Interaction of Melanosomal Proteins with Melanin

Abstract

Melanin is deposited in melanosomes upon a proteinaceous matrix enveloped by a melanosomal membrane. Since melanin is highly detergent insoluble, we hypothesized that the detergent solubility of proteins of the melanosomal matrix might be inversely related to the state of melanosomal melanization. Immunoblotting analyses were performed on extracts of albino and black melanocytes to test this hypothesis. The protein products of the silver (si) and the pink-eyed-dilution (p) loci as well as other matrix constituents were present at twofold higher levels in extracts of albino cells. When black cells were rendered amelanotic by growing cultures in the presence of the tyrosinase inhibitor phenylthiourea, the apparent levels of these proteins were also increased. To obviate the potential role of different levels of synthesis in contributing to these differences, we developed a cell-free melanosomal melanization assay. Upon incubation of a melanosome-rich fraction with the melanin precursor ~-3,4-dihydroxyphenylalanine (Dopa) followed by immunoblot analysis, the si locus protein, the p locus protein, and other putative matrix constituents became rapidly insoluble in SDS when compared with the members of the tyrosinaserelated family of melanosomal membrane proteins. Our results suggest that melanosomal proteins that interact with melanin may be identified by their relative insolubility in SDS under conditions of increasing melanization. In addition to the si locus protein and other putative melanosomal matrix proteins, the membrane-bound p locus protein may also interact closely with melanin.Keywords: melanosome ; tyrosinase ; pink-eyed dilution locus ; silver locus ; melanoma antigen.Melanocytes synthesize and deposit melanin pigments within a subcellular organelle, the melanosome. Melanosomes are highly specialized members of the endolysosomal lineage of organelles [l]. It is recognized that melanosomal biogenesis involves a bipartite pathway in which tyrosinase-containing coated vesicles fuse with premelanosomes containing a well-defined matrix (stage I1 melanosomes). This fusion is believed to initiate the complex process of melanization [2, 31. Although a family of membrane proteins involved in the synthesis of melanin and related to the prototypic enzyme tyrosinase has been well studied [4-61, little is known about other melanosomal proteins or their role in melanin synthesis and degradation. Recently, a melanosomal protein encoded by the pink-eyed-dilution (p) locus has been identified [7]. This 110-kDa integral membrane protein is unrelated to the tyrosinase-related protein (TRP) family, and its function remains unknown [7].The least well studied melanosomal proteins are those that comprise the internal structure of the melanosome, termed the matrix. It is upon this proteinaceous matrix that melanin polymers are deposited [l]. Even the partial disruption of the internal matrix of melanosomes requires extensive extraction with strongly chaotropic agents such as SDS [8, 91. Effective partial Abbreviafions. Dopa, ~...

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

“…Similar results were obtained in transiently transfected HeLa cells expressing OCA2, although processing in the Golgi seemed to be faster (Figure 1b). Note that processed OCA2 disappeared rapidly from detergent lysates in melanocytes, consistent with earlier results, suggesting close association with melanin (Donatien and Orlow, 1995), but much less rapidly in transfected HeLa cells. The reduction in M r resulting from PNGase F or endoH treatment of OCA2 was ϳ7 kDa, consistent with loss of two N-linked oligosaccharide chains.…”

“…OCA2 was originally thought to localize predominantly to mature melanized melanosomes based on subcellular fractionation of melanocytes (Rosemblat et al, 1994), poor extraction by detergent from melanized melanocytes relative to nonmelanized melanocytes (Donatien and Orlow, 1995), and interpretation of results from confocal immunofluorescence microscopy (IFM) analyses (Toyofuku et al, 2002). Tyrp1-containing compartments in melanocytes from OCA2-deficient mice are less acidic than in wild-type melanocytes, suggesting that OCA2 not only localizes to melanosomes but also modulates their pH (Puri et al, 2000), although this interpretation is disputed because Tyrp1 does not localize to melanosomes properly in OCA2-deficient melanocytes (Manga et al, 2001).…”

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

“…Indeed, the colocalization of Tyr and p in melan-c cells further strengthens our conclusions regarding the ER localization of p. However, it is still possible that a portion of total p may locate in other organelles. Previous experiments have shown that increasing melanin deposition is inversely proportional to p detection by Western blotting (Donatien and Orlow, 1995). Thus, mature melanosomes may still contain some p, which is not detectable by the techniques used in the current study.…”

“…Hypotheses regarding p function include the following: 1) p transports tyrosine into melanosomes (Lee et al, 1995). 2) p plays a structural role in melanosomes (Donatien and Orlow, 1995;Rosemblat et al, 1998) or is involved in melanosome biogenesis (Orlow and Brilliant, 1999). 3) p plays a role as a pH regulator in melanosomes and provides an optimal pH for Tyr activity (Puri et al, 2000;Brilliant, 2001).…”

“…This prompted us to reexamine the location of p. It was generally thought that p was located in melanosomes (Rosemblat et al, 1994;Donatien and Orlow, 1995). However, a combination of data from sucrose gradient and Percoll gradient fractionation as well as immunofluorescence microscopy reveals that the ER contains a significant amount of p protein.…”