Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells

Melanosomes and premelanosomes are lysosome-related organelles with a uniqu e structure and cohort of resident proteins. We have positioned these organ elles relative to endosomes and lysosomes in pigmented melanoma cells and m elanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separ ate vesicular structures that were distinct from those enriched in lysosoma l proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most e nriched along the intralumenal striations of premelanosomes, Increased pigm entation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosom al compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of f luid phase uptake and immunogold labeling, premelanosomal proteins segregat ed from endocytic markers within an unusual endosomal compartment. This com partment contained Pmel17, was accessed by BSA-gold after 15 min, was acidi c, and displayed a cytoplasmic planar coat that contained clathrin. Our res ults indicate that premelanosomes and melanosomes represent a distinct line age of organelles, separable from conventional endosomes and lysosomes with in pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanos omes and lysosomes are sorted.