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

Abstract

The function of signaling receptors is tightly controlled by their intracellular trafficking. One major regulatory mechanism within the endo-lysosomal system required for receptor localization and down-regulation is protein modification by ubiquitination and downstream interactions with the endosomal sorting complex responsible for transport (ESCRT) machinery. Whether and how these mechanisms operate to regulate endosomal sorting of mammalian G protein-coupled receptors (GPCRs) remains unclear. Here, we explore the involvement of ubiquitin and ESCRTs in the trafficking of OA1, a pigment cell-specific GPCR, target of mutations in Ocular Albinism type 1, which localizes intracellularly to melanosomes to regulate their biogenesis. Using biochemical and morphological methods in combination with overexpression and inactivation approaches we show that OA1 is ubiquitinated and that its intracellular sorting and down-regulation requires functional ESCRT components. Depletion or overexpression of subunits of ESCRT-0, -I, and -III markedly inhibits OA1 degradation with concomitant retention within the modified endosomal system. Our data further show that OA1 ubiquitination is uniquely required for targeting to the intralumenal vesicles of multivesicular endosomes, thereby regulating the balance between down-regulation and delivery to melanosomes. This study highlights the role of ubiquitination and the ESCRT machinery in the intracellular trafficking of mammalian GPCRs and has implications for the physiopathology of ocular albinism type 1.

Gprotein-coupled receptors (GPCRs) regulate important physiological processes through the coordinated action of their signaling pathways. These pathways are modulated by highly conserved mechanisms that initially involve receptor endocytosis and subsequent targeting to the lysosome for degradation (down-regulation) and/or recycling to the plasma membrane to restore cellular signaling responsiveness (1). OA1 (GPR143) is a pigment cell-specific glycoprotein with structural and functional features of GPCRs (2). Mutations in the OA1 gene underlie ocular albinism type 1 (3), an X-linked disorder that affects the number and the size of melanosomes, the lysosome-related organelles (LROs) of pigment cells devoted to melanin synthesis (4). The retinal pigment epithelium (RPE) and skin melanocytes of OA1 patients and corresponding mouse model display giant melanosomes (“macromelanosomes”; 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: It 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 melanin precursor l-DOPA (8) present in the lumen of the melanosome, triggers a signaling cascade from the organelle to the cytosol. Although this cascade remains poorly characterized, our recent studies highlighted that the macromelanosomes result from abnormal fusion/fission events at early steps of their biogenesis (9). OA1 function in melanogenesis is certainly regulated by a tight balance between its targeting to melanosomes and its down-regulation. However, the mechanisms that regulate OA1 trafficking within the endo-melanosomal system and thereby its localization and function remain undefined. Posttranslational modification by ubiquitination controls intracellular trafficking events within the endo-lysosomal system (10, 11). Ubiquitin-modified membrane proteins delivered from the endocytic or biosynthetic pathways can be recognized by components of the endosomal sorting complex responsible for transport (ESCRT) machinery for targeting to intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) and for subsequent lysosomal degradation (12). Although ubiquitination and ESCRT function were reported to regulate trafficking of GPCRs in yeast (13–15) and have been independently proposed for the down-regulation of selected mammalian GPCRs (16–18), it remains unclear how and where in the cell they operate (1). Moreover, the emerging evidences of ubiquitin-independent mechanisms involved in their down-regulation reflect the additional complexity of the endocytic sorting of mammalian GPCRs (19). Using a combination of light and electron microscopy (EM) and biochemical methods, we show that OA1 down-regulation is dependent on ESCRT function due to its postranslational modification by ubiquitination. Ubiquitination controls sorting to the intraluminal vesicles of MVBs for appropriate targeting within the endo-melanosomal network.

Results and Discussion

GPCR OA1 Is Postranslationally Modified by Ubiquitination.

A unique feature of OA1 as a GPCR is its intracellular localization to lysosomes and melanosomes in melanocytes, and to late endosomes/lysosomes when transiently expressed in nonmelanocytic cells (7, 9). In endosomes, OA1 is present both at the delimiting and internal membranes, in particular associated with intralumenal membrane vesicles (ILVs; ref. 9; Figs. S1 A and B and S2A) suggesting that its distribution within endosomal membranes might be a critical step in regulating its function and/or degradation. To investigate whether OA1 can be modified by ubiquitination we have used the approach described for cell surface associated GPCRs (20). As reported for these GPCRs, ubiquitinated forms of OA1 were not easily detected probably due to the small amounts of the total cellular complement of this GPCR that is ubiquitinated at steady state. Thus, we have coexpressed by transfection Flag-tagged OA1 and HA-tagged ubiquitin in the melanocytic cell line MNT1 and in HeLa cells. In a first step, OA1-Flag was immunoprecipitated from MNT-1 cell lysates in stringent conditions (20). Subsequent immunoblotting (IB) with anti-HA antibodies revealed ubiquitinated forms of OA1, with detectable bands from 68 kDa (monoubiquitin) to 92 kDa (four ubiquitins) and a smear at higher molecular weight corresponding to polyubiquitin chains (Fig. 1Aa). We confirmed the specificity of these bands by cotransfecting OA1-FLAG with a control HA-plasmid (Fig. 1Aa). Providing further evidence for ubiquitination in an heterologous cell system (Fig. 1Ab), the same experiments were performed in HeLa cells in which OA1 is correctly targeted to endosomes and lysosomes (Figs. S1 A and B and S2A; ref. 7). Ubiquitination occurs generally on lysine (Lys) residues (21). In support of these observations and as shown by immunofluorescence (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 the third cytosolic loop (K215, K225, K243, K248) and 3 Lys in the cytosolic tail (K325, K355, K391), all of them potentially involved as Ub acceptors. Substitution of all 7 (lysines) Lys (K) to Arg (R) (OA1^K1-7R) (Fig. S1C) abrogated OA1 ubiquitination in transfected HeLa cells (Fig. 1Bb) and also led to a mild increase in the unmodified OA1 protein levels (Fig. 1Ba). Overall, our results show that OA1 undergoes ubiquitination on Lysin residues 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 can be basally ubiquitinated, similarly to what was reported for the protease-activated receptor, PAR1 (22). Several mammalian GPCRs have been shown to be mono- or polyubiquitinated on lysine 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 for their internalization at the plasma membrane but also to direct membrane cargo, delivered from both endocytic and biosynthetic pathways, to the intraluminal vesicles within late endosomes/MVBs (reviewed in ref. 25). In mammalian cells, this process has been well described for the down-regulation of EGF receptor (26). Mammalian GPCRs do not appear to require ubiquitination for efficient endocytosis from plasma membrane but mainly for their trafficking after endocytosis (1). Given the exclusive intracellular localization of OA1, further analysis of its ubiquitin-dependent sorting offers a unique opportunity to dissect the function of this posttranslational modification within the endocytic system of a mammalian GPCR.

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 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 different 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) HeLa cells were transfected with Flag-tagged wt (OA1^wtFlag) or mutant OA1 (OA1^K1-7RFlag) constructs and either ubiquitin-HA or empty vector HA. Immunoprecipitation of OA1 and immunoblotting with anti-Flag (a) and with anti-HA antibodies (b) were performed as in A. Mutation of all lysine in OA1^K1-7R abrogated OA1 ubiquitination. Arrows and asterisk indicate bands and a smear, respectively, corresponding to ubiquitinated forms of OA1 missing in the ubiquitin-deficient mutant OA1^K1-7R.

Ubiquitination Is Required for the Targeting of OA1 to ILVs of Multivesicular Endosomes.

To underscore the consequences of ubiquitination in OA1 trafficking, we analyzed by immuno-electron microscopy (IEM) the localization in HeLa cells of the two OA1 constructs: wild type (OA1^wt-Flag) and the all lysine mutant OA1 (OA1^K1-7R-Flag). In these cells the bulk of OA1 localizes to CD63-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). Such effective sequestration of this GPCR in ILVs of MVBs is supported by quantitative evaluation of the distribution of OA1 wild type and OA1 Ub mutant on the limiting membrane and ILVs of MVBs (Fig. 2C). In agreement, OA1-Flag expressed in HeLa cells partially overlaps with structures positive for K63-linked ubiquitin chains (Fig. S2 D–F) that are known to act as a signal for protein sorting into the MVB pathway (27). Notably, 40% the MVBs retaining the Ub-defective OA1 mutant were considerably enlarged with heavily packed ILVs compared with those hosting the wild-type OA1. These observations suggest that retention of OA1 at the limiting membrane of MVBs is likely to modify the homeostasis and/or distribution of proteins involved in the biogenesis and maturation of these compartments. A similar enlargement of MVBs was also observed in dendritic cells upon expression of a Ub-mutant of MHC class II molecules (28). Reinforcing the essential requirement of OA1 ubiquitination in endosomal sorting, the all lysine mutant of MART-1 (MART-1^K1-6R; 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).

Fig. 2.

The ubiquitin-deficient mutant of OA1 accumulates at the limiting membrane of enlarged MVBs in HeLa cells. (A and B) Ultrathin cryosections of HeLa cells transfected with Flag-tagged wild type (OA1^wtFlag) or all lysine mutant OA1 (OA1^K1-7RFlag) were double-immunogold labeled for Flag (PAG 15) to detect OA1 and for CD63 (PAG 10). Whereas OA1^wt is mostly associated with the ILVs of CD63-positive MVBs (arrows) (A), the mutant OA1^K1-7R is present at the limiting membrane of enlarged MVBs packed with ILVs (arrows) (B). (C) Quantitative 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 OA1^K1-7R is retained at LM compared with wild-type OA1 (10%). (D) A similar quantification for the all-lysine mutant of MART-1 (MART-1^K1-6R; ref. 29), show that only 30% of MART-1 is retained at the limiting membrane of MVBs and 70% is still sorted to ILVs of MVBs whose morphology is not affected (E and F). (Scale bars, 150 nm.)

To further investigate how ubiquitination impacts on the trafficking of OA1 to lysosomes and melanosomes in melanocytic cells, we next analyzed by IEM in MNT1 cells the subcellular localization of the Flag-tagged OA1 wild type (OA1^wt) and all lysine mutant OA1 (OA1^K1-7R). The mutant OA1^K1-7R 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 OA1^K1-7R transfection. They were also similar in luminal content to those generated when the function of ESCRT components 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; quantification 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). Taken together, our observations indicate that ubiquitination of OA1 by regulating its down-regulation is likely to control the balance of receptor within the endo-melanosomal system and ensure its intracellular function.

Fig. 3.

The ubiquitin-deficient OA1 mutant is retained on MVBs and melanosomes in MNT-1 cells. (A and B) Ultrathin cryosections of MNT-1 cells transfected with Flag-tagged wildtype (OA1^wtFlag) or all-lysine mutant OA1 (OA1^K1-7R Flag) were double-immunogold labeled for Flag (PAG 15) to detect OA1 and Tyrp1 (PAG 10). OA1^K1-7R 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 of OA1 at the limiting membrane of mature melanosomes in OA1^wt and OA1^K1-7R expressing cells (arrowheads, A and B). (Scale bars, 200 nm.) (E) Histogram depicting a quantitative evaluation of OA1 labeling (gold particles) at the melanosome membrane of OA1^wt and OA1^K1-7R transfected cells.

Hrs (ESCRT-0) Is Required for the Endosomal Transport of OA1.

To elucidate the downstream molecular players involved in OA1 trafficking and down-regulation we next examined the requirement for the ESCRT machinery (12). Hrs is a component of the ESCRT-0 complex directly involved in recognition of ubiquitinated membrane cargo on endosomes (31). At steady state, OA1 partially overlaps with a small subset of Hrs positive endosomes in MNT1 cells (Fig. S2 J–L) and we have further provided biochemical evidence for an interaction between OA1 and Hrs (Fig. 4I). Hrs was detected by IB in lysates immunoprecipitated with anti-Flag (OA1) antibody of Hrs-myc and OA1-Flag-transfected MNT-1 cells, but not in lysates of MNT-1 cells cotransfected with Hrs-myc and a Flag empty vector. We next examined by IF and IEM the effect of overexpressing Hrs-YFP, one of the Hrs-tagged variants that function as dominant inhibitors of Hrs-mediated lysosomal sorting of transmembrane proteins including receptors (32, 33) and GPCRs (18). IF analysis indicates that overexpression of Hrs-YFP strongly increases localization of endogenous OA1 in the Hrs-positive endosomes (arrows, Fig. 4 A–C) and in the enlarged Hrs-positive clusters of endosomes that are generated in overexpressing cells (arrows, Fig. 4 D–F). These modified endosomes have been shown to trap ubiquitinated cargo destined to ILVs, as demonstrated for MART-1 in melanocytic cells (34) and also for another GPCR, PAR-2 (35). IEM revealed that both endogenous and transfected OA1 were retained at the limiting membrane of these compartments (arrows, Fig. 4G and Inset). In addition, as shown by IB (Fig. 4H), the protein levels of OA1 were significantly increased when coexpressed with Hrs-YFP compared with control cells expressing only YFP. Because nonubiquitinated proteins traversing early endosomes can also be trapped in the “Hrsosomes” (30), we further inactivated Hrs by RNAi in MNT-1 cells. Depletion of Hrs results in the accumulation of transiently expressed OA1-Flag in Hrs-depleted cells, as shown by IF (Fig. S3M, arrow; quantification in Fig. S3N). Similar observations were reported for a PAR2 construct, another GPCR that undergoes ubiquitination and subsequent interaction with Hrs (35). However, depletion of Hrs does not result in the accumulation of endogenous OA1 within the cell but rather results in its decreased expression 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 confirmed a reduction of both endogenous OA1 and MART-1 in the majority of siHrs-treated cells (arrows, Fig. S3 F–I) compared with control cells (Fig. S3 B–E). These observations suggest that the absence of functional Hrs impacts on their normal trafficking without fully impairing their degradation, as reported for the EGFR (36). In addition, we found by quantitative real-time PCR that the reduction of the endogenous OA1 and MART-1 proteins was certainly related to a down-regulation of their transcripts in siHrs-treated cells (Fig. S3 J–L). This observation indicates that Hrs depletion, could also consequently impact on the transcription of these melanosomal proteins. Taken together, our results indicate that the trafficking of OA1 requires its ubiquitination and functional Hrs. Such requirements slightly differ from what was previously reported for the DOR, the down-regulation of which appeared dependent on Hrs despite lack of ubiquitination (18).

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 cells 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 at the limiting membrane of the Hrs-positive (PAG 10) endosomes. Note the interconnected network of large endosomal vacuoles densely packed with small ILVs. (Scale bars, 200 nm.) (H) Total cell lysates of MNT-1 cotransfected with either Hrs-YFP or YFP and OA1-flag were analyzed by immunoblotting with the indicated antibodies. The arrow indicates the accumulation of OA1 upon coexpression with Hrs-YFP, but not with YFP alone. (I) MNT-1 cells were transfected with either OA1-Flag or Flag empty vector and Hrs-myc. Lysates were immunoprecipitated with monoclonal anti-Flag antibody, fractionated by SDS/PAGE, and immunoblotted with anti-myc (Hrs) or Flag (OA1). The arrow indicates a specific band detected by anti-myc antibody in OA1-immunoprecipitates but not in immunoprecipitates of empty-Flag vector.

OA1 Sorting and Degradation Requires ESCRT-I Function.

One of the downstream effectors of Hrs in the regulation of endosomal protein sorting is ESCRT-I. Tsg101 is a “core” component of this complex and can bind both ubiquitinated cargo and Hrs (37). Knockdown of Tsg101 has been shown to significantly inhibit lysosomal sorting and degradation of various membrane receptors including EGFR (38) and the GPCR GABA(B)-receptor (39). To investigate the involvement of ESCRT-I in OA1 sorting, we depleted Tsg101 by RNAi from MNT-1 cells. Efficient knockdown (>90%) of Tsg101 protein was confirmed by IB (Fig. 5A). Revealing a role for ESCRT-I in OA1 trafficking, depletion of Tsg101 interfered with OA1 degradation resulting in a significant 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 accumulates in enlarged vesicular structures throughout the cytoplasm 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 and MART-1 at their limiting membranes (Fig. 5J and Inset). OA1 was also found at the membrane of MART-1 containing autophagosome-like compartments generated in siTsg101 cells (30, 40) (arrows, Fig. 5K). We recently reported that MART-1 interacts with OA1 and acts as an escort for this GPCR at early steps of its biosynthetic pathway (9). Therefore, the accumulation of OA1 in Tsg101-depleted cells could be merely a consequence of the accumulation of MART-1 itself. To test whether the effect of Tsg101 on OA1 was mediated by MART-1, we concomitantly inactivated Tsg101 and MART-1 in MNT-1 cells. WB analysis revealed an accumulation of OA1 also in Tsg101/MART1-depleted cells (Fig. 5A), in agreement with a specific effect of Tsg101 on OA1 protein sorting and degradation independent of its association with MART-1. These results also strengthen a role for MART-1 in the maintenance of OA1 stability that precedes the involvement of Tsg101 in OA1 sorting (9). Consistent with a block in the trafficking that precedes OA1 delivery to lysosomes and degradation, colocalization of endogenous OA1 with the lysosomal protein LAMP1 was also reduced (Fig. S4 A-H). Overall, these observations indicate that OA1 degradation and sorting within the endo-lysosomal system requires both ESCRT-0 and ESCRT-I function alike other ubiquitinated cargoes (38) but unlike the DOR, for which degradation was dependent on Hrs but not Tsg101 (18). Upon Tsg101 inactivation, OA1 was also detected at the limiting membrane of intracellular vesicles, similar to 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 involved in recognition and sequestration of ubiquitinated cargoes in endosome membranes and in membrane bud formation, ESCRT-III subunits are essentially required for ILV formation and scission (41).

Fig. 5.

Knockdown of Tsg101 (ESCRT-I) affects OA1 sorting and degradation in MNT-1 cells. MNT-1 cells were treated with control siRNA (siCtrl) or siRNAs specific for Tsg101 (siTsg101), for Tsg101 and MART-1 (siTsg101/MART-1) and for MART-1 (siMART-1). (A) Whole cell lysates were analyzed by immunoblotting for OA1, Tsg101, MART-1, or tubulin as a control. Note the effective depletion 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) Cells treated with siRNAs targeting Tsg101 (siTsg101) (F–I) or Ctrl siRNAs (siCtrl) (B–E) were analyzed by IFM using antibodies to OA1 (B and F) and MART-1 (C and G). Overlays are shown in D and H. Boxed regions correspond to 5× magnified areas in E and I. (Scale bars, 10 μm.) (J and K) Ultrathin cryosections of Tsg101-depleted MNT-1 cells were immunogold labeled for OA1 (PAG 15) and MART-1 (PAG 10). OA1 localizes at the limiting membrane of MART-1 positive class E membranous structures (J) and aberrant MVBs (J Inset) and is also associated with autophagosomal-like compartments (K). (Scale bars, 200 nm.)

OA1 Sorting and Degradation Requires ESCRT-III Function.

To investigate 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 AAA ATPase Vps4 (Vps4E228Q), known to block the trafficking of ubiquitinated cargoes (43), also increased levels of endogenous OA1 relative to those observed in control cells by IB (Fig. S5E). Together, our results indicate that the degradation and sorting of OA1 within endo-lysosomal membranes requires not only ESCRT-0 and -I, but also requires ESCRT-III function.

Fig. 6.

Knockdown of Vps24 (ESCRT-III) affects OA1 sorting and degradation in MNT-1 cells. MNT-1 cells were treated with control siRNA (siCtrl) or siRNAs specific 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, and boxed regions are 2.5× magnifications. (D and H) Brightfield (BF) images of the 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, MART-1, Vps24, or tubulin, as indicated. Note the accumulation of OA1 and MART-1 in Vps24-depleted cells. Arrows point to the different maturation forms of OA1. (J and K) IEM analysis was performed in siVps24-treated cells with the indicated antibodies. In Vps24-depleted cells, OA1 (PAG 10) is found at the limiting membrane of MART-1 (PAG 15)-positive “coated” endosomes (arrows, J) and multivesicular endosomes (arrows, K). (Scale bars, 200 nm.)

Conclusion.

Taken together, our data uniquely highlight how the intracellular trafficking of a GPCR is regulated through ubiquitination and the ESCRT machinery. We show that the product of the Ocular Albinism type 1 gene, the GPCR OA1, is ubiquitinated, and that this ubiquitination is essential for its targeting to the intraluminal vesicles of MVBs in nonmelanocytic and melanocytic cells. Our results extend former studies by providing further evidence for the direct involvement of ubiquitination and the ESCRT machinery in the sequestration of a mammalian GPCR in ILVs of MVBs, a step essential in the regulation of the trafficking of several signaling receptors. In melanocytic cells, ubiquitination of OA1 is likely to control the balance between down-regulation and delivery to melanosomes, where this GPCR functions to maintain melanosome identity and composition (9). Our study has also important implications in the physiopathology of ocular albinism type 1, an X-linked genetic disorder caused by mutations 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 mutations could possibly generate additional acceptor sites of ubiquitin and consequently affect OA1 degradation. Importantly, an impairment of OA1 degradation might also affect OA1 signaling within the endomembrane system (2), which may be tightly related to the balance between down-regulation and localization to the melanosome. The OA1 downstream effectors are still obscure, but it is surely a challenge for the future to investigate how its intracellular trafficking and regulatory mechanisms impact in OA1 signaling and function.

Materials and Methods

Cell Culture, Transfection, and siRNA Depletion.

MNT-1 and HeLa cells were cultured and transfected with plasmids and oligonucleotides as described (9, 46, 47) using Lipofectamine 2000 or Oligofectamine (Invitrogen). Cells were analyzed 24–48 h (plasmids) or 72 h (oligos) after transfection.

Antibodies.

Polyclonal anti-human OA1, raised against the C terminus of the 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 antibodies are listed in SI Materials and Methods.

Plasmids and siRNAs.

The OA1-Flag plasmid was reported elsewhere (9). Flag-tagged plasmid encoding OA1 with single K to R substitutions, OA1^K1-6R, was obtained using the PCR-based QuikChange Lightning multi site-directed mutagenesis kit (Stratagene). Source of other plasmids and siRNAs are listed in SI Materials and Methods.

Quantitative Real-Time PCR.

Total RNA was extracted from siRNA and siCtrl transfected MNT-1 cells using RNeasy Mini kit (Quiagen). The same amount of cDNA was synthesized using SuperScript II (Invitrogen) and random primers. Real-time PCR was carried out with the GeneAmp 7000 Sequence Detection System (Applied Biosystems; ref. 9) and normalized using the ribosomal gene S26. Primers used for quantitative PCR of OA1, MART-1, and the reference gene S26 were as described.(9).

Biochemistry.

SDS/PAGE and immunoblotting were carried out by standard methods and as described (9). Immunoprecipitations from MNT-1 or HeLa cells were performed using Protein G-agarose beads (Invitrogen; ref. 9). For ubiquitin detection, MNT-1 cells (10⁸ cells) or HeLa cells (10⁶ cells) expressing OA1-Flag and HA-ubiquitin or HA-empty vector were lysed for 30 min on ice in 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 Diagnostic), 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 and MNT-1 cells cultured on coverslips were fixed with 4% paraformaldehyde in sodium phosphate buffer (PBS) and immunofluorescence was carried out as described (9). For immuno electron microscopy, cells were fixed with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer and processed for ultracryomicrotomy and immunogold labeling (48). Ultrathin cryosections were single- or double-immunogold labeled with antibodies and protein A coupled to 10 or 15-nm gold, as indicated. Sections were observed under a CM120 electron microscope (FEI, Eindoven), equipped with a KeenView camera (Soft Imaging System; SIS, Germany). For quantification of OA1 labeling, gold particles were counted in randomly selected intracellular compartments in each of two separate experiments. Data are presented as mean ± SD.