- The long-term goal of this proposed research is to elucidate the molecular basis of cutaneous melanization in order to understand the pathogenesis of human diseases affecting pigmentation and to devise treatments for them. By combining techniques of cellular and molecular biology with the power of mouse genetics, the basic mechanisms by which a melanocyte creates its specialized organelle, the melanosome, will be addressed through three specific aims over the next 5 years: (1) Elucidate the biogenesis of melanosomes. The distribution of known and newly identified melanosomal proteins will be examined in relation to other subcellular proteins. The hypothesis that melanosomes are constructed via a bipartite pathway wherein smooth ER-derived premelanosomes fuse with Golgi-derived vesicles will be tested. Immunomicroscopy, subcellular fractionation, metabolic labeling, and transfection will be used to assess how individual proteins arrive at melanosomes. By combining these techniques with the treatment of melanocytes with Agouti signal protein, which shifts production from the eu- to the pheo-melanosome, insight it is hoped will be gained into the fundamental differences between these two types of melanosomes; (2) explore the relationship between melanosomes and """"""""secretory lysosomes."""""""" The melanosome is a member of the lysosomal lineage of organelles which most closely resembles the """"""""secretory lysosome."""""""" Whether melanosomes have subsumed all lysosomal function in melanocytes will be investigated. The potential association of the AP-3 adaptor complex with melanosomes will be determined, as will its interaction with the cytosolic tails of melanosomal membrane proteins; (3) role of the buff (bf) locus and 65 kDa melanosomal protein. A 65 kDa peripheral membrane protein in melanocytes and in cells containing """"""""secretory lysosomes"""""""" has been identified which localizes to the cytosolic face of melanosomes. Levels of this protein are altered by mutation at the buff (bf) and beige (bg) loci, both known to affect the secretory lysosomal pathway. The protein will be sequenced and its relationship to the bg gene product determined. The cDNA encoding the protein will be cloned and, if it is not a splice form of bg, its chromosomal location will be mapped in the mouse. If it maps to bf or to another coat color locus, mutational analysis will be used to determine if it is encoded at that locus. The protein's synthesis, distribution, and its interactions with other proteins will be studied to elucidate its function.
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