We previously reported that treatment of B16 melanotic
melanoma cells with
reduced glutathione (GSH) converts them to amelanotic cells without any significant down-regulation of
tyrosinase activity. To characterize the cellular mechanism(s) involved, we determined the intracellular distribution of melanocyte-specific
proteins, especially in
melanin synthesis-specific organelles, termed melanosomes by subcellular fractionation followed by Western blotting and confocal
laser microscopy (CFLM). In the melanosome-rich large granule fraction and in highly purified melanosome fractions, while GSH-induced amelanotic B16 cells have significantly diminished levels of
protein/activity of
tyrosinase and
tyrosinase-related protein-1 compared with control melanized B16 cells, there was substantially no difference in the distribution and levels of
dopachrome tautomerase and the processed
isoform of Pmel17 (HMB45) between control melanized and GSH-induced amelanotic B16 cells. Analysis of merged images obtained by CFLM revealed that whereas
tyrosinase, Pmel17 and
dopachrome tautomerase colocalize with each other in the control melanized B16 cells,
tyrosinase does not colocalize with Pmel17 or its processed
isoform and with
dopachrome tautomerase in GSH-induced amelanotic B16 cells. The sum of these findings suggests that
reduced glutathione selectively disrupts the intracellular trafficking of
tyrosinase and
tyrosinase-related protein-1 but not
dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization, probably serving as a putative model for
oculocutaneous albinism type 4.