Open AccessIdentification and characterization of a melanocyte‐specific novel 65‐kDa peripheral membrane protein
Author(s) -
Samaraweera Preminda,
Donatien Philippe D.,
Qazi Sadia,
Kobayashi Takeshi,
Hearing Vincent J.,
Panthier JeanJacques,
Orlow Seth J.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00930.x
Subject(s) - melanosome , melanocyte , organelle , biology , cytosol , differential centrifugation , microbiology and biotechnology , intracellular , membrane protein , biochemistry , melanin , membrane , enzyme , melanoma , genetics
In order to study proteins of the melanosome, we developed a panel of antisera against various protein fractions of melanosomes from B16 melanoma cells. An antiserum raised against a Triton X‐100 insoluble fraction of melanosomes recognized a 65‐kDa protein in melanocytes from mice homozygous for the buff mutation, but not in their wild type counterparts. Further studies were 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 melanocyte origin. Density gradient centrifugation of subcellular organelles and indirect immunofluorescent cell staining, indicated that the protein was associated 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 and biochemical defects in the buff mouse, a potential role has been suggested for the 65‐kDa protein in intracellular membrane trafficking.