Protein Interactions Controlling ER/Golgi Transport

描述（由申请方提供）：正常的细胞功能和避免疾病需要亚细胞靶向以及转运囊泡与受体膜的融合。囊泡和受体膜之间的初始识别是由外周膜系链蛋白复合物介导的，它们的特异性融合是由整合膜SNARE复合物介导的。虽然这些蛋白质家族的一般功能的建立，蛋白质的相互作用介导的拴系事件的具体识别和融合前的膜粘附在很大程度上是未知的，我们的知识的监管关系连接和整合系链和陷阱功能是非常基本的。此外，细胞内膜融合步骤在许多情况下由管腔钙离子的释放调节，甚至依赖于管腔钙离子的释放，然而这些重要作用在特定转运步骤中的记录、介导钙释放的机制的鉴定以及连接钙与系链和SNARE功能的途径的阐明仍然难以捉摸。我们开发了一种体外重建系统，用于阐明分泌途径中第一个膜融合步骤中的系链和SNARE功能，即COPII囊泡的同型融合以产生VTC，这是以前未研究的步骤。我们将结合体内成像技术使用我们的体外重建来解决以下具体目标：1）测试同型COPII囊泡系链是涉及SNARE信号化系链募集和顺序作用多个系链分子的多层过程的假设。2)测试钙负调节同型COPII囊泡融合的假设。3)检验以下假设：每个细胞器固有的腔钙浓度是早期分泌途径中膜转运的重要决定因素。与公共卫生的关系：COPII囊泡的同型融合产生VTC，这是一种在分泌途径质量控制中具有核心作用的细胞器。多种疾病，包括严重的神经病变，可能是由于错误折叠的分泌蛋白的不适当的VTC运输，VTC最近被牵连在病毒颗粒的生物发生和其他功能。对VTC生物发生的坚定理解是理解VTC在致病状态中的作用的核心。

DESCRIPTION (provided by applicant): Proper cell function and avoidance of disease requires subcellular targeting and fusion of transport vesicles with acceptor membranes. The initial recognition between vesicles and acceptor membranes is mediated by peripheral membrane tether protein complexes, and their specific fusion is mediated by integral membrane SNARE complexes. Although the general function of each of these protein families is established, the protein interactions mediating a tethering event-the specific recognition and adhesion of membranes prior to fusion-are largely unknown and our knowledge of the regulatory relationships linking and integrating tether and SNARE function is very rudimentary. Furthermore, intracellular membrane fusion steps are in many cases regulated by and even dependent upon the release of luminal calcium ions, yet documentation of these important roles in specific transport steps, identification of the machinery mediating calcium release and elucidation of the pathways connecting calcium to tether and SNARE function remain elusive. We developed an in vitro reconstitution system for elucidation of tether and SNARE function in the first membrane fusion step in the secretory pathway, the homotypic fusion of COPII vesicles to generate VTCs, a step that was previously unstudied. We will use our in vitro reconstitution in conjunction with in vivo imaging techniques to address the following specific aims: 1) Test the hypothesis that homotypic COPII vesicle tethering is a multi-layered process involving SNARE-signaled tether recruitment and sequential action multiple tether molecules. 2) Test the hypothesis that calcium negatively regulates homotypic COPII vesicle fusion. 3) Test the hypothesis that the luminal calcium concentration intrinsic to each organelle is an important determinant of membrane transport in the early secretory pathway. RELEVANCE TO PUBLIC HEALTH: The homotypic fusion of COPII vesicles produces VTCs, an organelle with a central role in secretory pathway quality control. Multiple diseases, including severe neuropathies, can result from improper VTC trafficking of misfolded secretory proteins, and VTCs have recently been implicated in the biogenesis of viral particles and other functions. A firm understanding of VTC biogenesis is central to an understanding of the role of VTCs in pathogenic states.