Melanosomes are lysosome-related organelles of ocular and epidermal pigment cells within which melanins are synthesized and stored. Melanosomes are segregated from and coexist with endocytic organelles, and develop through four distinct morphological stages, each with characteristic contents. Eumelanins are deposited along intralumenal fibrils that begin to form in multivesicular endosomes that overlap with stage I melanosomes, and mature to a highly organized matrix within stage II melanosomes. A main constituent of the fibrils is the pigment cell-specific protein Pmel17;we have shown that Pmel17 expression in non-pigment cells generates fibrillar structures in multivesicular endosomes, and that a recombinant PmeU 7 fragment forms fibrils in vitro with structural hallmarks of amyloid. Mutations in Pmel17 disrupt fibril formation and affect melanosome morphology, melanocyte viability, and consequent pigmentation. Thus PmeU7 fibril formation is critical for pigmentation and is a physiological model system for the development of amyloid associated with pathologies such as Alzheimer's and prion diseases. We have shown that fibril formation requires PmeU 7 transport to internal membranes of multivesicular endosomes, but how these membranes or structural features of PmeU7 regulate fibril formation is not known. In this proposal, we will dissect the structural hallmarks of PmeU 7 that mark the transition from a non-aggregated integral membrane protein to an amyloid like structure and define the role of endosomal membranes in facilitating fibril formation. We hypothesize that unique structural features of several PmeU 7 domains regulate compartmentalization and fibril formation directly through membrane interactions, and that endosomal intermediates participate directly in fibril formation. We will test these hypotheses in the following specific aims. 1. To test whether the transmembrane domain influences PmeU 7 sorting to multivesicular endosomes. 2. To test whether structural features unique to the PmeU 7 PKD domain regulate sorting and fibril formation. 3. To test whether the lipid components of multivesicular stage I melanosomes contribute to fibril formation by directly binding to PmeU 7. PmeU 7 forms amyloid-like fibers within pigment cells. By defining the mechanism, we may uncover potential cures for albinism, hyperpigmented lesions, and/or neurodegenerative amyloid diseases like Alzheimer's.
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