Faculty Bibliography

Melanosomes, the subcellular site of melanin synthesis and deposition, may be related to the endolysosomal lineage of organelles. To determine if melanosomes contain lysosomal hydrolases, we examined the subcellular distribution of five of these enzymes in melanocytes cultured from C57BL/6J mice. Analyses of Percoll gradient density centrifugations demonstrated that beta-hexosaminidase, beta-galactosidase, beta-glucuronidase, and cathepsins B and L all co-sedimented with tyrosinase-rich densely sedimenting melanosomes. The melanosomal distribution of these enzymes was confirmed in studies of melanocytes cultured from albino mice and of melanocytes rendered amelanotic by transfection with the v-rasHa oncogene (which lack dense, melanized melanosomes). In these cells, only a less dense peak of activity for each hydrolase was present. The level of each hydrolase was elevated in black cells when compared with albino cells. Metabolic labeling studies confirmed that the increase in beta-glucuronidase in black versus albino cells resulted mainly from increased synthesis of this enzyme. The data suggest that melanosomes represent specialized lysosomes present within melanocytes, that they contain a broad array of lysosomal hydrolases, and that the levels of these hydrolases are elevated in cells actively engaged in pigment production

The pink-eyed dilution (p) locus in the mouse is critical to melanogenesis; mutations in the homologous locus in humans, P, are a cause of type II oculocutaneous albinism. Although a cDNA encoded by the p gene has recently been identified, nothing is known about the protein product of this gene. To characterize the protein encoded by the p gene, we performed immunoblot analysis of extracts of melanocytes cultured from wild-type mice with an antiserum from rabbits immunized with a peptide corresponding to amino acids 285-298 of the predicted protein product of the murine p gene. This antiserum recognized a 110-kDa protein. The protein was absent from extracts of melanocytes cultured from mice with two mutations (pcp and p) in which transcripts of the p gene are absent or greatly reduced. Introduction of the cDNA for the p gene into pcp melanocytes by electroporation resulted in expression of the 3.3-kb mRNA and the 110-kDa protein. Upon subcellular fractionation of cultured melanocytes, the 110-kDa protein was found to be present in melanosomes but absent from the vesicular fraction; phase separation performed with the nonionic detergent Triton X-114 confirmed the predicted hydrophobic nature of the protein. These results demonstrate that the p gene encodes a 110-kDa integral melanosomal membrane protein and establish a framework by which mutations at this locus, which diminish pigmentation, can be analyzed at the cellular and biochemical levels

The silver mutation in mice causes progressive graying of hair due to the loss of functional follicular melanocytes. Recently the silver locus gene (called Pmel 17) has been cloned; its encoded product shares homology with a chick melanosomal matrix protein and a bovine retinal pigment epithelial protein. Although the sequence of the silver gene and the correlation of its expression with pigment production have been reported, its function in melanogenesis is still unknown. In an effort to characterize that function, we have synthesized the predicted carboxyl-terminal peptide of the mouse Pmel 17 protein and generated a rabbit polyclonal antibody (alpha PEP13) to it; that antibody recognized the silver protein specifically. The immunoaffinity-purified silver protein lacked all of the known melanogenic catalytic activities which other tyrosinase-related proteins (TRP) have, nor did it appear to modulate any of those TRP activities. Metabolic labeling experiments demonstrated that the silver protein disappears in vivo within a few hours, indicating that it is rapidly degraded, or quickly processed to lose its carboxyl terminus. Cross-reactivity experiments showed that a recently reported anti-melanosomal matrix protein antibody (alpha MX) also recognizes the silver protein, although at a different epitope from that of alpha PEP13. Using Western immunoblotting, we analyzed subcellular fractions isolated from B16 F10 melanoma cells and found that the silver protein was rich in the melanosome fraction but was absent from coated vesicles which deliver TRPs to melanosomes. These results suggest that the silver locus product is a melanosomal matrix protein which may contribute to melanogenesis as a structural protein, although the possibility remains that it also has a novel catalytic function in melanogenesis

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight 'multimers' of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products

The lines of Blaschko represent a pattern followed by many skin disorders. We review the clinical and histologic features of X-linked, congenital/nevoid, and acquired skin diseases that follow these lines. We also include cutaneous disorders that have a linear distribution but do not follow Blaschko's lines. Finally, we differentiate Blaschko's lines from other patterns on the skin such as dermatomes and Langer's lines

To identify a broad spectrum of melanosomal proteins, antisera were raised in rabbits against melanosomal protein fractions separated on the basis of their solubility in the nonionic detergent Triton X-114. Antisera against the different fractions each recognized a distinct set of bands when used for immunoblotting analysis with extracts of melanocytes cultured from wild-type black mice. Immunoblotting with antisera to whole melanosomes or to Triton X-114-soluble melanosomal proteins that segregated with the detergent phase gave identical patterns with protein extracts from melanocytes from wild-type mice and from mice homozygous for the si (silver) coat color mutation. By contrast, an antiserum against Triton X-114 soluble melanosomal proteins that segregated in the aqueous phase recognized an 85-kDa protein that was present in extracts from wild-type melanocytes but was absent from si melanocytes. This suggested that the protein was encoded at the si (silver) locus. This was confirmed by employing an antiserum directed against the carboxyl terminus of the predicted murine silver protein sequence. The detergent solubility, biochemical characteristics, and immunologic properties of the 85-kDa protein and of the authentic si gene product were identical. Further analysis demonstrated that this protein corresponds to a melanosomal matrix glycoprotein that we recently described. Our results suggest that employing polyclonal antisera to fractionated organelles such as melanosomes, to screen tissues from mutant mice, a technique that we call 'organelle scanning', can serve as a powerful means of identifying new organellar proteins and their respective genes