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 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and 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 seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.
Kushimoto
T
T
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Basrur
V
V
Valencia
J
J
Matsunaga
J
J
Vieira
W D
WD
Ferrans
V J
VJ
Muller
J
J
Appella
E
E
Hearing
V J
VJ
D004586:Electrophoresis / Q000379:methods; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000032:analysis; D020460:Melanosomes / Q000737:chemistry* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000032:analysis; D018613:Microscopy, Confocal / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods; D014442:Monophenol Monooxygenase / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis*; D010088:Oxidoreductases; D011506:Proteins / Q000032:analysis; D014407:Tumor Cells, Cultured; D058951:gp100 Melanoma Antigen
D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D011506:Proteins; D058951:gp100 Melanoma Antigen; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase