The pink-eyed dilution locus (p) plays a central role in controlling mammalian pigmentation and encodes a 12-transmembrane domain protein lacking significant homology to any other vertebrate proteins. We took advantage of the ease of manipulation of S. cerevisiae as a tool for studying the function of p protein in greater detail.
In view of the resemblance of the 12-transmembrane structure of p to that of a transporter or channel (Gardner et al., 1992
; Rinchik et al., 1993
; Rosemblat et al., 1994
) and in light of the homology of p to the ArsB family of bacterial transporters, we assessed the response of p-expressing cells to arsenical compounds. We found unexpectedly that expression of p conferred increased sensitivity to arsenical compounds. Pentavalent arsenate in yeast is reduced to trivalent arsenite by glutathione (Mukhopadhyay et al., 2000
). The arsenite is subsequently detoxified by two mechanisms: transport out of the cell by Acr3p, an arsenite permease (Wysocki et al., 1997
) and by conjugation with GSH and transport into the vacuole by Ycf1p, a 12-transmembrane pump (Szczypka et al., 1994
; Ghosh et al., 1999
). The vacuolar localization of heterologously expressed p suggested to us that if p transported arsenite into the vacuole it should confer a resistant phenotype, yet we observed an exaggerated arsenite-sensitive phenotype. A possible explanation for the sensitive phenotype conferred by p expression was the sequestration or depletion of intracellular glutathione, needed for conjugation with arsenite as a prelude to its detoxification. That possibility was further supported by finding increased sensitivity of both p-expressing yeast cells and melanocytes to other compounds known to require GSH for detoxification. In addition, we found that p-expressing yeast cells exhibit greatly diminished levels of intracellular GSH compared with control cells confirmed by 5,5′ dithiobis-(2-nitrobenzoic acid)-glutathione coupled assay and fluorometry after labeling with monochlorobimane. The involvement of p in glutathione metabolism was further confirmed by the observation that Gsh1p, the rate-limiting enzyme in glutathione biosynthesis, completely reverses the arsenical sensitivity conferred by p expression in yeast.
We also found that p-expressing yeast cells and melanocytes are more sensitive to selenate, but not to selenite. We attribute this difference to the different mechanisms of action of these two metalloids in causing cell toxicity. Because selenate is a structural analog of sulfate, its toxic effect is believed to be due to its incorporation into analogs of sulfur-containing compounds such as cysteine, methionine, and glutathione (Reuveny, 1977
). It is possible that p could differentially influence the consumption of glutathione in different cellular pools and that could result in increased cell sensitivity to selenate. On the other hand, selenite toxicity is associated with oxidative stress, which causes DNA damage in the form of single-strand breaks, chromosome breaks, and spindle disturbances in mammalian cells (Sinha et al., 1996
) and in yeast (Pinson et al., 2000
). Murine B16 melanoma cells are more sensitive to selenite but not to selenate (Siwek et al., 1994
). It has been suggested that p is not transcribed in melanoma cells (Gardner et al., 1992
), again consistent with the hypothesis that increased sensitivity of p-expressing melanocytes to selenate could be due to a function of p in glutathione transport. The reported increased sensitivity to selenite in melanoma cells compared with the lack of a difference in sensitivity in melanocytes could be due to the differential response to oxidative damage in both cells.
To gain a better understanding of how the expression of p could result in the depletion of intracellular GSH, we treated yeast cells with acivicin, a drug that blocks the enzyme γGT known to initiate the breakdown of GSH in the yeast vacuole (Chittur et al., 2001
; Mehdi et al., 2001
). After treatment of yeast with acivicin for 24 h, the GSH levels in p-expressing cells and in the control cells were the same. These data are consistent with a model in which p facilitates GSH sequestration from the cytoplasm to the vacuole, where the glutathione is subsequently degraded (Figure ). In mammalian cells, p is localized to the ER, and we ascribe the vacuolar localization of p when expressed in protease-deficient yeast cells to the well-established default pathway for heterologous membrane proteins in yeast (Conibear and Stevens, 2002
; Murray et al., 2002
). In light of this difference in distribution, it is perhaps not at all surprising that we did not observe any significant difference in total glutathione levels in wild-type melan-a cells and p-null melanocytes. The degradation of glutathione in yeast is triggered by vacuolar accumulation; there is no evidence that such a system exists in the mammalian ER. In melanocytes, p could alter the sequestration of glutathione between different subcellular pools, resulting in altered detoxification of, and increased sensitivity to arsenicals, cisplatin, and doxorubicin.
Figure 10 Model for effect of p on GSH metabolism in yeast. Glutathione normally enters the yeast vacuole through Ycf1p in the form of glutathione conjugates or as free glutathione. The expression of p triggers additional transport of glutathione into the vacuole. (more ...)
In agreement with the observation that p modulates glutathione metabolism, we observed a decrease in red pigment formation in Δade2
cells as well as in cells additionally disrupted in the YCF1
gene, involved in the transport of pigment-conjugated glutathione to the vacuole (Chaudhuri et al., 1997
). These results suggest that glutathione plays other roles in red pigment formation in yeast in addition to that of transport of pigment precursors as GSH conjugates into the vacuole.
In confirmation of the assumption that p could be involved in glutathione transport, we performed fluorescence studies with the dye monochlorobimane (Shrieve et al., 1988
). The results in Figure revealed that p-expressing Δycf1
cells, which otherwise fail to accumulate this dye in their vacuole, exhibit intense vacuolar fluorescence, but only if they are pretreated with acivicin to prevent intravacuolar GSH degradation. Together, these data suggest that p alters intracellular GSH metabolism in yeast by transporting GSH into the vacuole where as a secondary phenomenon it is subsequently degraded in a γGT-dependent manner. We cannot exclude the possibility that p may transport GSH-conjugates, even although the expression of p
in the Δycf1
strain failed to complement the arsenic- and cadmium-sensitive phenotype of that strain, as had previously been achieved with the mammalian drug transporter MRP1
, another 12-transmembrane domain protein (Tommasini et al., 1996
). A Δycf1
strain transformed with either p or pΔ exhibited the same growth in the presence of cadmium, arsenite, and arsenate compared with cells transformed with YCF1
(our unpublished data).
Thiol compounds have been suspected to play a significant role in melanin pigmentation, but the exact mechanisms have remained unknown (Benedetto et al., 1981
; Jara et al., 1988
; del Marmol et al., 1993
; Ito, 1993
). Pheomelanin is synthesized from cysteinyl-DOPA building blocks, and the p
gene is not transcribed during the pheomelanic phase of the murine agouti hair cycle (Tamate et al., 1989
; Rinchik et al., 1993
). Low levels of reduced GSH have been found to be associated with eumelanin production (Benedetto et al., 1981
). Tyrosinase activity is also important in the eumelanin/pheomelanin switch: high tyrosinase activity had been associated with eumelanogenesis and lower tyrosinase activity with pheomelanogenesis (Ito, 1993
; Lamoreux et al., 1995
). Glutathione has been reported to have two effects on tyrosinase activity: first, a direct one by inhibition of tyrosinase activity via the reduction of copper atoms at the active site of the enzyme; and second, indirectly via interference with melanin formation by redox mechanisms (Jara et al., 1988
). The effect of p on the transport or intracellular sequestration of GSH directly or indirectly might alter intraorganellar levels of GSH in such a way as to increase the formation of eumelanin.
Roles for p as an anion transporter, in the control of melanosomal pH, or in regulating the pH of other intracellular compartments have been suggested (Puri et al., 2000
; Ancans et al., 2001
; Brilliant and Gardner, 2001
; Halaban et al., 2002
). We have shown previously that inhibitors of vacuolar-type ATPases such as bafilomycin A1 and concanamycin, the ionophore monensin, and ammonium chloride all restore pigmentation in p-null cells (Manga and Orlow, 2001
). In the current studies, we did not observe an effect of p-expression upon the pH of the yeast vacuole. However, we note that involvement of p in intracellular GSH metabolism might alter intracellular pH indirectly by redox mechanisms or directly by regulating the activity of V-ATPases as has been shown previously (Oluwatosin and Kane, 1997
). On the other hand, evidence exists to suggest that certain V-ATPases can themselves transport glutathione, because it has been shown that bafilomycin A1 decreases the transport of GSH in isolated yeast vacuoles (Mehdi et al., 2001
Glutathione is also known to be required for the folding of cysteine-rich proteins (Cuozzo and Kaiser, 1999
). Tyrosinase passage through the ER is much slower than for the related protein Tyrp1, suggesting that tyrosinase ER folding and processing are highly regulated (Branza-Nichita et al., 1999
). Tyrosinase as well as the other members of the tyrosinase-related protein family have 15 cysteine residues in well-conserved positions along the polypeptide chain (Hearing and King, 1993
; Spritz and Hearing, 1994
; King et al., 1995
) and may be especially vulnerable to oxidative damage. Data exist that folding and maturation of tyrosinase-related protein-1 are also regulated by formation of disulfide bonds (Negroiu et al., 2000
). Because glutathione is a major redox buffer in the secretory pathway (Hwang et al., 1992
; Frand et al., 2000
) it may play an important role in the proper folding and processing of tyrosinase. Recently, we have shown that abnormal ER processing of tyrosinase, followed by aberrant trafficking and release of the tyrosinase into the medium, occurs in the absence of p (Chen et al., 2002
). Together with our recent observations that in melanocytes the majority of p is found in the ER (Chen et al., 2002
), the current data suggest a possible role for p in regulating the folding of tyrosinase via control of glutathione.
Finally, our results have implications for chemotherapeutic approaches to melanoma. Melanoma cells are recognized to be resistant to many chemotherapeutic agents, including those known to be detoxified by glutathione-dependent mechanisms. We have found that resistance to chemotherapeutic agents such as arsenic trioxide (Dai et al., 1999
; Yang et al., 1999
; Zhang et al., 2001
) cisplatin (Eton et al., 2002
; Gibbs et al., 2002
), and doxorubicin (Cho et al., 2001
) is modulated by p expression. Agents that mimic p function in the cell might well increase the sensitivity of melanoma to useful chemotherapeutic agents.