RESUMEN
The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13-Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.
Asunto(s)
Retículo Endoplásmico/metabolismo , Membranas Mitocondriales/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/genética , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane-localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C-terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post-ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Quitina Sintasa/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quitina Sintasa/genética , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Current models suggest that TRAPP tethering complexes exist in two forms. Whereas the seven-subunit TRAPPI complex mediates ER-to-Golgi transport, TRAPPII contains three additional subunits (Trs65, Trs120 and Trs130) and is required for distinct tethering events at Golgi membranes. It is not clear how TRAPPII assembly is regulated. Here, we show that Tca17 is a fourth TRAPPII-specific component, and that Trs65 and Tca17 interact with distinct domains of Trs130 and make different contributions to complex assembly. Whereas Tca17 promotes the stable association of TRAPPII-specific subunits with the core complex, Trs65 stabilizes TRAPPII in an oligomeric form. We show that Trs85, which was previously reported to be a subunit of both TRAPPI and TRAPPII, is not associated with the TRAPPII complex in yeast. However, we find that proteins related to Trs85, Trs65 and Tca17 are part of the same TRAPP complex in mammalian cells. These findings have implications for models of TRAPP complex formation and suggest that TRAPP complexes may be organized differently in yeast and mammals.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Animales , Humanos , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.
Asunto(s)
Endocitosis/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Actinas/metabolismo , Animales , Membrana Celular/metabolismo , Clatrina/genética , Clatrina/metabolismo , Perfilación de la Expresión Génica , Análisis por Micromatrices , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Familia de Multigenes , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.
Asunto(s)
Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Activo , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Fúngicos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Familia de Multigenes , Complejos Multiproteicos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores del Factor de Conjugación/genética , Receptores del Factor de Conjugación/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte VesicularRESUMEN
The endosomal sorting complex required for transport-I (ESCRT-I) complex, which is conserved from yeast to humans, directs the lysosomal degradation of ubiquitinated transmembrane proteins and the budding of the HIV virus. Yeast ESCRT-I contains four subunits, Vps23, Vps28, Vps37, and Mvb12. The crystal structure of the heterotetrameric ESCRT-I complex reveals a highly asymmetric complex of 1:1:1:1 subunit stoichiometry. The core complex is nearly 18 nm long and consists of a headpiece attached to a 13 nm stalk. The stalk is important for cargo sorting by ESCRT-I and is proposed to serve as a spacer regulating the correct disposition of cargo and other ESCRT components. Hydrodynamic constraints and crystallographic structures were used to generate a model of intact ESCRT-I in solution. The results show how ESCRT-I uses a combination of a rigid stalk and flexible tethers to interact with lipids, cargo, and other ESCRT complexes over a span of approximately 25 nm.