Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes

In past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human gen es are expressed in melanocytes and encode proteins predicted to have 12 me mbrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosom al membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanos omal pH. Melanosomes (and their precursor compartments) were defined by ant iserum directed against the melanosomal marker tyrosinase related protein 1 . Acidic vesicles were identified by 3-(2,4-dinitroanilino)-3'-amino-N-meth yldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL /6(+/+) (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalizat ion of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3'-amino-N-m ethyldipropylamine, indicating that almost all melanosomes or their precurs ors were acidic. By contrast, only 7%-8% of the staining vesicles in p muta nt cell lines (p(J)/p(J) and p(cp)/p(6H)) showed colocalization of tyrosina se related protein 1 and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylam ine. Thus, without a functional p protein, most melanosomes and their precu rsors are not acidic. As mammalian tyrosinase activity in situ is apparentl y dependent on low pH, we postulate that in the absence of a low pH environ ment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melani n that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex.