Pmel17: controversial indeed but critical to melanocyte function

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Dear Sir: We read with interest the timely review of Theos et al. (2005b) regarding the intriguing protein termed Pmel17. The gene encoding that protein is officially known as SILV (as determined by the HUGO Gene Nomenclature Committee) and the protein product is officially known as Pmel17. We think most scientists involved with research on Pmel17 have their own perspectives of its role(s) in melanocytes and its putative catalytic, structural and immune functions, but all can agree that it is a critical component of melanogenesis. Without its correct processing and delivery, the transition of premelanosomes from stage I to II simply will not occur, which adversely impacts melanin formation and which has long-term implications to melanocytes with respect to toxicity and survival, as originally reported by Quevedo et al. (1981). We point out several considerations for readers to keep in mind about Pmel17 when comparing the literature: Consistency of findings – virtually all data reported from various groups (Adema, Hearing, Jimbow, Marks, Orlow, Pawelek, Raposo, Solano, etc.) are in complete accord, although the interpretation of those results does not always take the same direction. Differences between processing in murine and human melanocytes – the trafficking of Pmel17 has become a controversial issue. The initial theory for the direct sorting of Pmel17 from the endoplasmic reticulum (ER) to early melanosomes was based on studies of murine melanocytes where Pmel17 reaches a maximum size of c. 87 kDa, is fully EndoH-sensitive (Kobayashi et al., 1994; Orlow et al., 1993) and is not detected in the coated vesicle fraction (where TYR, TYRP1 and DCT are found). In contrast, Pmel17 has been reported to sort only to early melanosomes through the endosomal system (Berson et al., 2001; Marks and Seabra, 2001), but this route does not explain three critical observations: (i) two different glycoforms of Pmel17 with different glycan compositions have been identified, one of them in the cytoplasm and characterized by an abundance of high-mannose sugars (Maresh et al., 1994); (ii) the maturation of Pmel17 begins as a precursor form of c. 90 kDa, after which some is further glycosylated to a mature form of >100 kDa and is subsequently cleaved into two subunits of c. 85 and 26 kDa in melanocytic cells. The full glycosylation of Pmel17 occurs to a minor extent in human melanocytes, and even less in murine melanocytes (Berson et al., 2001; Kobayashi et al., 1994; Martinez-Esparza et al., 2000); and (iii) the proposal for sorting of Pmel17 only through the endosomal system and not the ER is mostly based on indirect evidence. It rationalizes that the partially glycosylated form of Pmel17 (labeled P1 in Figure 3A of Berson et al., 2001), which is fully sensitive to EndoH and is immunoprecipitated by HMB-50 even after a 4-h chase, is a 'slow-processed' form in the ER, despite the fact that their immuno-electron microscopy (EM) (Raposo et al., 2001) and immunofluorescence (Berson et al., 2001) results show that HMB-50 reactivity is neither detected in the ER nor does it have an ER pattern. Obviously, further work will be necessary to further resolve these issues. Differences between processing in melanocytes and non-melanocytes – studies dealing with models assessing the processing of melanosomal proteins (including Pmel17) in non-melanocytic cells (e.g. using transfection) need to be taken with caution as the presence of putative melanocyte-specific chaperones or chaperone-like accessory factors, can markedly affect the post-translational processing, sorting and distribution of those proteins. As one example, the sorting of Pmel17 transfected into non-melanocytic cells depends on the level of its expression: HeLa cells expressing low levels of transfected Pmel17 sort that exogenous protein via intraluminal vesicles to lysosomes en route for degradation, but overexpression of the alien protein results in its accumulation in multivesicular bodies (MVB). One should remember that the role of MVB in epithelial cells is to sequester and destroy foreign materials. Promiscuity in intracellular trafficking – although one tends to think of the processing and trafficking of proteins (including melanocyte-specific proteins) as following simple routes, the sorting of many cellular proteins can follow very diverse routes. As an example, consider tyrosinase, a relatively simple case. Tyrosinase actually has two distinct sorting signals (Simmen et al., 1999) and its trafficking depends on AP3 to a major extent (Huizing et al., 2001). Mutations in AP3 lead to the mis-sorting of tyrosinase and is associated with at least one form of Hermansky–Pudlak syndrome. However, recent evidence has shown that AP1 also plays an independent role in tyrosinase sorting through a distinct trafficking pathway (Theos et al., 2005a). Our recent study shows that Pmel17 also takes advantage of various AP proteins and takes various routes to reach early melanosomes (Valencia et al., 2006). Melanocyte-specific chaperones and modifiers – when tyrosinase is expressed via transfection of non-melanocytic cells, it can be processed, folded and sorted to post-Golgi organelles that can even become pigmented (Bouchard et al., 1989). However, in melanocytes, various proteins (such as P, TYRP1, MATP and even Pmel17) also affect the trafficking of tyrosinase to melanosomes and the dramatic effects of P, TYRP1 or MATP dysfunction can be evidenced in the dramatic alterations of pigmentation in OCA2, OCA3 and OCA4 (Costin et al., 2003; Solano et al., 2000; Toyofuku et al., 2001, 2002). In a similar manner, Pmel17 requires MART-1 for its correct processing (Hoashi et al., 2005) and MART-1 would not normally be present in non-melanocytic cells. We would leave open the question of whether other unknown factors play a physiological role in the trafficking of Pmel17. We think that consideration of the above constraints explains many of the apparent discrepancies in the literature. In human melanocytic cells, Pmel17 reaches the cis-Golgi compartment and from there only about 5–20% of the precursor protein is fully glycosylated, while the rest remains partially glycosylated and is EndoH-sensitive (Yasumoto et al., 2004). This result can also be noted in Figures published by the Marks' group (e.g. as shown in Figure 3 of Berson et al., 2001). In a recent study (Valencia et al., 2006), we show that Pmel17 uses several different APs and sorting routes to reach melanosomes. In normal melanocytes, which express the AP1B subunit found in polarized cells, Pmel17 uses both the secretory and endocytic pathways to reach stage I melanosomes. Various pigmentary diseases (such as Hermansky–Pudlak syndrome) affect the biogenesis and function of lysosome-related organelles, a family that includes lysosomes, melanosomes, platelet-dense bodies and synaptic vesicles. Stage IV melanosomes purified to homogeneity (relatively easy because of their density, Kushimoto et al., 2001) have significant levels of lysosomal enzymes (Orlow, 1995) as well as proteins normally considered as ER proteins, etc. (Basrur et al., 2003). A tempting assumption when considering subcellular fractionation is to assume that the presence of proteins associated with other organelles indicates contamination. However, many proteins are known as markers for the ER, Golgi, endosomes, etc., simply because they are abundant there and typically were first identified in those organelles. There is not yet a proteomics study published that has described an organelle-specific complement of proteins that are sequestered from other cellular components. The presence of ER markers in stage II melanosomes (as criticized by Theos et al., 2005b) is not a contamination in our view, but simply reflects the presence of some ER-associated proteins in the repertoire of melanosomal proteins. In fact, the detection of EEA1 (a marker of coated endosomes) along with Pmel17 in stage I melanosomes is another relevant example (Raposo et al., 2001). These data confirm that melanosomes are active organelles in constant exchange with the surrounding milieu (Orlow et al., 1992). In sum, great care needs to be taken in the interpretation of existing studies on the processing and sorting of melanosomal proteins as the type of cells used, the sorting machinery functional in those cells and various tissue-specific chaperones and sorting factors play critically important roles in determining how those processes take place.