OCA2 function and Tyr trafficking

What do my results imply about OCA2 function? In OCA2-deficient cells, an unusually high proportion of Tyr is sensitive to endoglycosidase H at steady state (Chen et al., 2002; Toyofuku et al., 2002; Chen et al., 2004). This has been interpreted as a defect in Tyr maturation and ER exit. Moreover, Tyr is abnormally cleaved and secreted from these cells, and is also found in small vesicles distributed throughout the cytoplasm (Manga et al., 2001; Manga and Orlow, 2001; Chen et al., 2002; Toyofuku et al., 2002;

Chen et al., 2004; Ni-Komatsu and Orlow, 2006). How does one explain these findings

in the context of our model?

4.4.1 OCA2 as a pH regulator

Sequence homology places OCA2 in the ArsB/NhaD family of transporters. The NhaD founder is a Na+/H+ exchanger, and since OCA2 is thought to regulate pH, some researchers have proposed that OCA2 might also be a proton transporter (Lee et al., 1995; Ancans et al., 2001a), though this hypothesis is disputed (Brilliant and Gardner, 2001; Brilliant, 2001). The regulation of lumenal pH appears to be important for melanin synthesis. Fluorescent assays show a higher steady-state pH in Black melanocytes than in Caucasian melanocytes (Fuller et al., 2001). Treatment of Caucasian melanocytes with chemicals that raise organellar pH stimulates melanin synthesis in these cells; a similar

induction in melanin synthesis is seen in OCA2-deficient melanocytes receiving the same treatments (Ancans et al., 2001a; Ancans et al., 2001b; Fuller et al., 2001; Manga and Orlow, 2001; Chen et al., 2002; Chen et al., 2004; Ni-Komatsu and Orlow, 2006). NHE activity has been demonstrated to be a determinant of the difference in melanin synthesis activity between Caucasian and Black melanocytes (Smith et al., 2004), and population genetics analyses consistently demonstrate strong linkage between OCA2 polymorphisms and racial differences in skin color (Shriver et al., 2003; Lao et al., 2007; Norton et al., 2007). However, the NHE activity that was seen to be a factor in the activity in the different melanocytes was sensitive to derivatives of the fairly promiscuous NHE inhibitor amiloride (Sarangarajan et al., 2001; Smith et al., 2004), and researchers noted that the amino acid sequence that confers EIPA sensitivity on an NHE is not present within OCA2 (Smith et al., 2004), making it unlikely to be the critical NHE. My unpublished results also suggest that OCA2 does not directly act as an NHE.

Heterologous expression of a plasma membrane-localized mutant of OCA2 (lacking active LL1 and LL2) in NHE-deficient fibroblasts was not sufficient to rescue these cells from death following intracellular acid loading, while cells expressing a bona fide NHE protein survived. pH-sensitive live-cell fluorescence assays gave similar results, showing that the same OCA2-expressing fibroblasts could not recover their pH after short-term acid loading in a flow chamber apparatus. These experiments do not rule out pH regulation by OCA2 via some other mechanism within melanocytes.

The proposed role of OCA2 as a pH regulator may also explain the appearance of the aberrant cytoplasmic Tyr vesicles seen in OCA2-deficient melanocytes. The origin of

these vesicles is not completely clear. Tyrosinase has been visualized in AP-3 and AP-1 coated endosomal buds near melanosomes in wild-type cells (Theos et al., 2005). If OCA2 acting within the endosomes/melanosomes is required to promote correct targeting of endosomally-derived Tyr-containing vesicles, then OCA2 deficiency could lead to the accumulation of vesicles that bud appropriately but are unable to fuse with their target compartments. The fact that the vesicles detected in OCA2-deficient cells are not

pigmented but stain positively for Tyr activity by classical DOPA histochemistry (Manga et al., 2001) indicates that the Tyr in these vesicles is inactive in vivo but likely already loaded with copper (if it were not copper loaded, excess copper would be needed during DOPA histochemistry; (Setty et al., 2008)). This might reflect a failure to strip copper in endosomes (Setty et al., 2008) in OCA2-deficient cells, such that copper remains bound in endosome-derived transport vesicles. Failure to strip copper in endosomes due to loss of OCA2-mediated pH regulation is consistent with the appearance of some induced melanin deposition in endosome-like structures in OCA2-deficient melanocytes that have been treated with the vATPase inhibitor bafilomycin A1 (Chen et al., 2004), though the majority of the induced activity was correctly localized to melanosomes.

Alternatively, the accumulated Tyr vesicles could originate from the TGN instead of endosomes. In this case, OCA2 might be predicted to function at the endosome to facilitate targeting and/or fusion of the incoming Tyr vesicles. In support of the idea that OCA2-mediated pH regulation could affect TGN-derived Tyr-containing vesicle fusion at the endosome, modulation of lumenal pH has previously been proposed to play a role in membrane fusion at the yeast vacuole (Ungermann et al., 1999), and mutations in the

endosomal Ca2+ transporter MCOLN3 affect endosomal acidification as well as the rate of homotypic endosomal fusion in vitro (Lelouvier and Puertollano, 2011). Interestingly, a gain-of-function mutation in MCOLN3 gives rise to the varitint-waddler mouse, which exhibits hypopigmentation among other phenotypes (Di Palma et al., 2002).

4.4.2 Could OCA2 act elsewhere?

Other researchers have suggested that OCA2 might facilitate transmembrane glutathione transport, since heterologous expression in yeast leads to localization of the transgene to the vacuolar membrane and a correlated reduction in cellular glutathione levels due to degradation following import into the vacuolar lumen (Staleva et al., 2002). Because the Tyr glycosylation and cleavage/secretion defects seen in OCA2-deficient melanocytes reflect problems at relatively early steps in Tyr biogenesis, Chen, Manga and Orlow have proposed that OCA2 acts within the endoplasmic reticulum to promote Tyr maturation (Chen et al., 2002). Tyr contains several disulfide bonds, and its maturation might thus be particularly sensitive to changes in the redox state of the ER (Wang and Hebert, 2006). If OCA2 were an ER-localized glutathione transporter for example, it could greatly affect Tyr biosynthesis. My results from rescue assays performed with ER-localized OCA2 mutants show that OCA2 must traffic to a post-Golgi compartment for melanin synthesis to take place. I speculate that substrate gradients generated by OCA2 in distal endosomal compartments might be transduced back to the ER and correct the early defects in Tyr maturation. However, another untested possibility is that OCA2 actually functions in both the ER and the endosome/melanosome. In the ER it could act to promote Tyr folding and glycosylation while in the endosome/melanosome it could facilitate fusion of Tyr-

containing vesicles or promote the lumenal conditions needed for melanin synthesis in melanosomes. Our rescue assay only examined the most distal phenotype, melanin synthesis. It would be informative to see whether ER-restricted OCA2 mutants could correct the observed differences in Tyr glycosylation, cleavage and secretion, or mislocalization to scattered cytoplasmic vesicles.