Allosteric Regulation of SNAREs and the Rab GEF Mon1 by Phosphoinositides

描述（由申请方提供）：真核细胞由膜结合细胞器分隔，这些细胞器通过转运货物囊泡相互通信，最终导致膜融合。我们使用从酿酒酵母中分离的空泡溶酶体来检查膜融合。极性融合需要一组调节脂质，包括磷酸肌醇、麦角固醇、磷脂酸和甘油二酯。融合发生在膜微区，其中调节脂质与催化融合的蛋白质相互依赖地组装，所述蛋白质包括SNARE、Rab GT3 Ypt 7、系链复合物HOPS和肌动蛋白。由于脂质双分子层通过脂质修饰进行不断的重塑，我们假设脂质物质的代谢相互转化与膜融合是一体化的。在这个建议中，我们研究液泡膜的脂质成分和SNARE的功能以及Rab 7/Ypt 7核苷酸交换因子Mon 1之间的关系。我们计划通过以下目标阐明脂质组成的作用和液泡融合的调节：1 -检查SNARE跨膜结构域在融合过程中的功能以及这些结构域如何受局部脂质环境的控制：2 -确定脂质组成对SNARE复合物形成的亲和力动力学的作用; 3 -检查脂质磷脂酰肌醇3-磷酸与Rab 7/Ypt 7核苷酸交换因子Mon 1的募集和激活之间的关系。公共卫生关系：膜融合对于细胞活力、神经传递、抗体和激素的分泌、血液凝固和微生物的破坏是必不可少的。脂质代谢/修饰的失调与许多疾病相关，包括夏科-玛丽-图思病、肥胖症、2型糖尿病和心脏病。在每种情况下，膜运输和融合受到不利影响，但连接脂质修饰和融合机制的调节机制仍然未知。这项研究将确定液泡膜的组成如何调节SNARE和Mon 1功能，并为我们提供有关脂质修饰与人类健康和疾病之间关系的线索。

DESCRIPTION (provided by applicant): Eukaryotic cells are compartmentalized by membrane-bound organelles, which communicate with each other through the transport of cargo vesicles that culminates in membrane fusion. We use vacuolar lysosomes isolated from the yeast Saccharomyces cerevisiae to examine membrane fusion. Vacuolar fusion requires a group of regulatory lipids that includes phosphoinositides, ergosterol, phosphatidic acid and diacylglycerol. Fusion occurs at membrane microdomains where regulatory lipids interdependently assemble with the proteins that catalyze fusion including SNAREs, the Rab GTPase Ypt7 the tethering complex HOPS, and actin. Because the lipid bilayer undergoes constant remodeling through lipid modification, we hypothesize that the metabolic interconversion of lipid species is integrally coupled to membrane fusion. In this proposal we examine the relationship between the lipid composition of vacuolar membranes and the function of SNAREs as well as the Rab7/Ypt7 nucleotide exchange factor Mon1. We plan to elucidate the role of lipid composition and the regulation of vacuole fusion by pursuing the following aims: 1 - Examine the function of SNARE trans-membrane domains during fusion and how these domains are controlled by the local lipid environment; 2 - Determine the role of lipid composition on kinetics of affinities of SNARE complex formation; 3 - Examine the relationship between the lipid phosphatidylinositol 3-phosphate and the recruitment and activation of the Rab7/Ypt7 nucleotide exchange factor Mon1. PUBLIC HEALTH RELAVANCE: Membrane fusion is essential for cell viability, neurotransmission, the secretion of antibodies and hormones, blood clotting, and the destruction of microbes. Dysregulation of lipid metabolism/modification is tied t many diseases including Charcot- Marie-Tooth disease, obesity, type-2 diabetes, and heart disease. In each case, membrane trafficking and fusion is deleteriously affected, yet the mechanisms that link lipid modification and the regulation of the fusion machinery remain unknown. The proposed study will define how the composition of the vacuolar membrane regulates SNARE and Mon1 function and will give us clues about the relationship between lipid modification and human health and disease.