Identification and characterization of a melanocyte-specific novel 65-kDa peripheral membrane protein

In order to study proteins of the melanosome, we developed a panel of antis era against various protein fractions of melanosomes from B16 melanoma cell s. An antiserum raised against a Triton X-100 insoluble fraction of melanos omes recognized a 65-kDa protein in melanocytes from mice homozygous for th e buff mutation, but not in their wild type counterparts. Further studies w ere conducted using a specific, second generation antiserum raised against the purified protein. The protein was also detected in melanocytes cultured from albino mice, but absent in cultured mouse cell lines not of melanocyt e origin. Density gradient centrifugation of subcellular organelles and ind irect immunofluorescent cell staining, indicated that the protein was assoc iated with melanosomes and vesicles. The protein on intact organelles could be made soluble using sodium carbonate, and digested with proteases in the absence of detergent suggesting that it was a peripheral membrane protein localized on the cytosolic face of organelle membranes. Metabolic labelling of cells and N-glycosidase F digestion of cell extracts indicated that the protein was not N-glycosylated. Based on its intracellular localization an d biochemical defects in the buff mouse, a potential role has been suggeste d for the 65-kDa protein in intracellular membrane trafficking.