Melanosomal pH, pink locus protein and their roles in melanogenesis.

To the editor: We read with interest the article byPuri et al (2000) that reports aberrant pH of melanosomes in pink-eyed dilution (p) mutant mouse melanocytes. Although it is recognized that the product of pink-eyed dilution locus (P protein) is important for melanogenesis its specific function has not yet been identified. In their paper Puri et al propose that the P-protein is involved in acidification of melanosomal pH and that this acidic pH is essential for melanogenesis. This proposal is based on the finding of a reduced localization of the melanogenic enzyme TRP-1 in acidic organelles. Whilst this could suggest that p-mutant mouse melanocytes have fewer acidic melanosomes there are other interpretations. For instance, it could be argued that in these melanocytes there is a misrouting of TRP-1 protein. It would indeed be a unique finding if, as Puri et al suggest, the p-locus product is an ionic transporter involved in acidification of melanosomes. If this was the case then it is difficult to explain how the P-protein, which does not utilize energy from ATP, could function against a proton gradient of up to 1000-fold (pH 7.2 in cytosol versus pH < 4 in melanosomes); however, this hypothesis can be easily tested. If, as postulated by the authors, melanogenesis only occurs in acidic melanosomes then it follows that neutralization of intracellular acidic organelles should lead to a reduction of melanogenesis in wild-type cells. The same change in p null mouse melanocytes should result in little if any decrease in melanogenesis as most melanosomes are already at a neutral pH. There are several well-characterized substances available that neutralize intracellular acidic organelles that could be used to test this hypothesis. These include the selective vacuolar type proton pump inhibitors, bafilomycinA1 (BafA1) and concanomycinA (ConA), the H+/Na + exchanger monensin, and ammonium chloride (NH4Cl). Although Puri et al used monensin in their experiments to demonstrate the specificity of their method to visualize acidic organelles, they made no mention of its effects on melanogenesis. This was surprising since there are reports that treatment of mouse melanoma cells with monensin or NH4Cl will cause immediate and large increases in melanogenesis (Saeki and Oikawa, 1985;Fuller et al, 1993). These findings are consistent with the view that mammalian tyrosinase has optimal conditions at neutral pH (Hearing and Ekel, 1976;Townsend et al, 1984) but contrasts with the hypothesis that acidic conditions favor melanogenesis (Devi et al, 1987). In fact we also have observations that support the view that melanin production is optimal at near neutral pH. We have reported that the proton pump inhibitors, BafA1 and ConA, induced melanogenesis in tyrosinase positive but amelanotic human and mouse melanoma cells (Ancans and Thody, 2000) and our observations have recently been confirmed (Fuller et al, 2001). Interestingly, one of the melanoma cell lines (FM3) used in our study failed to express mRNA for the P-protein. Melanogenesis was also restored in response to monensin and NH4Cl and in all cultures this correlated with neutralization of acidic organelle pH. We carried out similar experiments using the mouse melanocyte line Melan-p1 (pcp/p25H) as well as wild-type P locus cell lines Melan-a and Melan-b (kindly provided by Dr. D. Bennett). Neutralization of acidic organelles resulted in a 4–5-fold increase in the melanin content of Melan-p1 cells while there was no significant change with the Melan-a cells (wild type). Melan-b cells that have wild-type P locus but reduced melanogenesis, as a result of mutated b locus, responded with a slight increase in melanin content Figure 1. These results were not affected by the protein synthesis inhibitor cycloheximide suggesting an activation of pre-existing tyrosinase. Our findings are consistent with the idea that P protein has a role in regulation of melanosomal pH but argue against the hypothesis of acidification. We would suggest the alternative hypothesis that the P-locus protein has a role in the neutralization of melanosomal pH and that this change facilitates tyrosinase activity. P-locus protein is homologous to the E. coli Na+/H + antiporter and in the light of our findings it is reasonable to propose that the P-protein functions as a channel that acts to reduce the proton concentration inside the melanosome (as these organelles are related to lysosomes). The observation made by Puri et al that both p null melanocyte lines had a 25% increase in the total number of acidic vesicles would be in keeping with this. Finally, our hypothesis does not contradict the fact that melanosomes can be acidic; however, we would propose that stages of melanin formation that require tyrosinase enzymatic activity will proceed optimally at near neutral pH and that it is the p-locus product that functions to provide these conditions. It is possible that the P-protein creates a neutral local microenvironment and this is important in maintaining the high molecular weight melanogenic complex of tyrosinase, TRP-1 and -2. This would explain the absence of this complex in p-locus mutated mouse cells (Lamoreux et al, 1995).