PROTEIN INTERACTIONS CONTROLLING ER/GOLGI TRANSPORT

转运囊泡与靶膜的对接和融合似乎是由囊泡和靶膜SNAP受体(SNARs)之间强烈的寡聚体蛋白复合体介导的。哺乳动物内质网到高尔基体转运的陷阱机制还不是很清楚。了解内质网/高尔基体圈套蛋白的相互作用及其功能作用将促进我们对细胞如何处理任何膜转运事件的一般知识。它还将提供特定的知识和试剂，可能被用来操纵ER到高尔基体的运输。在某些情况下，阻断或减少分泌可能在医学上是有益的，例如在病毒感染和肿瘤分泌自刺激剂期间。拟议的研究将描述哺乳动物ER/高尔基圈套合成素5、GOS-28、膜蛋白、rsec22b、rbet1等之间的蛋白质相互作用。使用可溶的、纯化的重组圈套的结合分析将用于确定这组圈套中的所有直接结合事件。将绘制每个交互作用的结构决定因素图。这些陷阱之间的结合协作性模式将表明哪些蛋白质子集同时组装成指导融合事件的更高阶功能复合体。这项工作将表征内质网/高尔基体复合体(ES)的亚基组成和化学计量比。这项工作还试图确定内质网/高尔基体陷阱相互作用的功能角色(S)。将开发抑制或破坏特定套圈套相互作用的试剂。破坏相互作用的生理后果将使用体外内质网到高尔基体运输重建来探索。需要不同陷阱相互作用的运输阶段将通过光学显微镜和亚细胞分离来表征。

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.