PROTEIN INTERACTIONS CONTROLLING ER/GOLGI TRANSPORT

运输囊泡与靶膜的对接和融合似乎是由囊泡和靶膜SNAP受体（SNARES）之间的强寡聚蛋白复合物介导的。哺乳动物内质网向高尔基体转运的SNARE机制尚不清楚。了解内质网/高尔基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认识。它还将提供特定的知识和试剂，可以潜在地用于操纵内质网到高尔基转运。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身刺激剂期间。拟建的研究将表征哺乳动物ER/高尔基SNARES syntaxin 5、GOS-28、membrin、rsec22b、rbet1等蛋白之间的相互作用。结合试验采用可溶性的、纯化的重组SNAREs，将用于确定这组SNAREs之间的所有直接结合事件。每个相互作用的结构决定因素将被映射。这些SNARES之间的结合协同模式将提示哪些蛋白质亚群同时组装成指导融合事件的高阶功能复合物。所提出的工作将表征ER/高尔基网罗复合物的亚基组成和化学计量学。这项工作还试图确定内质网/高尔基网相互作用的功能作用。将开发抑制或破坏特定SNARE相互作用集的试剂。破坏相互作用的生理后果将探讨使用体外内质网高尔基转运重建。转运过程中需要不同SNARE相互作用的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.