IDENTIFICATION OF A MELANOSOMAL MEMBRANE-PROTEIN ENCODED BY THE PINK-EYED DILUTION (TYPE-II OCULOCUTANEOUS ALBINISM) GENE

The pink-eyed dilution (p) locus in the mouse is critical to melanogen esis; mutations in the homologous locus in humans, P, are a cause of t ype II oculocutaneous albinism. Although a cDNA encoded by the p gene has recently been identified, nothing is known about the protein produ ct of this gene. To characterize the protein encoded by the p gene, we performed immunoblot analysis of extracts of melanocytes cultured fro m wild-type mice with an antiserum from rabbits immunized with a pepti de corresponding to amino acids 285-298 of the predicted protein produ ct 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 (p(cp) and p) in which transcripts of the p gene a re absent or greatly reduced. Introduction of the cDNA for the p gene into p(cp) melanocytes by electroporation resulted in expression of th e 3.3-kb mRNA and the 110-kDa protein. Upon subcellular fractionation of cultured melanocytes, the 110-kDa protein was found to be present i n melanosomes but absent from the vesicular fraction; phase separation performed with the nonionic detergent Triton X-114 confirmed the pred icted hydrophobic nature of the protein. These results demonstrate tha t the p gene encodes a 110-kDa integral melanosomal membrane protein a nd establish a framework by which mutations at this locus, which dimin ish pigmentation, can be analyzed at the cellular and biochemical leve ls.