Dopa and Cysteine Incorporation into Premelanosomes: Effects of Cycloheximide and Gene Substitution

Electron microscopic autoradiography was used to investigate the relationship between melanin formation and thiol incorporation in premelanosomes of genetically defined chick embryos. 3H-dopa was used as the indicator of melanogenic capacity, 35S-cysteine as a representative thiol compound, and 3H-leucine for control purposes since the latter is not specifically incorporated into melanoprotein. 3H-dopa was selectively incorporated into wild type (e+ / e+) melanocytes and was localized in melanogenic organelles as determined by comparison with 3H-leucine incorporation, 35S-cysteine also showed specificity for premelanosomes when compared to 3H-leucine, but its incorporation was not as great as that of 3H-dopa. Cycloheximide reduced the mean number of 35S-cysteine grains per μ2 of premelanosome although specificity was maintained for premelanosomes, probably due to non-peptide bond formation between cysteine molecules and melanin intermediates. Genetic substitution showed that both 3H-dopa and 35S-cysteine were incorporated in conjunction with premelanosome production. When premelanosome synthesis was high (E/E tissue), both dopa and cysteine incorporation were high. When premelanosome synthesis was low (ey/ey tissue), both dopa and cysteine incorporation were low. Of the cysteine molecules incorporated into premelanosomes, some reacted with melanin intermediates as “free” cysteines, while some became incorporated into the premelanosomal proteins. These autoradiographic results support several previous reports which suggest that thiol-bearing premelanosomal polypeptides provide attachment sites for melanin intermediates through thioether bonds. These thiol polypeptides may be the mechanism by which melanin is securely linked to premelanosomes.