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1.
Traffic ; 14(2): 219-32, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23167963

RESUMO

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo-lysosomal pathway of human cells. By expressing hemagglutinin (HA)-tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)-mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal-lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells.


Assuntos
Endossomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fusão de Membrana/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia , Catepsina B/metabolismo , Endocitose , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Proteólise , RNA Interferente Pequeno , Proteínas de Transporte Vesicular/genética
2.
Traffic ; 10(10): 1528-42, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19682328

RESUMO

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide-loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen-loaded DCs, which is triggered by cognately interacting antigen-specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent-resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Exossomos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Animais , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Exossomos/imunologia , Exossomos/ultraestrutura , Antígenos de Histocompatibilidade Classe II/genética , Imunoprecipitação , Lisossomos/imunologia , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Corpos Multivesiculares/imunologia , Corpos Multivesiculares/ultraestrutura , Transporte Proteico , Baço/citologia , Ubiquitinação
3.
Exp Cell Res ; 315(9): 1584-92, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18930046

RESUMO

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.


Assuntos
Antígenos CD/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Complexo 3 de Proteínas Adaptadoras/metabolismo , Animais , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Superfície/metabolismo , Cavéolas/metabolismo , Exossomos/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Neutrófilos/metabolismo , Células Parietais Gástricas/metabolismo , Transporte Proteico/fisiologia , Sinteninas/metabolismo , Linfócitos T/metabolismo , Tetraspanina 30
4.
Nat Commun ; 4: 1361, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23322049

RESUMO

Targeted delivery of lysosome-associated membrane proteins is important for lysosome stability and function. Here we identify a pathway for transport of lysosome-associated membrane proteins directly from the trans-Golgi network to late endosomes, which exists in parallel to mannose 6-phosphate receptor and clathrin-dependent transport of lysosomal enzymes to early endosomes. By immunoelectron microscopy we localized endogenous LAMP-1 and -2 as well as LAMP-1-mGFP to non-coated, biosynthetic carriers at the trans-Golgi network and near late endosomes. These LAMP carriers were negative for mannose 6-phosphate receptor, adaptor-protein complex-1, secretory albumin and endocytic markers, but contained the homotypic fusion and protein sorting complex component hVps41 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein VAMP7. Knockdown of hVps41 or VAMP7 resulted in the accumulation of lysosome-associated membrane protein carriers, whereas knockdown of hVps39 or hVps18 did not, indicating that the effect of hVps41 is independent of CORVET/HOPS. Mannose 6-phosphate receptor carriers remained unaffected upon hVps41 or VAMP7 knockdown, implicating that hVps41 and VAMP7 are specifically involved in the fusion of trans-Golgi network-derived lysosome-associated membrane protein carriers with late endosomes.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Animais , Clatrina/metabolismo , Endossomos/ultraestrutura , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Células Hep G2 , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Modelos Biológicos , Transporte Proteico , Ratos , Receptor IGF Tipo 2/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Rede trans-Golgi/ultraestrutura
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