Melanosomes are morphologically unique lysosome-like organelles in which melanin pigments are made and stored within melanocytes and retinal pigment epithelial cells. Melanosome precursors, premelanosomes, are characterized by intralumenal striations upon which melanin is subsequently deposited. These striated structures likely facilitate melanin sequestration, storage, transfer to keratinocytes, and perhaps detoxification of melanin intermediates and retinal metabolites. The mechanisms leading to striation formation are not known, but are likely similar to those involved in the biogenesis of other lysosome- like organelles and similarly affected by diseases such as Hermansky-Pudlak and Chediak-Higashi syndromes. Pmel17 is a constituent of the premelanosomal striations, and our preliminary evidence suggests that expression of Pmel17 alone in non- melanocytic cells results in the formation of striation-like structures within multivesicular endosomes. It is not known how Pmel17 induces formation of these striations, or whether Pmel17 is necessary for striation formation under physiological conditions. Furthermore, the poor fidelity of striation generation in non-melanocytic cells expressing Pmel17 suggests that other factors contribute to the efficiency with which these structures are formed in melanocytic cells. We hypothesize that Pmel17 is essential for the formation of premelanosomal striations, that features of Pmel17 primary structure, proteolytic processing and association with intralumenal membranes contribute to their formation, and that additional premelanosomal components improve the efficiency with which striations are formed.
The Specific Aims are designed to address these hypotheses, and to gain insight into how striation formation can be regulated and affected by genetic diseases. Specifically, we will: 1. Determine how a natural Pmel17 defect affects the biogenesis of melanosomes. 2. Determine the role of proteolytic processing and other primary structural features of Pmel17 in striation formation. 3. Determine the molecular form of Pmel17 found within striation- containing premelanosomes. 4. Determine whether additional premelanosomal components contribute to striation formation.
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