BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.     

AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type–specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1– and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type–specific positioning of endosomes that facilitate endosome–LRO contacts and are required for organelle maturation.     

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome‐related organelle that is commonly defective in Hermansky–Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC‐1, ‐2, ‐3, or AP‐1, ‐3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A‐depletion phenotype resembles Rab38/32‐inactivated or BLOC‐3‐deficient melanocytes, suggesting that Rab9A works in line with BLOC‐3 and Rab38/32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/32 or its effector VARP, or BLOC‐3‐deficiency in melanocytes decreased the length of STX13‐positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co‐regulatory GTPases control STX13‐mediated cargo delivery to maturing melanosomes.     

A BLOC-1–AP-3 super-complex sorts a cis-SNARE complex into endosome-derived tubular transport carriers

Membrane transport carriers fuse with target membranes through engagement of cognate vSNAREs and tSNAREs on each membrane. How vSNAREs are sorted into transport carriers is incompletely understood. Here we show that VAMP7, the vSNARE for fusing endosome-derived tubular transport carriers with maturing melanosomes in melanocytes, is sorted into transport carriers in complex with the tSNARE component STX13. Sorting requires either recognition of VAMP7 by the AP-3δ subunit of AP-3 or of STX13 by the pallidin subunit of BLOC-1, but not both. Consequently, melanocytes expressing both AP-3δ and pallidin variants that cannot bind their respective SNARE proteins are hypopigmented and fail to sort BLOC-1–dependent cargo, STX13, or VAMP7 into transport carriers. However, SNARE binding does not influence BLOC-1 function in generating tubular transport carriers. These data reveal a novel mechanism of vSNARE sorting by recognition of redundant sorting determinants on a SNARE complex by an AP-3–BLOC-1 super-complex.     

BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.     

Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.     

It takes two to tango to the melanosome

The role of clathrin adaptor proteins in sorting cargo in the biosynthetic and recycling routes is an area of intense research. In this issue, Delevoye et al. (2009. J. Cell Biol. doi:10.1083/jcb.200907122) show that a close interaction between the clathrin adaptor AP-1 and a kinesin motor KIF13A is essential for delivering melanogenic enzymes from recycling endosomes to nascent melanosomes and for organelle biogenesis.Melanosomes, along with platelet-dense granules and lung type II alveolar cell lamellar bodies, are lysosome-related organelles (LROs), compartments that originate from endosomes but are distinct from and usually coexist with lysosomes (Fig. 1). The most characteristic features of melanosomes are their ability to synthesize and store melanin and their presence in specialized pigmented cells such as skin melanocytes and iris and retinal pigment epithelial cells (Raposo and Marks, 2007; Wasmeier et al., 2008). In this issue, Delevoye et al. (see p. 247) report a melanogenic role for the clathrin adaptor AP-1 that involves interactions between the adaptor and the plus end kinesin motor KIF13A. An impressive set of data support a scenario in which the adaptor and the motor tightly interact, like in tango, to position donor recycling endosomes (REs) near nascent melanosomes at the cell periphery and to generate tubulovesicular intermediates that deliver newly synthesized pigmenting enzymes to melanosomes.Open in a separate windowFigure 1.Role of clathrin adaptor proteins in melanosome biogenesis. Post-Golgi trafficking routes of three melanosome cargoes (Pmel17, tyrosinase, and Tyrp1) in melanocytes are shown. Newly synthesized Pmel17 is transported to the limiting membrane and intraluminal vesicles of stage I melanosomes/early sorting endosomes via the plasma membrane. This process (depicted by a question mark) might involve clathrin and AP-2. From these EEA1-positive vacuolar endosomes, Pmel17 is sorted away from the late endosome/multivesicular body pathway into stage II melanosomes. Little is known as to how the enzymes essential for melanin synthesis, tyrosinase and Tyrp1, are sorted from the TGN to early REs, and it is likely that clathrin and its adaptors are involved in this process. Tyrosinase, which binds both AP-1 and -3, is transported to stage III melanosomes from tubular regions of REs, containing Tf/TfR and Rab11, by two distinct routes: one regulated by AP-3 and the other regulated by BLOC-1, BLOC-2, and perhaps AP-1. However, Tyrp1 binds only AP-1 and not AP-3, indicating a divergence of sorting mechanisms between tyrosinase and Tyrp1. Delevoye et al. (2009) now show that AP-1 interacts with the kinesin motor KIF13A to transport recycling endosomal domains to the melanocytic cell periphery. The close apposition of Tyrp1-containing tubules with melanosomes allows cargo transfer and biogenesis of stage III and IV melanosomes. Although Tf is found in these peripheral endosomal tubules, there appears to be a filtering mechanism that sorts it out before the tubules fuse with melanosomes. It is likely, although not yet confirmed, that BLOC-1 and -2 act in concert with AP-1 to transport Tyrp1. The tissue-specific Rabs, Rab32 and Rab38, might function in any or all of these pathways.Extensive studies have shown that melanosome biogenesis occurs in two waves that correspond to four morphologically distinct stages (Fig. 1; Marks and Seabra, 2001; Raposo and Marks, 2007). The first wave (stages I and II) is the formation of immature, pigment-free ellipsoidal melanosomes from vacuolar domains of early sorting endosomes. This process requires Pmel17, an integral membrane protein that likely reaches sorting endosomes by clathrin-dependent endocytosis from the plasma membrane. Upon proteolysis in the sorting endosomes/stage I melanosomes, Pmel17 forms intraluminal proteinaceous fibrils with characteristics of amyloid. The second wave starts with the post-Golgi transport of enzymes involved in melanin synthesis such as tyrosinase and tyrosinase-related protein 1 (Tyrp1) to nascent melanosomes. Melanin deposition occurs on Pmel17 fibrils and leads to the biogenesis of mature (stages III and IV) melanosomes. The clathrin adaptors AP-1 and -3 have partially redundant functions in sorting cargo proteins to melanosomes. Melanosomal cargo proteins have dileucine motifs that are recognized differentially by AP-1 and -3 in post-Golgi endosomes (Huizing et al., 2001; Theos et al., 2005). Nascent tyrosinase is found in distinct endosomal buds that contain either AP-3 or -1 in normal melanocytes and loss of AP-3 results only in a partial mislocalization of the enzyme. As these adaptors also mediate sorting from endosomes to other compartments, additional machinery, such as biogenesis of LRO complex 1 (BLOC-1), BLOC-2, and the tissue-specific small GTPases Rab32 and Rab38, regulate cargo delivery to melanosomes. Mutations in components of this melanosomal targeting machinery result in a variety of well-studied pigmentation defects in humans and animals such as Hermansky–Pudlak syndrome (Wei, 2006).Delevoye et al. (2009) show that knockdown of AP-1 in melanocytic MNT-1 cells decreases melanin content, demonstrating that AP-1 has a role in melanogenesis. Only late-stage (III/IV) melanosomes are decreased in number; unpigmented (stage I/II) melanosomes are unaffected, indicating that AP-1 functions selectively in the second wave of melanosome biogenesis. In AP-1–depleted cells, the melanosome cargo protein Tyrp1 is retained in vacuolar endosomes in a manner similar to that seen in BLOC-1–deficient melanocytes (Setty et al., 2007). Using immunofluorescence to monitor markers of various endosomal compartments, Delevoye et al. (2009) show that AP-1 performs its melanogenic function in early REs. Interestingly, additional data show that AP-1–containing REs have a peripheral distribution in MNT-1 cells, which is strikingly different from the perinuclear localization observed in other cells. Furthermore, siRNA-mediated knockdown of AP-1, but not of AP-3, relocates RE to a pericentriolar location.How might AP-1 influence endosome position? One possibility is by its association with the plus end–directed kinesin motor KIF13A (Fig. 1). Nakagawa et al. (2000) have previously shown that a subunit of AP-1 binds the C-terminal domain of KIF13A, mediating TGN to plasma membrane transport of the mannose 6-phosphate receptor. Indeed, Delevoye et al. (2009) show that KIF13A partially colocalizes with AP-1 in MNT-1 cells and coimmunoprecipitates with both AP-1 and Tyrp1. Furthermore, knockdown of KIF13A replicates the phenotype seen with AP-1 depletion: pericentriolar clustering of RE, accumulation of Tyrp1 in vacuolar endosomes, and reduction in mature melanosomes and melanin content. Delevoye et al. (2009) go on to show that the peripheral RE localization facilitates sorting of melanosomal proteins but decreases the efficiency of transferrin (Tf) receptor (TfR) recycling to the plasma membrane. They also show the converse; i.e., the pericentriolar localization of RE decreases the efficiency of melanosomal targeting and increases the efficiency of TfR recycling. Thus, the position of REs, determined by the interaction between a clathrin adaptor and a kinesin, is key for specific sorting functions of this organelle (like TfR recycling) and also regulates the biogenesis of another organelle (the melanosome). This is a novel and exciting finding and is an emerging theme in cell biology. It was recently reported that AP-1 interacts with another plus end–directed kinesin, KIF5, which helps transport endosomes to the cell periphery (Schmidt et al., 2009).The next question that Delevoye et al. (2009) approach is what is the nature of the carriers that transport melanosomal proteins from peripheral REs to immediately adjacent stage III/IV melanosomes? Live imaging experiments showed a dynamic network of Tf-containing RE tubules that extend and retract, making contact with melanosomes for at least 30 s. Double-tilt 3D electron tomography of thick (350–400 nm) sections of cells preserved by high pressure freezing and freeze substitution, a technique recently adapted to the study of melanosomes by Hurbain et al. (2008), revealed that some of these tubular elements are continuous with the melanosomal limiting membrane and that their lumens are often connected. Collectively, these results indicate that peripheral RE domains serve to deliver biosynthetic cargo to maturing melanosomes by the coordinated actions of AP-1 and KIF13A and that the mechanism involves tubular connections rather than vesicular transport (Fig. 1).The study by Delevoye et al. (2009) beautifully demonstrates the power of carefully chosen morphological and live imaging techniques, in combination with siRNA-mediated knockdown of molecules under study, to elucidate important details of cellular sorting processes. As always, several questions emerge from their results. Does this type of mechanism also operate in perinuclear REs, which were recently shown to cooperate with adjacent TGN in biosynthetic trafficking to the plasma membrane (Cancino et al., 2007; Gravotta et al., 2007)? Do newly synthesized melanosomal enzymes move from the TGN to REs using vesicular trafficking and clathrin adaptors or, rather, result from “maturation” of REs from the TGN? What is the role of clathrin in melanosome maturation? Are AP-1 and KIF13A essential for tubulogenesis from REs as the authors speculate? How are RE proteins (e.g., TfR) prevented from incorporating into melanosomes through the tubular connections? What is the mechanism that regulates docking and fusion of RE tubules with melanosomes? Likely, Rab32 and Rab38 participate in this process, as these proteins localize to tubulovesicular endosomal structures, and their loss causes mislocalization of tyrosinase and Tyrp1 (Wasmeier et al., 2006), but the SNAREs (if any) that participate in the mechanism are still unknown. Lastly, another intriguing aspect of this study is how adaptors sort proteins by differential recognition of dileucine motifs. Tyrp1 also has a dileucine motif that exclusively binds AP-1, but not AP-3, in melanocytic cells (Theos et al., 2005), whereas tyrosinase has dileucine motifs that bind AP-1 and -3, indicating that not all dileucine motifs are equal in the eyes of the adaptor.     

Characterization of BLOC-2, a complex containing the Hermansky-Pudlak syndrome proteins HPS3, HPS5 and HPS6

Hermansky–Pudlak syndrome (HPS) defines a group of at least seven autosomal recessive disorders characterized by albinism and prolonged bleeding due to defects in the lysosome-related organelles, melanosomes and platelet-dense granules, respectively. Most HPS genes, including HPS3, HPS5 and HPS6 , encode ubiquitously expressed novel proteins of unknown function. Here, we report the biochemical characterization of a stable protein complex named Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2), which contains the HPS3, HPS5 and HPS6 proteins as subunits. The endogenous HPS3, HPS5 and HPS6 proteins from human HeLa cells coimmunoprecipitated with each other from crude extracts as well as from fractions resulting from size-exclusion chromatography and density gradient centrifugation. The native molecular mass of BLOC-2 was estimated to be 340 ± 64 kDa. As inferred from the biochemical properties of the HPS6 subunit, BLOC-2 exists in a soluble pool and associates to membranes as a peripheral membrane protein. Fibroblasts deficient in the BLOC-2 subunits HPS3 or HPS6 displayed normal basal secretion of the lysosomal enzyme β-hexosaminidase. Our results suggest a common biological basis underlying the pathogenesis of HPS-3, -5 and -6 disease.     

The dark side of lysosome-related organelles: specialization of the endocytic pathway for melanosome biogenesis

Melanosomes are lysosome-related organelles within which melanin pigments are synthesized and stored in melanocytes and retinal pigment epithelial cells. Early ultrastructural studies of pigment cells revealed that melanosomes consist of a complex series of organelles; more recently, these structures have been correlated with cargo constituents. By studying the fate of melanosomal and endosomal cargo in melanocytic cells, the effects of disease-related mutations on melanosomal morphology, and the genes affected by these mutations, we are beginning to gain novel insights into the biogenesis of these complex organelles and their relationship to the endocytic pathway. These insights demonstrate how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

Fission of SNX-BAR–coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1

Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR–coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.     

Melanoregulin, Product of the dsu Locus, Links the BLOC-Pathway and Oa1 in Organelle Biogenesis

Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg^dsu gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg^dsu locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1^ko/ko mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.     

SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27–Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27–Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.

Coat complexes coordinate cargo recognition with biogenesis of transport carriers during integral membrane protein trafficking. Mechanistic study of the function and evolution of the SNX27:Retromer:ESCPE-1 assembly provides new insight into pathway defects associated with neurodegenerative disease and an interesting comparison with the yeast pentameric Retromer.     

Assembly of the Biogenesis of Lysosome-related Organelles Complex-3 (BLOC-3) and Its Interaction with Rab9

The Hermansky-Pudlak syndrome (HPS) is a genetic hypopigmentation and bleeding disorder caused by defective biogenesis of lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies. HPS arises from mutations in any of 8 genes in humans and 16 genes in mice. Two of these genes, HPS1 and HPS4, encode components of the biogenesis of lysosome-related organelles complex-3 (BLOC-3). Herein we show that recombinant HPS1-HPS4 produced in insect cells can be efficiently isolated as a 1:1 heterodimer. Analytical ultracentrifugation reveals that this complex has a molecular mass of 146 kDa, equivalent to that of the native complex and to the sum of the predicted molecular masses of HPS1 and HPS4. This indicates that HPS1 and HPS4 interact directly in the absence of any other protein as part of BLOC-3. Limited proteolysis and deletion analyses show that both subunits interact with one another throughout most of their lengths with the sole exception of a long, unstructured loop in the central part of HPS4. An interaction screen reveals a specific and strong interaction of BLOC-3 with the GTP-bound form of the endosomal GTPase, Rab9. This interaction is mediated by HPS4 and the switch I and II regions of Rab9. These characteristics indicate that BLOC-3 might function as a Rab9 effector in the biogenesis of LROs.     

BLOC-2 subunit HPS6 deficiency affects the tubulation and secretion of von Willebrand factor from mouse endothelial cells

Hermansky-Pudlak syndrome(HPS) is a recessive disorder with bleeding diathesis, which has been linked to platelet granule defects. Both platelet granules and endothelial Weibel-Palade bodies(WPBs)are members of lysosome-related organelles(LROs) whose formation is regulated by HPS protein associated complexes such as BLOC(biogenesis of lysosome-related organelles complex)-1,-2,-3, AP-3(adaptor protein complex-3) and HOPS(homotypic fusion and protein sorting complex). Von Willebrand factor(VWF) is critical to hemostasis, which is stored in a highly-multimerized form as tubules in the WPBs. In this study, we found the defective, but varying, release of VWF into plasma after desmopressin(DDAVP) stimulation in HPS1(BLOC-3 subunit), HPS6(BLOC-2 subunit), and HPS9(BLOC-1 subunit)deficient mice. In particular, VWF tubulation, a critical step in VWF maturation, was impaired in HPS6 deficient WPBs. This likely reflects a defective endothelium, contributing to the bleeding tendency in HPS mice or patients. The differentially defective regulated release of VWF in these HPS mouse models suggests the need for precise HPS genotyping before DDAVP administration to HPS patients.     

ESCRT-I Function is Required for Tyrp1 Transport from Early Endosomes to the Melanosome Limiting Membrane

Melanosomes are lysosome-related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky-Pudlak syndrome that lack BLOC-1, melanosomal proteins such as tyrosinase-related protein 1 (Tyrp1) accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here, we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverse early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT-I component, Tsg101, or inhibition of ESCRT function by dominant-negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT-dependent, ubiquitylated cargoes such as MART-1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101-depleted cells suggests that ESCRT-I functions downstream of BLOC-1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT-I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome-related organelle.     

Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.     

Biogenesis of melanosomes - the chessboard of pigmentation

Melanosomes are lysosome-related organelles in retinal pigment epithelial cells and epidermal melanocytes in which melanin pigments are synthesized and stored. Melanosomes are generated by multistep processes in which an immature unpigmented organelle forms and then subsequently matures. Such maturation requires inter-organellar transport of protein cargos required for pigment synthesis but also recruitment of effector proteins necessary for the correct transport of melanosomes to the cell periphery. Several studies have started to unravel the main pathways and mechanisms exploited by melanosomal proteins involved in melanosome structure and melanin synthesis. A major unexpected finding seen early in melanosome biogenesis showed the similarities between the fibrillar sheets of premelanosomes and amyloid fibrils. Late steps of melanosome formation are dependent on pathways regulated by proteins encoded by genes mutated in genetic diseases such as the Hermansky-Pudlak Syndrom (HPS) and different types of albinism. Altogether the findings from the past recent years have started to unravel how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

The late endosomal adaptor protein LAMTOR2/p14 is essential for tissue homeostasis by controlling MAPK and mTOR signaling, which in turn regulate cell growth and proliferation, migration and spreading. Moreover, LAMTOR2 critically controls architecture and function of the endocytic system, including epidermal growth factor receptor (EGFR) degradation in lysosomes, positioning of late endosomes and defense against intracellular pathogens. Here we describe the multifaceted ultrastructural phenotype of the endo/lysosomal system of LAMTOR2‐deficient mouse embryonic fibroblasts. Quantitative (immuno‐)electron microscopy of cryo‐fixed samples revealed significantly reduced numbers of recycling tubules emanating from maturing multivesicular bodies (MVB). Instead, a distinct halo of vesicles surrounded MVB, tentatively interpreted as detached, jammed recycling tubules. These morphological changes in LAMTOR2‐deficient cells correlated with the presence of growth factors (e.g. EGF), but were similarly induced in control cells by inactivating mTOR. Furthermore, proper transferrin receptor trafficking and recycling were apparently dependent on an intact LAMTOR complex. Finally, a severe imbalance in the relative proportions of endo/lysosomes was found in LAMTOR2‐deficient cells, resulting from increased amounts of mature MVB and (autophago)lysosomes. These observations suggest that the LAMTOR/Ragulator complex is required not only for maintaining the homeostasis of endo/lysosomal subpopulations but also contributes to the proper formation of MVB‐recycling tubules, and regulation of membrane/cargo recycling from MVB.