Pigmentation of human skin is the result of a complex process of pigment synthesis by epidermal melanocytes and transport of pigment granules to keratinocytes. Melanin pigment is synthesized within melanosomes. Melanosomes are specialized, cell-type specific organelles present in melanocytes. Biogenesis of melanosome presumably requires sorting and intracellular transport of melanosomal proteins to appropriate precursor structures. Biochemical studies on the biogenesis of melanosome have not been possible due to a lack of available molecular probes to melanosomal proteins. The most well characterized melanosomal membrane proteins are tyrosinase and a 75,000 dalton glycoprotein, gp75. Tyrosinase catalyzes the initial steps in melanin synthesis. Genes encoding mouse and human tyrosinase have been isolated and mapped to mouse c (albino) locus. Expression of cDNA encoding human tyrosinase in mouse fibroblasts leads to pigment synthesis by fibroblasts. In human pigmented melanocytic cells gp75 is the most abundant glycoprotein. We have purified gp75 from human melanoma cells, cloned the CDNA for gp75 and expressed in mouse fibroblasts. Nucleotide sequence analysis showed that the gp75 gene is the human homologue (90% amino acid sequence identity) of a mouse gene that maps to the b (brown) locus, and its a member of a family of (tyrosinase-related) proteins that are highly conserved between mouse and human. Brown locus in mouse determines coat color by influencing the type of melanin produced. Thus, the expression, intracellular sorting, and transport of tyrosinase and gp75 to melanosomal precursors are critical steps in melanosome biogenesis and skin pigmentation.
The aim of the present proposal is to devise, utilizing gp75 and tyrosinase as markers, cellular and molecular strategies to understand the biogenesis of melanosomes. Specifically, the objectives of the present proposal are to: 1. Identify intracellular compartments through which gp75 and tyrosinase are transported to melanosomes, by using subcellular fractionation and immunoelectron microscopic methods. 2. Investigate the role of Asn-linked oligosaccharides in transport of gp75 and tyrosinase to melanosomes. 3. Express gp75 and tyrosinase genes in tyrosinase and gp75 negative human melanoma cells by transfection with expression vectors containing the complete coding sequences of tyrosinase and gp75. 4. Identify the signals necessary for transport of gp75 and tyrosinase to melanosomes by deletion and site-directed mutagenesis.
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