IDENTIFICATION OF A MELANOSOMAL MATRIX PROTEIN ENCODED BY THE MURINE SI (SILVER) LOCUS USING ORGANELLE SCANNING

To identify a broad spectrum of melanosomal proteins, antisera were ra ised in rabbits against melanosomal protein fractions separated on the basis of their solubility in the nonionic detergent Triton X-114. Ant isera against the different fractions each recognized a distinct set o f bands when used for inmunoblotting analysis with extracts of melanoc ytes cultured from wild-type black mice. Immunoblotting with antisera to whole melanosomes or to Triton X-114-soluble melanosomal proteins t hat segregated with the detergent phase gave identical patterns with p rotein extracts from melanocytes from wild-type mice and from mice hom ozygous for the si (silver) coat color mutation. By contrast, an antis erum against Triton X-114 soluble melanosomal proteins that segregated in the aqueous phase recognized an 85-kDa protein that was present in extracts from wild-type melanocytes but was absent from si melanocyte s. This suggested that the protein was encoded at the si (silver) locu s. This was confirmed by employing an antiserum directed against the c arboxyl terminus of the predicted murine silver protein sequence. The detergent solubility, biochemical characteristics, and immunologic pro perties of the 85-kDa protein and of the authentic si gene product wer e identical. Further analysis demonstrated that this protein correspon ds to a melanosomal matrix glycoprotein that we recently described. Ou r results suggest that employing polyclonal antisera to fractionated o rganelles such as melanosomes, to screen tissues from mutant mice, a t echnique that we call ''organelle scanning'', can serve as a powerful means of identifying new organellar proteins and their respective gene s.