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To Identify a broad sperm of melanosomal proteins, antisera were raised In rabbits against melanosomal protein fractions separated on the basi oftheir soubilit in the onic detergent Triton X-114. Antisera METHODS Subceflular Fractionation and Prparation of Antisera. Melanosomes were purified as described (7) from B16 cells grown as tumors subcutaneously in C57BL/6J mice. Stage III-IV melanosomes made up >90%6 ofthe organelles in the purified fraction as assessed by electron microscopy (7). To further fractionate melanosomes, they were solubilized in 0.1 M sodium phosphate buffer (pH 6.8) containing 1% (vol/vol) Triton X-114 at 40C for 10 min. Detergent-insoluble material was then removed by centrifugation at 10,000 x g for 10 min. The Triton X-114-soluble proteins were subjected to separation into aqueous and detergent phases [adapted from the method of Bordier (6)] as described (7). The protein content ofeach fraction was determined, and rabbits were immunized monthly with 0.2 mg of protein emulsified with complete Freund's adjuvant initially and subsequently in incomplete adjuvant as described (5). Rabbits were bled from the ear vein, the blood was allowed to coagulate at room temperature, and sera were isolated by centrifugation at 10,000 x g for 30 min. Final bleeds were obtained at 6 months. The production of antiserum to the Triton-insoluble melanosomal matrix has been described (5).The polyclonal antibody aPEP13 was produced in rabbits (8) using procedures as detailed (3). Briefly, a 15-amino-acid sequence representing the predicted carboxyl terminus ofthe cloned murine si-encoded protein (8) was synthesized (BioSynthesis, Denton, TX) and conjugated to bovine serum albumin SuperCarrier (Pierce). Immunization and sera collection from the rabbit and the titration and specificity of antibody production, as determined by ELISA, was as reported (3).Immunoblotting. Melanocyte lines were established from C57BL/6J mice, either wild type ("melan-a" cells), homozygous for the silver (si) mutation ("melan-si-l" cells), or homozygous for the albino (c) mutation ("melan-c" cells),

The two major types of mammalian melanin are pheomelanin (yellow or red pigment) and eumelanin (black or brown). The agouti (A) and extension (E) loci determine whether follicular melanocytes will deposit pheomelanin or eumelanin within their melanosomes. Mutations at the murine pinkeyed-dilution (P) locus cause a striking reduction in deposition of eumelanic, but not pheomelanic, pigment. The mRNA encoded at the P locus is not expressed in skin that exclusively produces pheomelanic pigment as a result of mutation at the agouti locus. We have suggested, based upon both genetic and biochemical evidence, that three key melanogenic proteins--tyrosinase, tyrosinase-related-protein-1 (TRP-1), and TRP-2, encoded at the albino (C), brown (B), and slaty (Slt) loci, respectively--form a high-molecular-weight "melanogenic complex" within the melanosome. High-molecular-weight forms of tyrosinase, TRP-1 and TRP-2, are absent from eumelanic ocular tissues of p(un)/p(un) mice that fail to produce normal P-locus transcript, even though these mice are genetically normal at the loci that regulate production of the three melanogenic proteins. We have hypothesized that the presence of the p-locus protein is important for the integrity of the melanogenic complex and for the levels of members of the TRP family. We show here that the yellow skins of mice mutant at the agouti or extension loci, as well as the nonyellow skins of pinkeyed-unstable (p(un)/p(un)) mice, demonstrate greatly diminished levels of tyrosinase, TRP-1 and TRP-2, and an absence or markedly decreased proportion of high-molecular-weight forms of melanogenic proteins. We conclude that normal levels of wild-type P-locus protein are necessary for eumelanogenesis and that the absence of this protein may be necessary, but is not sufficient to cause the melanosome to switch to the production of pheomelanin. We discuss the implications of our results in relation to the interacting genetic controls regulating melanogenesis.

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Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promotor fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.