Protein Interactions Controlling ER/Golgi Transport

描述(由申请人提供)：正确的细胞功能和避免疾病需要亚细胞靶向和运输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留蛋白复合体介导的，它们的特异性融合是由完整的膜圈套复合体介导的。尽管这些蛋白质家族的一般功能已经确定，但调节系链事件的蛋白质相互作用-融合前膜的特定识别和黏附-在很大程度上是未知的，我们对连接和整合系链和圈套功能的调控关系的了解非常初级。此外，在许多情况下，细胞膜融合步骤受钙离子释放的调控，甚至依赖于腔钙离子的释放，但这些重要的作用在特定的运输步骤中的记录、介导钙释放的机制的鉴定以及钙与系绳和圈套功能之间的联系仍然是未知的。我们建立了一个体外重建系统，用于阐明在分泌途径的第一个膜融合步骤中的系绳和圈套功能，这是以前没有研究过的一个步骤，即COPII囊泡的同型融合产生VTC。我们将结合我们的体外重建和体内成像技术来解决以下具体目标：1)测试同型COPII囊泡拴系是一个多层过程的假设，该过程涉及陷阱信号的系留招募和顺序作用的多个系留分子。2)检验钙负调控同型COPII囊泡融合的假说。3)验证这一假设，即每个细胞器固有的腔内钙浓度是早期分泌途径中膜运输的重要决定因素。与公共健康相关：COPII囊泡的同型融合产生VTCs，这是一种在分泌途径质量控制中起核心作用的细胞器。多种疾病，包括严重的神经病，可能是由于VTC错误地运输错误折叠的分泌蛋白而引起的，VTC最近被认为与病毒颗粒的生物发生和其他功能有关。对VTC生物发生的确切理解是理解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.