RESUMO
Hermansky-Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2-deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2-deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.
Assuntos
Endossomos/metabolismo , Melanossomas/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Endocitose , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Melanócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredutases/metabolismo , Transporte Proteico , Pigmentação da PeleRESUMO
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.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Transporte/metabolismo , Lectinas/metabolismo , Melanossomas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Dipeptídeos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Melanócitos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Ligação Proteica , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmelâ»/â»). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmelâ»/â» melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation.
Assuntos
Melaninas/biossíntese , Melanossomas/metabolismo , Pigmentação/genética , Antígeno gp100 de Melanoma/genética , Antígeno gp100 de Melanoma/metabolismo , Alelos , Animais , Células Cultivadas , Células Epidérmicas , Epiderme/metabolismo , Cor de Cabelo/genética , Células HeLa , Humanos , Melaninas/genética , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mutação , Oxirredutases/metabolismo , Fenótipo , Pele/metabolismoRESUMO
PMEL is a pigment cell-specific protein that forms physiological amyloid fibrils upon which melanins ultimately deposit in the lumen of the pigment organelle, the melanosome. Whereas hypomorphic PMEL mutations in several species result in a mild pigment dilution that is inherited in a recessive manner, PMEL alleles found in the Dominant white (DW) chicken and Silver horse (HoSi)--which bear mutations that alter the PMEL transmembrane domain (TMD) and that are thus outside the amyloid core--are associated with a striking loss of pigmentation that is inherited in a dominant fashion. Here we show that the DW and HoSi mutations alter PMEL TMD oligomerization and/or association with membranes, with consequent formation of aberrantly packed fibrils. The aberrant fibrils are associated with a loss of pigmentation in cultured melanocytes, suggesting that they inhibit melanin production and/or melanosome integrity. A secondary mutation in the Smoky chicken, which reverts the dominant DW phenotype, prevents the accumulation of PMEL in fibrillogenic compartments and thus averts DW-associated pigment loss; a secondary mutation found in the Dun chicken likely dampens a HoSi-like dominant mutation in a similar manner. We propose that the DW and HoSi mutations alter the normally benign amyloid to a pathogenic form that antagonizes melanosome function, and that the secondary mutations found in the Smoky and Dun chickens revert or dampen pathogenicity by functioning as null alleles, thus preventing the formation of aberrant fibrils. We speculate that PMEL mutations can model the conversion between physiological and pathological amyloid.
Assuntos
Amiloide/biossíntese , Melaninas/biossíntese , Pigmentação/genética , Antígeno gp100 de Melanoma/genética , Antígeno gp100 de Melanoma/metabolismo , Sequência de Aminoácidos , Amiloide/genética , Animais , Células Cultivadas , Galinhas , Células HeLa , Cavalos , Humanos , Melaninas/genética , Melanócitos/ultraestrutura , Melanossomas/genética , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína/genética , Homologia de Sequência de AminoácidosRESUMO
The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membranas Intracelulares/metabolismo , Miosina Tipo I/metabolismo , Forma das Organelas , Rede trans-Golgi/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Miosina Tipo I/genética , Interferência de RNA , Receptor IGF Tipo 2 , Receptor de Fator de Crescimento Neural/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Toxinas Shiga/metabolismo , Fatores de Tempo , Transfecção , Rede trans-Golgi/ultraestruturaRESUMO
Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.
Assuntos
Benzamidas/farmacologia , Benzodiazepinonas/farmacologia , Citoproteção , Transporte Proteico , Ricina/antagonistas & inibidores , Tiofenos/farmacologia , Administração Intranasal , Animais , Benzamidas/química , Benzodiazepinonas/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Proteínas Qa-SNARE/metabolismo , Ricina/administração & dosagem , Ricina/toxicidade , Toxinas Shiga/antagonistas & inibidores , Toxinas Shiga/toxicidade , Tiofenos/química , Rede trans-Golgi/metabolismoRESUMO
Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans-Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes-TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069-7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.
Assuntos
Clatrina/metabolismo , Endossomos/metabolismo , Transporte Proteico , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Células HeLa , Humanos , Toxina Shiga/metabolismo , Nexinas de Classificação , Proteínas de Transporte Vesicular/metabolismoRESUMO
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.
Assuntos
Endossomos/metabolismo , Cinesinas/metabolismo , Melanossomas/metabolismo , Fator de Transcrição AP-1/metabolismo , Linhagem Celular , Endossomos/ultraestrutura , Humanos , Cinesinas/genética , Melanossomas/ultraestrutura , Microscopia Eletrônica , RNA Interferente Pequeno/genética , Fator de Transcrição AP-1/genéticaRESUMO
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.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/metabolismo , Melaninas/biossíntese , Melanócitos/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Microscopia de Fluorescência , Oxirredutases/biossíntese , Oxirredutases/genética , Fosfoproteínas/genética , Transporte Proteico , Fatores de Transcrição/genética , TransfecçãoRESUMO
Copper is a cofactor for many cellular enzymes and transporters. It can be loaded onto secreted and endomembrane cuproproteins by translocation from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes Menkes disease and Wilson disease, respectively. Endomembrane cuproproteins are thought to incorporate copper stably on transit through the trans-Golgi network, in which ATP7A accumulates by dynamic cycling through early endocytic compartments. Here we show that the pigment-cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the trans-Golgi network of mouse melanocytes. To catalyse melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a biogenesis of lysosome-related organelles complex-1 (BLOC-1)-dependent manner. These results indicate that cell-type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, because BLOC-1 subunits are mutated in subtypes of the genetic disease Hermansky-Pudlak syndrome, these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in Hermansky-Pudlak syndrome.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Melanossomas/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Tirosina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Cobre/farmacologia , ATPases Transportadoras de Cobre , Endossomos/metabolismo , Humanos , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanócitos/enzimologia , Melanócitos/metabolismo , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Camundongos , Especificidade de Órgãos , Proteínas Qa-SNARE/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.
Assuntos
Amiloide/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Amiloide/genética , Anticorpos/química , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/genética , Endossomos/ultraestrutura , Epitopos/genética , Epitopos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Melanossomas/genética , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Antígeno gp100 de MelanomaRESUMO
Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.
Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Toxina Shiga/metabolismo , Toxina Shiga/farmacologia , Animais , Endossomos/química , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Camundongos , Transporte Proteico/efeitos dos fármacos , Shigella dysenteriaeRESUMO
Previous studies have indicated a role for clathrin, the clathrin adaptors AP1 and epsinR, and the retromer complex in retrograde sorting from early/recycling endosomes to the trans Golgi network (TGN). However, it has remained unclear whether these protein machineries function on the same or parallel pathways. We show here that clathrin and the retromer subunit Vps26 colocalize at the ultrastructural level on early/recycling endosomes containing Shiga toxin B-subunit, a well-studied retrograde transport cargo. As previously described for clathrin, we find that interfering with Vps26 expression inhibits retrograde transport of the Shiga toxin B-subunit to the TGN. Under these conditions, endosomal tubules that take the Shiga toxin B-subunit out of transferrin-containing early/recycling endosomes appear to be stabilized. This situation differs from that previously described for low-temperature incubation and clathrin-depletion conditions under which Shiga toxin B-subunit labeling was found to overlap with that of the transferrin receptor. In addition, we find that the Shiga toxin B-subunit and the transferrin receptor accumulate close to multivesicular endosomes in clathrin-depleted cells, suggesting that clathrin initiates retrograde sorting on vacuolar early endosomes, and that retromer is then required to process retrograde tubules. Our findings thus establish a role for the retromer complex in retrograde transport of the B-subunit of Shiga toxin, and strongly suggest that clathrin and retromer function in consecutive retrograde sorting steps on early endosomes.
Assuntos
Clatrina/metabolismo , Endossomos/metabolismo , Transporte Proteico , Endossomos/ultraestrutura , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Toxina Shiga/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
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.
Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Vacúolos/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Endocitose , Endossomos/ultraestrutura , Humanos , Lisossomos/ultraestrutura , Melaninas/biossíntese , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/metabolismo , Proteínas Mutantes/metabolismo , Oxirredutases/química , Pigmentação/fisiologia , Transporte Proteico , Proteínas Qa-SNARE/metabolismo , Vacúolos/ultraestruturaRESUMO
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.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Antígenos CD/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Células HeLa , Humanos , Imunoprecipitação , Melanócitos/citologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Biológicos , Oxirredutases/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Transporte Proteico , Tetraspanina 30RESUMO
Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.
Assuntos
Endocitose , Retículo Endoplasmático/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação/genética , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células HeLa , Humanos , Melanossomas/ultraestrutura , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Antígeno gp100 de MelanomaRESUMO
Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.
Assuntos
Endossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Organelas/metabolismo , Transporte Proteico , Vesículas Transportadoras/metabolismo , Antígenos de Neoplasias , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Antígeno MART-1 , Melanossomas/metabolismo , Glicoproteínas de Membrana/genética , Morfogênese , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Antígeno gp100 de MelanomaRESUMO
Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.
Assuntos
Endossomos/metabolismo , Miosina Tipo I/metabolismo , Citoesqueleto de Actina/metabolismo , Citocalasina B/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Humanos , Melanossomas/ultraestrutura , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão , Frações SubcelularesRESUMO
Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.
Assuntos
Complexo 1 de Proteínas Adaptadoras/fisiologia , Complexo 3 de Proteínas Adaptadoras/fisiologia , Endossomos/metabolismo , Melanossomas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Melanócitos/metabolismo , Melanócitos/ultraestrutura , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Transfecção , Células Tumorais CultivadasRESUMO
Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.