Pink-eyed Dilution Protein Modulates Arsenic Sensitivity and Intracellular Glutathione Metabolism

Abstract

Mutations in the mouse p (pink-eyed dilution) and human P genes lead to melanosomal defects and ocular developmental abnormalities. Despite the critical role played by the p gene product in controlling tyrosinase processing and melanosome biogenesis, its precise biological function is still not defined. We have expressed p heterologously in the yeast Saccharomyces cerevisiae to study its function in greater detail. Immunofluorescence studies revealed that p reaches the yeast vacuolar membrane via the prevacuolar compartment. Yeast cells expressing p exhibited increased sensitivity to a number of toxic compounds, including arsenicals. Similarly, cultured murine melanocytes expressing a functional p gene were also found to be more sensitive to arsenical compounds compared with p-null cell lines. Intracellular glutathione, known to play a role in detoxification of arsenicals, was diminished by 50% in p-expressing yeast. By using the glutathione-conjugating dye monochlorobimane, in combination with acivicin, an inhibitor of vacuolar gamma-glutamyl cysteine transpeptidase, involved in the breakdown of glutathione, we found that p facilitates the vacuolar accumulation of glutathione. Our data demonstrate that the pink-eyed dilution protein increases cellular sensitivity to arsenicals and other metalloids and can modulate intracellular glutathione metabolism.

Mouse coat color mutations: From fancy mice to functional genomics

“…The exact function of the P protein is enigmatic, and the idea that it regulates melanosomal pH is inconsistent with the results of Toyofuku et al (2002) and Chen et al (2002), who showed that P protein is involved in processing and sorting of tyrosinase, which is retained in the endoplasmic reticulum in mutant melanocytes. It is also inconsistent with the observation of Staleva et al (2002), who showed that the P protein may be involved in glutathione metabolism. Regardless of the exact role of the P protein in melanocytes, it is clear that the melanosome membrane plays an important role in melanosome biology.…”

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

“…For example, alterations in intralumenal ion balance or pH can affect cellular membrane fusion events (Peters and Mayer, 1998;Ungermann et al, 1999;Pryor et al, 2000), perhaps explaining the accumulation of melanosomal cargo in vesicular structures in OCA2-deficient melanocytes (Manga et al, 2001). Moreover, disruption of OCA2 transport activity across the melanosomal membrane may indirectly alter ion concentrations or pH in the cytosol; for example, exogenous expression of OCA2 in Saccharomyces cerevisiae led to a depletion of cytoplasmic glutathione due to glutathione transport into the vacuole (Staleva et al, 2002). Alterations in substrate levels could in turn have downstream effects on ion transport between the cytosol and other compartments, such as the ER.…”

Allele variations in the OCA2 gene (pink-eyed-dilution locus) are associated with genetic susceptibility to melanoma

“…This is supported by the fact that the pink-eyed dilution protein increases cellular sensitivity to arsenicals and other metalloids by modulating the intracellular glutathione metabolism that is involved in the detoxification of reactive free radicals of oxygen. 11 Loss-of-function OCA2 mutant melanocytes The P-value corresponds to the removal of one risk factor.…”

“…6 -8 Tyrosinase-positive oculocutaneous albinism (OCA, type II), the most prevalent type of albinism, 9 is an inherited autosomal recessive disorder due to germline mutations of the P gene (occuloalbinism 2 (OCA2)). OCA2 maps to chromosome 15q11.2 -12, consists of 25 exons, encoding a vacuolar 838-amino-acid protein 10,11 that is the human homologue of the mouse pink-eyed dilution locus, p.…”