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

Abstract

To identify a broad spectrum of melanosomal proteins, antisera were raised in rabbits against melanosomal protein fractions separated on the basis of their solubility in the nonionic detergent Triton X-114. Antisera against the different fractions each recognized a distinct set of bands when used for immunoblotting analysis with extracts of melanocytes cultured from wild-type black mice. Immunoblotting with antisera to whole melanosomes or to Triton X-114-soluble melanosomal proteins that segregated with the detergent phase gave identical patterns with protein extracts from melanocytes from wild-type mice and from mice homozygous for the si (silver) coat color mutation. By contrast, an antiserum 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 melanocytes. This suggested that the protein was encoded at the si (silver) locus. This was confirmed by employing an antiserum directed against the carboxyl terminus of the predicted murine silver protein sequence. The detergent solubility, biochemical characteristics, and immunologic properties of the 85-kDa protein and of the authentic si gene product were identical. Further analysis demonstrated that this protein corresponds to a melanosomal matrix glycoprotein that we recently described. Our results suggest that employing polyclonal antisera to fractionated organelles such as melanosomes, to screen tissues from mutant mice, a technique that we call "organelle scanning", can serve as a powerful means of identifying new organellar proteins and their respective genes.