PROTEIN INTERACTIONS CONTROLLING ER/GOLGI TRANSPORT

控制内质网/高尔基体运输的蛋白质相互作用

• 批准号: 6181475
• 负责人: JESSE C HAY
• 金额: $ 18.19万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181475
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; endoplasmic reticulum; enzyme linked immunosorbent assay; high performance liquid chromatography; immunoprecipitation; laboratory mouse; laboratory rabbit; light microscopy; membrane permeability; monoclonal antibody; nucleic acid sequence; polymerase chain reaction; protein binding; protein isoforms; protein protein interaction; protein structure function; protein transport; stoichiometry; syntaxin; western blottings

Transport vesicle docking and fusion with target membranes appears to be mediated by strong, oligomeric protein complexes between vesicle and target membrane SNAP receptors (SNARES). SNARE mechanisms in mammalian ER to Golgi transport are not well known. Understanding ER/Golgi SNARE protein interactions and their functional roles will advance our general knowledge about how cells handle any membrane trafficking event. It will also provide specific knowledge and reagents that can potentially be used to manipulate ER to Golgi transport. In some instances it may be medically beneficial to block or reduce secretion, for example during viral infection and secretion of autostimulatory agents by tumors. The proposed studies will characterize protein interactions among the mammalian ER/Golgi SNARES syntaxin 5, GOS-28, membrin, rsec22b, rbet1 and others. Binding assays employing soluble, purified recombinant SNAREs will be used to define all of the direct binding events among this set of SNARES. Structural determinants for each interaction will be mapped. The pattern of binding cooperativity among these SNARES will suggest which subsets of proteins assemble simultaneously into higher-order functional complexes that direct fusion events. The proposed work will characterize the subunit composition and stoichiometry of ER/Golgi SNARE complex(es). The work also seeks to determine the functional role(s) of ER/Golgi SNARE interactions. Reagents will be developed that inhibit or disrupt particular sets of SNARE interactions. The physiological consequences of disrupting the interactions will be explored using an in vitro ER to Golgi transport reconstitution. The stage in transport at which different SNARE interactions are required will be characterized by light microscopy and subcellular fractionation.

转运囊泡对接和与靶膜的融合似乎是由囊泡和靶膜捕捉受体（SNARES）之间的强，寡聚蛋白复合物（SNARES）介导的。哺乳动物ER到高尔基体运输的圈圈机制尚不清楚。 了解ER/Golgi Snare蛋白相互作用及其功能作用将提高我们对细胞如何处理任何膜运输事件的一般知识。 它还将提供特定的知识和试剂，这些知识和试剂有可能用于操纵高尔基运输。 在某些情况下，阻塞或减少分泌可能在医学上是有益的，例如在病毒感染和通过肿瘤对自动刺激剂的分泌。 拟议的研究将表征哺乳动物ER/Golgi Snares语法5，GOS-28，膜，RSEC22B，RBET1等之间的蛋白质相互作用。 使用可溶的，纯化的重组网罗的结合测定将用于定义这组网罗中的所有直接结合事件。将映射每种相互作用的结构决定因素。 这些小网罗之间结合合作的模式将表明哪些蛋白质的亚集同时组装成直接融合事件的高阶功能复合物中。 提出的工作将表征ER/Golgi Snare Complex（ES）的亚基组成和化学计量。 这项工作还旨在确定ER/Golgi Snare相互作用的功能作用。 将开发抑制或破坏特定的网罗相互作用集的试剂。 破坏相互作用的生理后果将使用高尔基运输重建的体外探索。 需要不同的圈圈相互作用的转运阶段将以光学显微镜和亚细胞分级为特征。