. Taken together, our results fi indicate that the trafficking of OA1 requires its ubiquitination and interfered with OA1 degradation resulting in a signi cant increase in OA1 levels relative to control cells (Fig. 5A). As a control and in agreement with previous observations (30), MART-1 levels also increased upon Tsg101 down-regulation (Fig. 5A). IF analysis showed that in Tsg101-depleted cells endogenous OA1 accumu- lates in enlarged vesicular structures throughout the that were mostly overlapping with those positive for MART-1 (Fig. 5 B–I). IEM revealed characteristic aberrant membranous structures (class E compartments, ref. 38) depicting both OA1

Fig. 4. OA1 interacts with Hrs and Hrs overexpression traps OA1 at the limiting membrane of endosomes. (A–F) Immunofluorescence analysis of MNT-1 cells transfected with fluorescent Hrs-YFP (A and D) and labeled for endogenous OA1 (B and E). C and F represent merged images from the two left panels. Insets are 2.5× magnifications of boxed regions. Arrows indicate area of colocalization. (Scale bar, 10 μm.) (G) Ultrathin cryosections of MNT-1 Fig. 5. Knockdown of Tsg101 (ESCRT-I) affects OA1 sorting and degradation cells transfected with Hrs-YFP alone (Inset) or in combination with OA1-Flag in MNT-1 cells. MNT-1 cells were treated with control siRNA (siCtrl) or siRNAs (G) were double immunogold labeled with anti-GFP (Hrs) and anti-OA1 specific for Tsg101 (siTsg101), for Tsg101 and MART-1 (siTsg101/MART-1) and (endogenous OA1) (Inset) or GFP (Hrs) and anti-Flag (transfected OA1) (G), for MART-1 (siMART-1). (A) Whole cell lysates were analyzed by immuno- respectively. Both endogenous and transfected OA1 (PAG 15) are retained at blotting for OA1, Tsg101, MART-1, or tubulin as a control. Note the effective the limiting membrane of the Hrs-positive (PAG 10) endosomes. Note the depletion of Tsg101 and the enrichment of OA1 and MART-1 in siTsg101- interconnected network of large endosomal densely packed with treated cells. The amount of OA1 is significantly increased also in siTsg101/ small ILVs. (Scale bars, 200 nm.) (H) Total cell lysates of MNT-1 cotransfected MART-1-treated cells. Arrows point to the 60-kDa form of OA1. (B–I) Cells with either Hrs-YFP or YFP and OA1-flag were analyzed by immunoblotting treated with siRNAs targeting Tsg101 (siTsg101) (F–I) or Ctrl siRNAs (siCtrl) (B– with the indicated antibodies. The arrow indicates the accumulation of OA1 E) were analyzed by IFM using antibodies to OA1 (B and F) and MART-1 (C and upon coexpression with Hrs-YFP, but not with YFP alone. (I) MNT-1 cells were G). Overlays are shown in D and H. Boxed regions correspond to 5× magnified transfected with either OA1-Flag or Flag empty vector and Hrs-myc. Lysates areas in E and I. (Scale bars, 10 μm.) (J and K) Ultrathin cryosections of Tsg101- were immunoprecipitated with monoclonal anti-Flag antibody, fractionated depleted MNT-1 cells were immunogold labeled for OA1 (PAG 15) and MART- by SDS/PAGE, and immunoblotted with anti-myc (Hrs) or Flag (OA1). The 1 (PAG 10). OA1 localizes at the limiting membrane of MART-1 positive class E arrow indicates a specific band detected by anti-myc antibody in OA1- membranous structures (J) and aberrant MVBs (J Inset) and is also associated immunoprecipitates but not in immunoprecipitates of empty-Flag vector. with autophagosomal-like compartments (K). (Scale bars, 200 nm.)

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103381108 Giordano et al. Downloaded by guest on September 30, 2021 and MART-1 at their limiting membranes (Fig. 5J and Inset). melanocytic cells. Our results extend former studies by providing OA1 was also found at the membrane of MART-1 containing further evidence for the direct involvement of ubiquitination and autophagosome-like compartments generated in siTsg101 cells the ESCRT machinery in the sequestration of a mammalian (30, 40) (arrows, Fig. 5K). We recently reported that MART-1 GPCR in ILVs of MVBs, a step essential in the regulation of the interacts with OA1 and acts as an escort for this GPCR at early trafficking of several signaling receptors. In melanocytic cells, steps of its biosynthetic pathway (9). Therefore, the accumulation ubiquitination of OA1 is likely to control the balance between of OA1 in Tsg101-depleted cells could be merely a consequence down-regulation and delivery to melanosomes, where this GPCR of the accumulation of MART-1 itself. To test whether the effect functions to maintain melanosome identity and composition (9). of Tsg101 on OA1 was mediated by MART-1, we concomitantly Our study has also important implications in the physiopathology inactivated Tsg101 and MART-1 in MNT-1 cells. WB analysis of ocular albinism type 1, an X-linked genetic disorder caused by revealed an accumulation of OA1 also in Tsg101/MART1-de- mutations in the OA1 gene (44). Notably, two “gain of lysines” pleted cells (Fig. 5A), in agreement with a specific effect of Tsg101 on OA1 protein sorting and degradation independent of its as- mutations (T232K, E233K) were reported in OA1 patients (45). sociation with MART-1. These results also strengthen a role for In view of our data, it is tempting to speculate that these mu- MART-1 in the maintenance of OA1 stability that precedes the tations could possibly generate additional acceptor sites of ubi- involvement of Tsg101 in OA1 sorting (9). Consistent with a block quitin and consequently affect OA1 degradation. Importantly, in the trafficking that precedes OA1 delivery to lysosomes and an impairment of OA1 degradation might also affect OA1 sig- degradation, colocalization of endogenous OA1 with the lyso- naling within the (2), which may be tightly somal protein LAMP1 was also reduced (Fig. S4 A-H). Overall, related to the balance between down-regulation and localization these observations indicate that OA1 degradation and sorting to the melanosome. The OA1 downstream effectors are still ob- within the endo-lysosomal system requires both ESCRT-0 and scure, but it is surely a challenge for the future to investigate how ESCRT-I function alike other ubiquitinated cargoes (38) but its intracellular trafficking and regulatory mechanisms impact in unlike the DOR, for which degradation was dependent on Hrs but OA1 signaling and function. not Tsg101 (18). Upon Tsg101 inactivation, OA1 was also detected at the limiting membrane of intracellular vesicles, similar Materials and Methods to those in which Tyrp1 traffics for delivery to melanosomes (Fig. Cell Culture, Transfection, and siRNA Depletion. MNT-1 and HeLa cells were B S5 ; ref. 30) explaining the reduced delivery to melanosomes cultured and transfected with plasmids and oligonucleotides as described (9, D (Fig. S5 ). Whereas ESCRT-0, -I, and -II are primarily involved 46, 47) using Lipofectamine 2000 or Oligofectamine (Invitrogen). Cells were in recognition and sequestration of ubiquitinated cargoes in analyzed 24–48 h (plasmids) or 72 h (oligos) after transfection. endosome membranes and in membrane bud formation, ESCRT- CELL BIOLOGY III subunits are essentially required for ILV formation and scis- Antibodies. Polyclonal anti-human OA1, raised against the C terminus of the sion (41). human OA1 (9), MART-1 7c10 (ABCAM), monoclonal anti Flag M2, polyclonal anti Flag, and polyclonal anti HA were from SIGMA-Aldrich. Sources of other OA1 Sorting and Degradation Requires ESCRT-III Function. To in- antibodies are listed in SI Materials and Methods. vestigate the possible implication of ESCRT-III as downstream effectors of OA1 down-regulation, we depleted Vps24 by RNAi in MNT1 cells. We then analyzed OA1 protein expression in siVps24-treated cells by IB. Similarly to Tsg101 depletion, Vps24 depletion led to an accumulation of OA1, as well as MART-1, compared with control cells (Fig. 6I). IF analysis showed that depletion of Vps24 affects OA1 distribution in a manner similar to MART-1, resulting in a dispersed localization of both proteins throughout the cell (Fig. 6 A–H). IEM showed that the OA1 and MART-1 positive structures correspond to small endosomes (Fig. 6J). OA1 was also retained at the limiting membrane of small MVBs (arrows, Fig. 6K), which is typically generated when hVps24 is depleted in HeLa cells (42). Together these findings indicate that OA1 degradation and sorting to ILVs of MVBs is also affected by Vps24 depletion. Similarly to what was observed for the all lysine mutant of OA1 (Fig. 3 B and E) and MART-1 (29) depletion of Vps24 did not affected OA1 localization to melanosomes (Fig. S5 C and D). Moreover, and differently from control cells in which OA1 is also detected in small ILVs inside melanosomes (Fig. S5A Inset) and (9), in Vps24-depleted cells OA1 was detected almost exclusively at their limiting membrane (Fig. S5C). These observations suggest that OA1 ubiquitination and ESCRT-III components could be involved in an inward budding step also at the melanosomal membrane. Reinforcing the involvement of the ESCRT machinery in OA1 intracellular trafficking, overexpression of a dominant negative version of the fi AAA ATPase Vps4 (Vps4E228Q), known to block the traf ck- Fig. 6. Knockdown of Vps24 (ESCRT-III) affects OA1 sorting and degradation ing of ubiquitinated cargoes (43), also increased levels of en- in MNT-1 cells. MNT-1 cells were treated with control siRNA (siCtrl) or siRNAs dogenous OA1 relative to those observed in control cells by IB fi – E speci c for Vps24 (siVps24). (A H) Cells were analyzed by IFM using anti-OA1 (Fig. S5 ). Together, our results indicate that the degradation (A and E) and anti-MART-1 (B and F) antibodies. Overlay is shown in D, and and sorting of OA1 within endo-lysosomal membranes requires boxed regions are 2.5× magnifications. (D and H) Brightfield (BF) images of the not only ESCRT-0 and -I, but also requires ESCRT-III function. same cells. (Insets) Overlays of BF and OA1-MART-1 fluorescent signals. (Scale bars, 10 μm.) (I) Whole cell lysates were analyzed by immunoblotting for OA1, Conclusion. Taken together, our data uniquely highlight how the MART-1, Vps24, or tubulin, as indicated. Note the accumulation of OA1 and intracellular trafficking of a GPCR is regulated through ubiq- MART-1 in Vps24-depleted cells. Arrows point to the different maturation uitination and the ESCRT machinery. We show that the product forms of OA1. (J and K) IEM analysis was performed in siVps24-treated cells of the Ocular Albinism type 1 gene, the GPCR OA1, is ubiq- with the indicated antibodies. In Vps24-depleted cells, OA1 (PAG 10) is found uitinated, and that this ubiquitination is essential for its targeting at the limiting membrane of MART-1 (PAG 15)-positive “coated” endosomes to the intraluminal vesicles of MVBs in nonmelanocytic and (arrows, J) and multivesicular endosomes (arrows, K). (Scale bars, 200 nm.)

Giordano et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Plasmids and siRNAs. The OA1-Flag plasmid was reported elsewhere (9). Flag- Immunofluorescence (IF) and Immuno Electron Microscopy (IEM). HeLa and tagged plasmid encoding OA1 with single K to R substitutions, OA1K1-6R, was MNT-1 cells cultured on coverslips were fixed with 4% paraformaldehyde in obtained using the PCR-based QuikChange Lightning multi site-directed sodium phosphate buffer (PBS) and immunofluorescence was carried out as mutagenesis kit (Stratagene). Source of other plasmids and siRNAs are listed described (9). For immuno electron microscopy, cells were fixed with a mix- in SI Materials and Methods. ture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer and processed for ultracryomicrotomy and immunogold labeling (48). Ultrathin Quantitative Real-Time PCR. Total RNA was extracted from siRNA and siCtrl cryosections were single- or double-immunogold labeled with antibodies transfected MNT-1 cells using RNeasy Mini kit (Quiagen). The same amount of and protein A coupled to 10 or 15-nm gold, as indicated. Sections were cDNA was synthesized using SuperScript II (Invitrogen) and random primers. observed under a CM120 electron microscope (FEI, Eindoven), equipped with Real-time PCR was carried out with the GeneAmp 7000 Sequence Detection fi System (Applied Biosystems; ref. 9) and normalized using the ribosomal gene a KeenView camera (Soft Imaging System; SIS, Germany). For quanti cation S26. Primers used for quantitative PCR of OA1, MART-1, and the reference of OA1 labeling, gold particles were counted in randomly selected in- gene S26 were as described.(9). tracellular compartments in each of two separate experiments. Data are presented as mean ± SD. Biochemistry. SDS/PAGE and immunoblotting were carried out by standard methods and as described (9). Immunoprecipitations from MNT-1 or HeLa ACKNOWLEDGMENTS. We are grateful to G. Strouss, G. Hassink, S. Urbé, cells were performed using Protein G-agarose beads (Invitrogen; ref. 9). For D. Rimoldi, R. Tsapis, H. Stenmark, E. Santonico, and V. Marigo for helpful ubiquitin detection, MNT-1 cells (108 cells) or HeLa cells (106 cells) expressing suggestions and for generous gifts of reagents. We thank our colleagues G. van Niel, C. Delevoye, M. Romao, D. Tenza, and I. Hurbain for discussions OA1-Flag and HA-ubiquitin or HA-empty vector were lysed for 30 min on ice · during the course of this work. We also thank V. Fraisier and L. Sengmanivong in 1% Triton X-100 (50 mM Tris HCl/150 mM NaCl/10 mM EDTA, pH 7.2/0.1% for assistance with deconvolution processing and W. Faigle for help with mass SDS) supplemented with complete protease inhibitors mixture (Roche Di- spectrometry. This work was supported by Institut Curie, Centre National de la agnostic), 20 mM N-ethylmaleimide (NEM) and in stringent conditions (20). Recherche Scientifique, and the Association pour la Recherche contre le Can- OA1-Flag was immunoprecipitated as detailed in SI Materials and Methods. cer. F.G. was a fellow from the Fondation pour la Recherche Médicale.

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