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ABSTRACT: Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for lysosome-related organelle (LRO) biogenesis. PMEL-a component of melanocyte LROs (melanosomes)-is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.
Developmental cell 09/2011; 21(4):708-21. · 13.36 Impact Factor
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ABSTRACT: The function of signaling receptors is tightly controlled by their intracellular trafficking. One major regulatory mechanism within the endo-lysosomal system required for receptor localization and down-regulation is protein modification by ubiquitination and downstream interactions with the endosomal sorting complex responsible for transport (ESCRT) machinery. Whether and how these mechanisms operate to regulate endosomal sorting of mammalian G protein-coupled receptors (GPCRs) remains unclear. Here, we explore the involvement of ubiquitin and ESCRTs in the trafficking of OA1, a pigment cell-specific GPCR, target of mutations in Ocular Albinism type 1, which localizes intracellularly to melanosomes to regulate their biogenesis. Using biochemical and morphological methods in combination with overexpression and inactivation approaches we show that OA1 is ubiquitinated and that its intracellular sorting and down-regulation requires functional ESCRT components. Depletion or overexpression of subunits of ESCRT-0, -I, and -III markedly inhibits OA1 degradation with concomitant retention within the modified endosomal system. Our data further show that OA1 ubiquitination is uniquely required for targeting to the intralumenal vesicles of multivesicular endosomes, thereby regulating the balance between down-regulation and delivery to melanosomes. This study highlights the role of ubiquitination and the ESCRT machinery in the intracellular trafficking of mammalian GPCRs and has implications for the physiopathology of ocular albinism type 1.
Proceedings of the National Academy of Sciences 07/2011; 108(29):11906-11. · 9.68 Impact Factor
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Steven T Truschel, Sabrina Simoes,
Subba Rao Gangi Setty,
Dawn C Harper,
Danièle Tenza,
Penelope C Thomas,
Kathryn E Herman,
Sara D Sackett,
David C Cowan,
Alexander C Theos,
Graça Raposo,
Michael S Marks
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ABSTRACT: 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.
Traffic 07/2009; 10(9):1318-36. · 4.92 Impact Factor
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ABSTRACT: TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrP(Sc), a conformationally altered isoform of the normal prion protein (PrP(C)), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a).
The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome-like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome-like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs.
The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.
Biology of the Cell 05/2008; 100(10):603-15. · 3.60 Impact Factor
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ABSTRACT: Exosomes are membrane vesicles that are released by cells upon fusion of multivesicular bodies with the plasma membrane. Their molecular composition reflects their origin in endosomes as intraluminal vesicles. In addition to a common set of membrane and cytosolic molecules, exosomes harbor unique subsets of proteins linked to cell type-associated functions. Exosome secretion participates in the eradication of obsolete proteins but several findings, essentially in the immune system, indicate that exosomes constitute a potential mode of intercellular communication. Release of exosomes by tumor cells and their implication in the propagation of unconventional pathogens such as prions suggests their participation in pathological situations. These findings open up new therapeutic and diagnostic strategies.
Journal of Biochemistry 08/2006; 140(1):13-21. · 2.37 Impact Factor
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ABSTRACT: Exosomes are membrane vesicles that are released by cells upon fusion of multivesicular bodies with the plasma membrane. Their molecular composition reflects their origin in endosomes as intraluminal vesicles. In addition to a common set of membrane and cytosolic molecules, exosomes harbor unique subsets of proteins linked to cell type– associated functions. Exosome secretion participates in the eradication of obsolete pro-teins but several findings, essentially in the immune system, indicate that exosomes constitute a potential mode of intercellular communication. Release of exosomes by tumor cells and their implication in the propagation of unconventional pathogens such as prions suggests their participation in pathological situations. These findings open up new therapeutic and diagnostic strategies. Multivesicular bodies (MVBs), and their intraluminal vesi-cles (ILVs), are involved in the sequestration of proteins destined for degradation in lysosomes (1). An alternative fate of MVBs is their exocytic fusion with the plasma mem-brane leading to the release of the 50–90 nm ILVs into the extracellular milieu (Fig. 1). The secreted ILVs are then called exosomes (reviewed in Refs. 2 and 3). After their initial description as vesicles of endosomal origin secreted by reticulocytes during differentiation (4), vesicles with the hallmarks of exosomes appeared to be released by other cells. Exosomes are present in the culture superna-tant of several cell types of hematopoietic origin [B cells (5), dendritic cells (6), mast cells (7), T cells (8) and platelets (9)] and of non hematopoietic origin [intestinal epithelial cells (10), tumor cells (11), Schwann cells (12) and neuronal cells (13)]. In addition there is increasing evidence for the presence of exosomes in physiological fluids such as plasma (14), malignant and pleural effusions (15, 16) and urine (17). As a consequence of proteins and lipids sorting at the limiting membrane of endosomes during the formation of the ILVs in MVBs, exosomes harbour a specific set of molecules. The sorting process and the generation of ILVs require the recognition of cargo proteins by a series of multiprotein complexes that form the ESCRT machinery [for review (18)] (Fig. 2). There is increasing evidence, how-ever, that cargo sorting and MVB generation is not solely dependent on ESCRT components. The mechanisms lead-ing to the fusion of MVBs with the plasma membrane and the consequent release of exosomes are unknown,
Journal of Biochemistry 01/2006; 140:13-21. · 2.37 Impact Factor
