Reduce, reuse, recycle: a retrieval transport pathway for the membrane fusion machinery involved in melanosome biogenesis.

Abstract

The formation of the melanosome – the organelle responsible for synthesizing the melanin pigment found in hair, skin, and eyes – has long been studied as a model for the specialized development of organelles. In part this is due to the relative ease in identifying genes that impact pigmentation based on subtle to severe forms of albinism. The melanosome belongs to a family of lysosome-related organelles (LROs) that share some characteristics including an acidic lumen, part of their protein content, and biogenesis mechanisms. Underscoring the similar biogenesis of LROs, syndromic forms of albinism such as Hermansky–Pudlak syndrome (HPS) display hypopigmentation due to melanosome defects and other manifestations caused by deficiency of additional LROs. For example, HPS patients also present with excessive bleeding as a result of deficiency in platelet-dense granules, LROs important for platelet function in hemostasis. The melanosome, while being the best characterized LRO, is only one type of the many specialized LROs found in distinct cell types. The simultaneous deficiency of multiple types of LROs in HPS underlies the similarities of these different LROs, but also highlights the importance of understanding melanosome biogenesis. Hermansky–Pudlak syndrome is not one disease but a group of autosomal recessive disorders each of them defined by a specific gene. Characterization of different forms of HPS has uncovered that the proteins encoded by the affected genes organize into several large, stable complexes: biogenesis of lysosome-related organelles complex-1, biogenesis of lysosome-related organelles complex-2, biogenesis of lysosome-related organelles complex-3 (BLOC-1, BLOC-2, BLOC-3) and the tetrameric adaptor protein-3 (AP-3) complex that coordinate to facilitate melanosome biogenesis (Figure 1). Interactions of several of these protein complexes with many other proteins mediate the anterograde trafficking of melanogenic enzymes from recycling endosomes to melanosomes in vesicles or extended endosomal tubules (Figure 1; Dennis et al., 2015; Di Pietro et al., 2006; Theos et al., 2005). While some uncertainty still exists, the critical roles of BLOC-1, BLOC-2, AP-3, and the related AP-1 complex and their interacting partners in anterograde trafficking from endosomes to melanosomes are well established (Figure 1; Dennis et al., 2015; Di Pietro et al., 2006; Theos et al., 2005). Defects in anterograde transport result in deficient delivery of melanosomal proteins needed for pigment synthesis and cause albinism. ‘the retrograde pathway depends on BLOC-3 function, … the least understood [of the] protein complexes involved in HPS’ In stark contrast, no retrograde pathway from melanosomes back to endosomes has been described that would allow recycling of the trafficking and fusion machinery as observed in other membrane transport pathways. Remarkably, the recently published article by Dennis et al. in The Journal of Cell Biology uncovered a retrograde pathway departing from melanosomes (Figure 1). Moreover, the study found that the retrograde pathway depends on BLOC-3 function, thus shedding light on one of the least understood protein complexes involved in HPS (Figure 1). This work also refined our understanding of the BLOC-1-mediated anterograde trafficking to melanosomes. Most importantly, the authors focused on VAMP7, a v-SNARE previously shown to be needed for melanosome biogenesis and present on melanosomes (Figure 1). The authors also study two melanosome residents: tyrosinase-related protein-1 (TYRP1), an enzyme used for the synthesis of melanin from tyrosine, and the oculocutaneous albinism type 2 protein (OCA2), a transmembrane chloride channel that regulates pigment synthesis. Employing quantitative live-cell fluorescence microscopy and immuno-electron microscopy, the authors investigate the trafficking pathways for the anterograde and retrograde transport to and from melanosomes that use distinct tubular transport intermediates (Figure 1). At steady state, TYRP1 and OCA2 accumulate and remain in melanosomes, where they act in the production of the melanin pigments. In contrast to TYRP1 and OCA2, Dennis et al. hypothesize that after facilitating fusion of vesicles or tubules with the melanosome membrane, VAMP7 is trafficked back to endosomes using a distinct, novel retrograde tubular trafficking mechanism first characterized in The Journal of Cell Biology article. Consistent with a fusogenic role of VAMP7 in melanosome biogenesis, the authors use live-cell microscopy to demonstrate that VAMP7-depleted melanocytes have defects in TYRP1 and OCA2 delivery to melanosomes, melanin production, and aberrant subcellular localization of TYRP1. These results are consistent with previous findings regarding VAMP7 function in melanosome biogenesis. The data are also in line with previous reports showing that defective anterograde transport to melanosomes results in cargo such as TYRP1 retained in recycling endosomes and mistrafficked to the plasma membrane and lysosomes(Di Pietro et al., 2006). The authors then focus on studying the major proteins responsible for the anterograde and retrograde transport mechanisms. Using a transient and stable ‘rescue approach’ in BLOC-1-deficient melanocytes, the authors confirm and extend previous findings that TYRP1, OCA2, and VAMP7 are transported to melanosomes from endosomes in tubules that rely upon a BLOC-1-dependent mechanism (Figure 1). While TYRP1/OCA2 and VAMP7 depend, at least in part, on a BLOC-1-dependent anterograde transport to melanosomes, TYRP1/OCA2 and VAMP7 have different fates post-melanosome fusion. The authors observe both anterograde tubular transport from recycling endosomes to melanosomes and retrograde tubular compartments departing from melanosomes, which are differentiated by distinct protein components. Syntaxin 13 (STX13), a pan-early endosomal SNARE protein, labels the melanosome anterograde tubules, and the VPS9-ankyrin-repeat protein (VARP), a VAMP7 interaction partner that holds VAMP7 in an inactive conformation, identifies the tubules generated from melanosomes (and likely destined for recycling endosomes). Consistent with the notion of fusion machinery recycling, tubules exiting melanosomes contain VAMP7 but no TYRP1 or OCA2 (Figure 1). Importantly, the authors show that the formation of retrograde tubules depends on a BLOC-3-dependent mechanism. BLOC-3 was previously identified as a guanine nucleotide exchange factor (GEF) required for the recruitment to melanosomes and activation of small GTPases Rab32 and Rab38, which have also been characterized as interaction partners of VARP (Figure 1). Consistent with a possible role for BLOC-3, Rab32/38, and VARP in retrograde transport of VAMP7, the authors show colocalization of Rab38/VARP and Rab38/VAMP7 on melanosomes (Figure 1). Interestingly, previous reports on the roles of Rab32, Rab38, and VARP suggested that these proteins function in the anterograde transport to melanosomes (Figure 1), which would explain localization to melanosomes as resulting from delivery to melanosomes in anterograde vesicles or tubules(Bultema et al., 2012; Wasmeier et al., 2006). To provide mechanistic evidence of a VARP/Rab38 function in retrograde transport, the authors utilized previously reported knowledge on the VARP-Rab38 and VARP-VAMP7 binding sites to create specific VARP mutants that selectively inhibit binding of Rab38, VAMP7, or both binding partners. Supporting a role in retrograde transport, the authors find that mutation of VAMP7 or Rab38 binding sites results in partial reduction in VARP localization to melanosomes and that mutation of both binding sites results in further decrease in melanosome localization. To determine order of recruitment to melanosomes, the authors performed rescue experiments in BLOC-3-deficient cells. As expected, deficiency of BLOC-3 resulted in decreased melanosomal localization of Rab38 and VARP, and, consistent with a Rab38-dependent recruitment of VARP to melanosomes, this defect was resolved in BLOC-3-rescued cells. Interestingly, localization of VAMP7 and TYRP1 to melanosomes was not significantly altered in BLOC-3-deficient cells, which suggests that TYRP1 and VAMP7 anterograde traffic to melanosomes is primarily Rab38-independent. However, Rab32/38 likely functions in both anterograde and retrograde transport. Supporting this possibility, previous studies have identified partial colocalization of Rab32/38 with BLOC-2, AP-3, and AP-1 and cargo proteins at early endosome domains where BLOC-2, AP-3, and AP-1 function in anterograde trafficking to melanosomes (Figure 1). Rab32/38 could be activated by a GEF different from BLOC-3 to mediate forward transport. That Rab38 has functions independent of BLOC-3 is suggested by the more severe pigmentation phenotype displayed by Rab38-mutant mice (chocolate) compared with BLOC-3 mutant mice (pale ear and light ear). Chocolate mice display a generalized pigment dilution, whereas pale ear and light ear show a near-normal coat pigmentation except in the ears and tail. The distinct pigmentation phenotype is all the more striking considering that chocolate mice are hypomorphic rather than null mutants and the known compensatory effect of Rab32 (i.e., Rab32/Rab38 double deficiency enhances the melanosome biogenesis problem caused by Rab38 single deficiency; Bultema et al., 2012; Wasmeier et al., 2006). Additionally, a function of Rab32/38 in forward transport to melanosomes better explains the phenotypes observed with cells deficient in another effector of these Rab proteins, MYO5C. Taken together, the previously published data and the study by Dennis et al. suggest Rab32 and Rab38 have functions both in forward and retrograde transport during melanosome biogenesis (Figure 1). Finally, the authors observe that BLOC-3-deficient cells show a reduction in the length of retrograde transport tubules from melanosomes and that rescue with BLOC-3 results in a return to wild-type retrograde tubule length. Loss of retrograde VAMP7 transport tubules in BLOC-3 mutant cells suggests a role for Rab32/38 in recycling tubule function (Figure 1). As discussed above, the proposed VAMP7 recycling mechanisms with BLOC-3 and Rab32/38 likely correspond to functions of Rab32/38 in addition to the forward trafficking to melanosomes. A potential explanation provided by Dennis et al. highlights the importance of VAMP7 for cargo fusion with melanosomes and suggests that cargo mistrafficking and hypopigmentation phenotypes observed in BLOC-3/Rab32/Rab38 depletion/mutant melanocytes results from downstream defects caused by diminished retrograde VAMP7 transport from melanosomes to endosomes, rather than a direct anterograde trafficking defect. In other words, defective recycling of VAMP7 in BLOC-3/Rab32/Rab38-deficient cells would then deplete the anterograde transport tubules from the v-SNARE needed for fusion with the maturing melanosome (Figure 1). Dennis et al. further hypothesize that phenotypic differences in hypopigmentation and melanosome size observed in different pigmented cell types in Rab38 and BLOC-3 mutants could be explained by differences in the expression levels and requirements for VAMP7 in retrograde transport from melanosomes to endosomes. The possibility that different pigment-producing cells also use subtly different pathways for the biogenesis and secretion of melanosomes could also underlie different phenotypes observed in different pigmented tissues of HPS patients and corresponding mutant mice. The precise mechanisms by which Rab32/38 and their effectors function in melanosome biogenesis are complex, and these proteins likely function at multiple distinct steps in anterograde and retrograde transport to and from melanosomes. Owen and colleagues demonstrated that the VAMP7 fusogenic activity, which is regulated by its Longin domain, is maintained in an inhibited state by binding to VARP or AP-3. When and how is the fusogenic activity of VAMP7 unleashed or maintained in an inhibited state is not clear. In any case, the recent publication by Dennis et al. described for the first time the existence of a BLOC-3-dependent retrograde transport mechanism used by melanosomes and allows the authors to convincingly posit new hypotheses on the intracellular trafficking mechanisms and SNARE recycling used in melanosome biogenesis. These questions and the degree to which these systems are also employed for the biogenesis of other LROs warrant additional study.