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.

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.

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.)

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.

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.

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.)

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.)