Protein Interactions Controlling ER/Golgi Transport

控制 ER/高尔基体运输的蛋白质相互作用

• 批准号: 7935882
• 负责人: JESSE C HAY
• 金额: $ 11.47万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935882
• 关键词: Address; Adhesions; Back; Biochemical; Biogenesis; Calcium; Calcium ion; Cell physiology; Complex; Confusion; Disease; Docking; Documentation; Drops; Environment; Event; Golgi Apparatus; Imaging Techniques; In Vitro; Individual; Intracellular Membranes; Intrinsic factor; Knowledge; Link; Mammals; Mediating; Membrane; Membrane Fusion; Neuropathy; Organelles; Pathway interactions; Peripheral; Physiological; Play; Process; Protein Family; Protein Secretion; Proteins; Public Health; Pus; Quality Control; Regulation; Role; SNAP receptor; Signal Transduction; Site; Specific qualifier value; Staging; System; Testing; Transmembrane Transport; Transport Vesicles; Tubular formation; Vesicle; Viral; in vivo; interest; particle; prevent; protein complex; protein transport; reconstitution; secretory protein; sensor; trafficking

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.

描述（由申请人提供）：适当的细胞功能和避免疾病需要亚细胞靶向和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体膜SNARE复合物介导的。尽管这些蛋白家族的一般功能已经确定，但介导系绳事件的蛋白相互作用——在融合之前对膜的特异性识别和粘附——在很大程度上是未知的，我们对系绳和SNARE功能连接和整合的调节关系的了解非常初级。此外，细胞膜内融合步骤在许多情况下受腔内钙离子释放的调节，甚至依赖于腔内钙离子的释放，但这些在特定运输步骤中的重要作用的文献记录，介导钙释放的机制的鉴定以及钙与系绳和SNARE功能连接的途径的阐明仍然难以捉摸。我们开发了一个体外重构系统来阐明tether和SNARE在分泌途径的第一个膜融合步骤中的功能，即COPII囊泡产生VTCs的同型融合，这一步骤以前未被研究过。我们将使用体外重构和体内成像技术来解决以下具体目标：1)验证假设，即同型COPII囊泡系泊是一个涉及snare信号系泊募集和顺序作用多个系泊分子的多层过程。2)验证钙负调控COPII同型囊泡融合的假说。3)验证各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的假设。与公共卫生相关：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，包括严重的神经病变，可由错误折叠的分泌蛋白的VTC运输不当引起，VTCs最近被认为与病毒颗粒的生物发生和其他功能有关。对VTC生物发生机制的坚定理解对于理解VTC在致病状态中的作用至关重要。