We show here for the first time that Rab38 and Rab32 act in a functionally redundant way in regulating skin melanocyte pigmentation. These Rab proteins control an important post-Golgi step in the trafficking of key melanogenic enzymes and are therefore critical for melanosome maturation.
The identification of the cht
mutation as Rab38G19V
implicated Rab38 in the regulation of pigment biogenesis (Loftus et al., 2002
), but the effect of this mutation on Rab38 function remained unclear. Although not an invariant residue within the Rab family, glycine 19 is conserved in >50% of Rabs and is situated within the generally highly conserved GTP binding pocket. The corresponding Rab5 mutant, Rab5A30V
, showed increased activity (Li and Liang, 2001
). The biochemical properties of Rab38G19V
, however, are not compatible with a functionally active Rab, and we therefore regard the cht
mouse as equivalent to a Rab38-null mutant.
In comparison with other mouse pigmentation mutants (Li et al., 2004
), the cht
phenotype is very mild. The similarity in coat color, and reduced Tyrp1 levels in cht
melanosomes, led to the suggestion that the cht
mouse may be a phenocopy of the brown
mouse, with the cht
phenotype arising from a defect in Tyrp1 trafficking (Loftus et al., 2002
). We now demonstrate that pigmentation in cht
melanocytes is dependent on Rab32. The dramatic loss of pigment in the absence of both Rab38 and Rab32 is consistent with a critical role not only in the trafficking of Tyrp1 but also of tyrosinase, the key enzyme in melanin synthesis, which requires the melanosomal environment for catalytic activity (Watabe et al., 2004
; Hearing, 2005
). On the basis of these observations, we would predict a Rab38/Rab32 double-knockout mouse to show severe hypopigmentation. Compensation by Rab32 may also explain why mutations in Rab38 have not been identified in human patients with oculocutaneous albinism (Suzuki et al., 2003
). However, the presence of a detectable coat color phenotype in the cht
mouse may indicate subtle functional differences between the two Rab proteins, possibly accounting for the differential expression patterns observed in some specialized cell types.
Functional redundancy between mammalian Rab38 and Rab32 is consistent with the presence of only a single homologue in other species, such as Rab-RP1 in Drosophila melanogaster
(Fujikawa et al., 2002
) or Glo-1 in Caenorhabditis elegans
(Hermann et al., 2005
). Rab-RP1 is mutated in lightoid
, a D. melanogaster
eye color phenotype exhibiting defects in pigment granule synthesis (Ma et al., 2004
), and C. elegans
Glo-1 mutants lack lysosome-like gut granules (Hermann et al., 2005
). These observations suggest an evolutionarily conserved role for the Rab38/Rab32-related subgroup of Rab proteins in the biogenesis of specialized lysosome-related organelles (LROs) such as gut granules in C. elegans
, eye pigment granules in D. melanogaster
, and mammalian melanosomes.
Deficiencies in the biogenesis of LROs are also characteristic of HPS and the mouse models for HPS, with melanocytes and platelets most severely affected. The corresponding proteins (AP-3, Vps33, and the BLOC subunits), however, are expressed ubiquitously, suggesting more general roles in the biogenesis of lysosomes (Li et al., 2004
; Pietro and Dell'Angelica, 2005
). In contrast, Rab38 and Rab32 (Jager et al., 2000
; Osanai et al., 2001
; Cohen-Solal et al., 2003
) were restricted to cell types characterized by the presence of LROs, a morphologically and functionally diverse group of organelles that includes melanosomes, platelet-dense granules, mast cell granules, lamellar bodies of lung epithelial cells, lytic granules of cytotoxic T-lymphocytes, and MHC class II compartments of antigen-presenting cells (Marks and Seabra, 2001
; Stinchcombe et al., 2004
). This further supports a highly specialized role for Rab38 and Rab32 in LRO function.
Recent studies investigating the intracellular trafficking of melanosomal integral membrane proteins like Pmel17, tyrosinase, and Tyrp1 have revealed much about the formation of melanosomes and the complex sorting pathways involved (Raposo and Marks, 2002
; Theos et al., 2005a
; Valencia et al., 2006
). Premelanosomes appear to be largely derived from endosomal precursors, which progress to stage II organelles through the Pmel17-dependent formation of lumenal striations. These provide the fibrillar matrix for the subsequent deposition of melanin, initiated by the delivery of melanogenic enzymes, i.e., tyrosinase and tyrosinase-related proteins. The differential enrichment for Pmel17 in early-stage organelles and tyrosinase and Tyrp1 in later-stage pigmented structures provides evidence for the existence of distinct sorting pathways to premelanosomes and to more mature organelles (Raposo et al., 2001
). Our results suggest that Rab38 and Rab32 are not required for the formation of early-stage melanosomes. Trafficking of tyrosinase and Tyrp1 to these organelles, on the other hand, is dependent on Rab38 or Rab32.
The subcellular localization of these Rab proteins argues for their recruitment to post-TGN transport vesicles and a role in regulating the subsequent delivery of such vesicles to maturing melanosomes. Given the loss of tyrosinase in the absence of Rab38 and Rab32, this may represent a tissue-specific trafficking route critical for diverting proteins destined for LROs away from the degradative pathway to lysosomes. The TGN localization of tyrosinase in Rab38/Rab32-deficient cells is consistent with the recruitment of these Rabs to carrier vesicles derived directly from the TGN. Melanosomal targeting of tyrosinase is thought to involve transit through an early endosomal compartment (Theos et al., 2005a
), which may implicate Rab38/Rab32 in TGN to endosome trafficking. Alternatively, tyrosinase may traverse the endosome normally in the absence of Rab38 and Rab32, but may require their subsequent recruitment to endosome-derived transport vesicles. The latter scenario is supported by the continued presence of Rab38 on or in close proximity to mature melanosomes. A detailed analysis of the different subpopulations making up the endosomal system should prove informative with regard to pinpointing the precise site of action of Rab38 and Rab32.
Morphological studies of melanosomes in a range of HPS mutants documented disruption of organelle maturation at distinct stages in different mutants (Nguyen et al., 2002
; Nguyen and Wei, 2004
; Wei, 2006
). The molecular mechanisms and sites of action of most of the corresponding proteins, in particular the BLOC components, are still unknown. However, aberrant localization of tyrosinase and Tyrp1 is seen in many forms of HPS (Gwynn et al., 2004
; Richmond et al., 2005
). A more detailed comparison of melanosomal protein trafficking in the different mutants should shed light on the potential interplay between Rab38/Rab32 and other HPS proteins during melanosome biogenesis.
D. melanogaster lightoid
(Rab38/Rab32 homologue) and ruby
(AP-3) double mutants show considerably reduced eye pigmentation compared with the corresponding single mutants, indicating that lightoid
may function in an AP-3–independent pathway (Ma et al., 2004
). The association of Rab38 with Tyrp1 trafficking (Loftus et al., 2002
) and the mistargeting of tyrosinase but not Tyrp1 observed in melanocytes lacking AP-3 (Huizing et al., 2001
) raised the possibility that tyrosinase may traffic via an AP-3–dependent pathway, whereas Tyrp1 followed a separate Rab38-dependent route. Our results, however, demonstrate a role for Rab38/Rab32 in tyrosinase trafficking as well. The lack of colocalization between Rab38/Rab32 and AP-3 (unpublished data) is consistent with the regulation of distinct steps. The plasticity of the sorting pathways involved, though (Theos et al., 2005a
), could certainly result in additive effects upon removal of two components that normally act sequentially within the same pathway. Melanosome targeting of tyrosinase was more severely impaired than that of Tyrp1 upon loss of Rab38/Rab32, possibly indicating that the latter can access alternative pathways more efficiently. For a better understanding of Rab38/Rab32 function and the trafficking pathways regulated by this subfamily of Rab proteins in melanocytes and other cell types, the identification of interacting partners for Rab38 and Rab32 will be of great interest.