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Correspondence to Miguel C. Seabra: m.seabra{at}imperial.ac.uk
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The study of mouse mutants displaying alterations in coat color has identified a large number of gene products that affect pigmentation (Bennett and Lamoreux, 2003). Many of these are involved in melanosome function. They include the enzymes required for melanin synthesis, primarily tyrosinase, as well as tyrosinase-related protein (Tyrp/TRP) 1 and Dct (TRP2; Hearing, 2005), and molecules with structural roles in melanosome formation, such as Pmel17 (Theos et al., 2005b). Others are involved in the regulation of intracellular protein trafficking and organelle biogenesis. Most mouse models for Hermansky-Pudlak syndrome (HPS), a heterogeneous group of disorders associated with albinism in humans, fall into this category. They include subunits of the biogenesis of lysosome-related organelles complexes (BLOCs), of adaptor protein 3 (AP-3), or of the homotypic vacuolar protein sorting complex (Li et al., 2004; Pietro and Dell'Angelica, 2005; Wei, 2006). Others again are required for correct intracellular distribution of melanosomes, for example, Rab27a, melanophilin, and myosinVa (Seabra and Coudrier, 2004).
Analysis of the coat color mutant "chocolate" (cht) identified the small GTPase Rab38 as another gene involved in the regulation of pigmentation (Loftus et al., 2002). The Rab family of proteins consists of >60 members in mammalian cells (Pereira-Leal and Seabra, 2001). Some are ubiquitously expressed, whereas others are highly tissue specific, reflecting more specialized roles. Each Rab protein shows a characteristic subcellular distribution and may thus represent an important determinant of organelle identity (Pfeffer, 2001; Munro, 2002; Seabra and Wasmeier, 2004). Rabs serve as key regulators of vesicular trafficking between subcellular compartments. They have been implicated in the formation of vesicles at the donor membrane and in the tethering, docking, and fusion of vesicles or organelles with their target membranes (Segev, 2001; Zerial and McBride, 2001). They are also involved in a variety of other processes, for example, the regulation of early endosome motility by Rab5 (Nielsen et al., 1999), the capture of melanosomes at the cell periphery by Rab27a (Seabra and Coudrier, 2004), or the remodeling of the cytoskeleton by Rab8 (Peranen et al., 1996). The regulatory capacity of Rab proteins is based on their ability to act as GTP-dependent molecular switches, with activation coupled to reversible association with intracellular membranes. In this way, Rabs can control the recruitment of downstream effectors to specific subcellular compartments in an activation-dependent way (Seabra and Wasmeier, 2004).
Rab38 is predominantly expressed in melanocytes and retinal pigment epithelial cells and is localized to pigmented melanosomes. In the cht mouse, a recessive Gly19 to Val point mutation was identified in Rab38. The resulting coat color phenotype resembles that of the brown mouse, which carries a mutation in Tyrp1, and reduced levels of Tyrp1 were reported in melanosomes of cultured cht melanocytes, suggesting an involvement of Rab38 in Tyrp1 transport (Loftus et al., 2002). The present study characterizes Rab38 in the cht mutant and defines a role for Rab38 in melanosome biogenesis. We demonstrate that Rab38 and the closely related Rab32 are important, functionally redundant regulators of melanosomal protein trafficking and melanocyte pigmentation.
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In control cht cells, Tyrp1 and tyrosinase localized to vesicles distributed throughout the cytoplasm and along the cell periphery, with an additional pool in the Golgi region. In contrast, in Rab38/Rab32-deficient cells, Tyrp1 and tyrosinase were almost exclusively restricted to the perinuclear region (Fig. 8). Little Tyrp1, and virtually no tyrosinase, was detected on peripheral structures in these cells. This change in subcellular distribution suggests that the loss of pigmentation induced by Rab32 knockdown is the result of a disruption in the intracellular trafficking of Tyrp1 and, more important, tyrosinase.
The perinuclear pool of tyrosinase in control cht cells as well as tyrosinase in Rab38/Rab32-deficient cells showed a high degree of colocalization with TGN38, a marker for the TGN (Fig. 9). Post-Golgi sorting of tyrosinase is thought to involve trafficking through an endosomal compartment (Theos et al., 2005a). In Rab38/Rab32-deficient cells, some additional tyrosinase-positive structures were frequently observed in close proximity to the TGN38-labeled compartment. However, these did not appear to possess either AP-3 or the transferrin receptor (unpublished data); thus, it is not clear if they could represent such endosomes.
To distinguish between newly synthesized protein in transit through the Golgi stack and protein retained in this compartment because of a block in transport, protein synthesis was inhibited with cycloheximide for 3 h. In control cells, this resulted in depletion of the perinuclear pool of tyrosinase, whereas protein in peripheral melanosomes was not affected, and no substantial changes in overall levels of tyrosinase were observed. In contrast, the majority of Rab38/Rab32-deficient cells (
70% of depigmented cells compared with <10% of control cells) showed drastically reduced tyrosinase labeling after incubation with cycloheximide (Fig. 9), indicating degradation of the protein after its exit from the TGN. Any residual tyrosinase seen in these cells remained largely colocalized with TGN38, with no obvious accumulation in a post-TGN compartment. This argues for a critical Rab38/Rab32-dependent step in the trafficking of tyrosinase from the TGN to the melanosome.
Cht cells were transfected with Rab32 siRNA to induce loss of pigmentation and were subsequently infected with virus introducing either EGFP or EGFP-Rab38. Expression of EGFP alone did not affect levels of pigmentation or the characteristic perinuclear localization of tyrosinase. In contrast, in cells expressing EGFP-Rab38, trafficking of tyrosinase to cytoplasmic and peripheral vesicles was restored, concomitant with the cells recovering normal levels of pigmentation (Fig. 10 A). Approximately 59% of EGFP-expressing cells in this experiment were depigmented and showed restriction of tyrosinase to the TGN region, whereas only 10% of EGFP-Rab38–positive cells had not recovered both pigmentation and a normal subcellular distribution of tyrosinase (Fig. 10 B). This further supports the conclusion that Rab38 can rescue melanosome biogenesis in Rab38/Rab32-deficient cells and that it restores pigment biosynthesis by mediating tyrosinase transport from a perinuclear compartment to the melanosome.
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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,b; 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 or garnet (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.
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Tissue and cell preparations and subcellular fractionation
Mouse tissues were collected from C57BL/6J mice perfused with PBS and were homogenized in 3 volumes of 50 mM Hepes, pH 7.2, 10 mM NaCl, 1 mM dithiothreitol, and protease inhibitor cocktail (Roche). Homogenates were centrifuged at 1,000 g for 10 min at 4°C to sediment unbroken cells and nuclei. Cultured cells were pelleted and disrupted by sonication in the aforementioned buffer. Protein concentrations of postnuclear supernatants or whole cell lysates were determined using the Bio-Rad protein assay (Bio-Rad Laboratories). For subcellular fractionation, cells were disrupted as above. Membranes were pelleted by centrifugation at 100,000 g for 1 h at 4°C. Alternatively, Triton X-114 (Calbiochem) was added to 1%, samples were incubated on ice for 10 min, followed by 5 min at 37°C, and centrifuged at 16,000 g for 3 min at room temperature. Aqueous and detergent phases were recovered, and the detergent phase was reextracted once with buffer. All fractions were adjusted to the same volume, and equal volumes were analyzed by SDS-PAGE and immunoblotting.
Antibodies
To generate polyclonal antibodies to Rab38 and Rab32, the C-terminal hypervariable regions of rat Rab38 (residues 162–211) or mouse Rab32 (residues 175–223) were fused to GST and purified on glutathione–Sepharose beads as previously described (Hume et al., 2002). Sera from rabbits immunized with these fusion proteins were preabsorbed against GST and affinity purified using the same antigens immobilized on AminoLink Coupling Gel (Pierce Chemical Co.) as described previously (Seabra et al., 1995). Anti-Rab27a monoclonal antibody 4B12 was described previously (Hume et al., 2001). Rabbit polyclonal anti-tyrosinase PEP7 (IF, 1:150) was a generous gift from V. Hearing (National Institutes of Health, Bethesda, MD). Other antibodies were as follows: rabbit anti-tyrosinase (EM; provided by A. Theos and M. Marks, University of Pennsylvania, Philadelphia, PA; Theos et al., 2005a), mouse anti-Tyrp1 TA99 (IF, 1:200 [ID Labs]; EM [American Type Culture Collection]), mouse anti-Pmel17 HMB45 (IF, 1:50 [DakoCytomation]; EM [Labvision]), sheep anti-TGN38 (1:100; Serotec), rabbit anti-EGFP (EM; Invitrogen), mouse anti-EGFP (immunoblot; 1:1,000; Roche), mouse anti–
tubulin and mouse anti–
tubulin (1:1,000; Sigma-Aldrich).
Immunoblotting
Samples were fractionated on 13% SDS-PAGE gels and transferred to polyvinylidene difluoride membrane (Millipore). Membranes were blocked in PBS/0.1% Tween-20 (PBS/T) with 4% nonfat dried milk, incubated with primary antibody in PBS/T, washed four times in blocking solution, and incubated with horseradish peroxidase–conjugated secondary antibody (anti–rabbit or anti–mouse; 1:5,000; DakoCytomation) followed by washing in PBS/T. Bound antibody was detected using the ECL Plus Western blotting detection system (GE Healthcare).
Plasmid constructs
pEGFP-Rab27a, -Rab38, and -Rab32 were generated by subcloning cDNA for rat Rab27a, rat Rab38, or human Rab32 into pEGFP (CLONTECH Laboratories, Inc.), using standard cloning techniques. pEGFP-Rab38G19V was generated from pEGFP-Rab38 using the QuikChange site-directed mutagenesis system (Stratagene) to introduce a single nucleotide substitution in codon 19 (GGT to GTT). For mRFP-Rab38, Rab38 was amplified from pEGFP-Rab38 by PCR using Pfu polymerase (Stratagene) and was subcloned into pRFP-C (monomeric red fluorescent protein; a gift from R. Tsien, University of California, San Diego, La Jolla, CA).
Transfection of cells with plasmids and oligonucleotides
For the introduction of plasmid constructs, cells were transfected with Fugene 6 (Roche) according to the manufacturer's recommendations. Per 16-mm well, 0.3 µg DNA and 1 µl transfection reagent were used. Cells were analyzed after 48 h. For siRNA oligonucleotides, mouse melanocytes were transfected with Oligofectamine (Invitrogen). Per 16-mm well, 0.625 µl of reagent was used, and the final concentration of siRNA oligos was 100 nM. For most experiments shown, cells were subjected to two rounds of transfection on days 0 and 5, were passaged as required, and were analyzed on day 10 or 11. siRNAs were purchased from Dharmacon. Oligo 32-1 corresponded to nucleotides 261–280 and oligo 32-2 to nucleotides 602–621 of mouse Rab32. As control, a pool of four nontargeting control siRNAs (Dharmacon) was used.
Lentivirus production and infection of melanocytes
The lentivirus vector pHR'SIN-cPPT-SEW (a gift from A. Thrasher, Institute of Child Health, London, UK) expresses the EGFP gene under the control of the spleen focus forming virus U3 promoter/enhancer and carries a modified Woodchuck posttranscriptional regulatory element to improve virus titre, and the central polypurine tract sequence of HIV-1 to aid nuclear entry (Demaison et al., 2002). EGFP-Rab38 was amplified from pEGFP-Rab38 by PCR using Pfu polymerase and was subcloned into this plasmid using standard techniques. Lentivirus particles were produced and titred as previously described (Waddington et al., 2004). Cells were plated in RPMI-1640 with 5% FCS (5 x 104 cells per well) and were infected with virus at an approximate MOI of 50 for MNT-1 cells and MOI of 100 for mouse melanocytes.
Melanin assay
Cells were disrupted by sonication in 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, and protease inhibitors. Pigment was pelleted at 20,000 g for 15 min at 4°C, rinsed once in ethanol/ether (1:1), and dissolved in 2 M NaOH/20% dimethylsulfoxide at 60°C. Melanin content was measured as optical density at 492 nm.
IF microscopy
Cells were grown on glass coverslips (coated with polylysine for MNT-1 cells) and transfected with plasmid constructs where indicated. For inhibition of protein synthesis, cycloheximide was added to the growth medium to 100 µg/ml, and cells were incubated for a further 3 h. Cells on coverslips were rinsed in PBS, fixed with 3% PFA in PBS for 30 min, rinsed again, and permeabilized with 0.05% saponin in PBS. Coverslips were blocked for 30 min, incubated with primary antibodies for 1.5 h, washed four times and incubated with Alexa 488– or Alexa 568–conjugated anti–mouse, anti–rabbit or anti–sheep secondary antibodies (1:200; Invitrogen), and washed again. All incubations and wash steps were in PBS/BSA/0.01% saponin. Coverslips were mounted in Mowiol (Calbiochem) mounting medium and viewed on a microscope (DM-IRBE; Leica) with a PL Fluotar 40x 1.0 oil objective. Images were acquired using a confocal system (TCS NT; Leica) and processed with Photoshop (Adobe).
EM
For conventional EM, cells grown on coverslips were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 24 h. After several washes with 0.1 M cacodylate buffer, the cells were postfixed with 2% OsO4, dehydrated in ethanol, and embedded in Epon while on the coverslips. Ultrathin sections were prepared and counterstained with uranyl acetate and lead citrate before observation. For immunogold labeling, cells were fixed with 2% PFA or with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Cells were processed for ultracryomicrotomy as described previously (Raposo et al., 1997). Ultrathin sections were prepared with an Ultracut FCS ultracryomicrotome (Leica), and single- or double-immunogold labeled with antibodies and protein A coupled to 5 or 10-nm gold, as indicated in the figure legends. Sections were observed and photographed under an electron microscope (Philips CM120; FEI Company). Digital acquisitions were made with a numeric camera (Keen View; Soft Imaging System).
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Acknowledgments |
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This work was supported by grants from the Biotechnology and Biological Sciences Research Council (C19747) and the Wellcome Trust (075498).
Submitted: 19 June 2006
Accepted: 19 September 2006
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