Identification of a melanosomal matrix protein encoded by the murine si (silver) locus using "organelle scanning".

Abstract

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),

A model for melanosome biogenesis based on the purification and analysis of early melanosomes

“…The two antibodies that recognize GP100 (i.e., ␣PEP13 and HMB45) show radically different reactivity patterns. The epitope for HMB45 is on an interior motif of the protein whereas ␣PEP13 recognizes the carboxyl terminus (5,6,33). Previous metabolic labeling and pulse-chase analysis combined with sucrose density gradient subcellular fractionation previously showed that processing of GP100 is very quick compared with the TYRPs, and that it is not processed through the trans-Golgi network, but follows a route distinct from the TYRPs to be sorted to melanosomes (5).…”

“…These include tyrosinase (TYR), the tyrosinase-related proteins-1 and -2 (TYRP1͞TRP1 and DCT͞TRP2, respectively; refs. 1-3), GP100͞Pmel17͞silver (4)(5)(6), and Pink (7)(8)(9). In addition, another protein, termed MART1 (melanoma antigen recognized by T lymphocytes), has been cloned that localizes in melanosomes (10,11), but its function there remains unclear.…”

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

“…For immunofluoresence analysis, ␣Pep7 was further affinity purified using the Pep7 peptide linked to an Affigel15 column (Bio-Rad, Hercules, CA). The polyclonal antibody ␣Pep13 was generated against the C terminus of the mouse silver protein (Zhou et al, 1994). The following antibodies were also used: GRP78/bip, syntaxin 6, and Vti1b (Transduction Laboratories, Lexington, KY); ribophorin (obtained from Dr. G. Kreibich, New York University Medical Center, New York, NY); anti-V5 antibody (Invitrogen, San Diego, CA); Mel-5 (Signet, Dedham, MA), and Calnexin (StressGen, Victoria, British Columbia, Canada).…”

“…Tyr distribution was biphasic with a predominant peak in the melanosome fractions 1-3 and a minor peak in the Golgi/ER fractions. To determine whether p is located in premelanosomes, we used ␣Pep13 against the silver protein as a premelanosome marker (Zhou et al, 1994;Raposo et al, 2001). The premelanosomes were found between fractions 9 and 11, in a single peak and one fraction lighter than p. Thus, most p is not present in premelanosomes.…”

Retrotransposon insertion in SILV is responsible for merle patterning of the domestic dog

“…Although this gene is known to have a central role in pigmentation, the precise function of SILV remains controversial (24). Multiple studies have provided data to suggest that SILV is involved in the biogenesis of premelanosomes (25)(26)(27). Significant expression of the gene is almost exclusive to the skin and eye, providing further evidence to support a role in pigmentation (24).…”