The ocular albinism 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 protein modification by ubiquitination and down- brane proteins 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 cell-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 CELL BIOLOGY 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 endosomes, 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 ocular albinism type 1. lysosomes and melanosomes in melanocytes, and to late endo- somes/lysosomes when transiently expressed in nonmelanocytic GPR143 | melanosome | multivesicular endosome | 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 lysosome 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 gene 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 organelles (LROs) of a first step, OA1-Flag was immunoprecipitated from MNT-1 cell pigment cells devoted to melanin 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 organelle to the cytosol. 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,