New insights on the structure of the mouse silver locus and on the function of the silver protein

The melanosomal proteins encoded by the silver locus pla); important roles in melanogenesis. The human locus yields two proteins, PMEL17 and GP100, by alternative mRNA splicing. The mouse si locus was reported to encode a Pme l17 protein, and later gp87, a GP100 homologue. When we re-examined the pro ducts of wild-type and silver-mutant mouse si loci, RT-PCR of wild-type RNA and genomic DNA sequence accounted for gp87 but excluded the occurrence of Pmel17. Analysis of cDNA from the silver (si/si) melanocyte Line, melan-si , showed that the pathogenic mutation is a G to A substitution at nt 1808, which yields a premature stop codon and a predicted protein truncated in th e C-terminus. This was confirmed hv reaction of a specific anti-gp87 antise rum with si/si melanocyte extracts. To further explore gp87 function, we compared the DHICA oxidase activity of extracts from B16, melan-si (heterozygous for the brown mutation and homoz ygous for the silver mutation and Cloudman S91 cells (homozygous for the br own mutation), since both TRP1 and gp87 are thought to be involved in DHICA oxidation/polymerization. Cloudman extracts do not oxidize significantly D HICA and its methyl ester, supporting the involvement of native mouse TRP1 in DHICA oxidation. Extracts from B16 and melan-si do not show significant differences for the oxidation of free acid and methylated dihydroxyindoles, indicating that the mechanism is not decarboxylative. Melan-si extracts ar e very efficient in catalyzing dihydroxyindole oxidation, in spite of bring heterozygous for the TRP1 mutation, consistent with a stablin effect for t he wild-type gp87 protein. On the other hand, aggregated and degraded forms of that mutant gp87 protein are found in the cytosolic fraction of melan-s i, suggesting that misrouting and aberrant professing of the gp87 and tyros inase ma! also be related to the high DHICA oxidase activity of these melan ocytes.