A model for melanosome biogenesis based on the purification and analysis of early melanosomes

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 mela noma cells after disruption and initial separation by sucrose density gradi ent centrifugation. Low-density sucrose fractions were found by electron mi croscopy to be enriched in stage I and stage II melanosomes, and these frac tions were further separated and purified by free flow electrophoresis. Tyr osinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) pr oteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cle aved and inactivated in stage I melanosomes. Proteolytic cleavage was criti cal for the refolding of GP100 within the melanosomal milieu, and subsequen t reorganization of amorphous stage I melanosomes into fibrillar, ovoid, an d highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features see n during melanosome biogenesis, and they yield further clues as to the phys iological regulation of pigmentation.