Allosteric Regulation of SNAREs and the Rab GEF Mon1 by Phosphoinositides

磷酸肌醇对 SNARE 和 Rab GEF Mon1 的变构调节

• 批准号: 8460003
• 负责人: RUTILIO A FRATTI
• 金额: $ 27.94万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460003
• 关键词: 1,2-diacylglycerol; 1-Phosphatidylinositol 3-Kinase; Actins; Affect; Affinity; Allosteric Regulation; Antibodies; Binding; Biochemical; Blood Clot; Blood coagulation; Cell Survival; Cell physiology; Charcot-Marie-Tooth Disease; Chimeric Proteins; Communicable Diseases; Complex; Controlled Environment; Coupled; Defect; Development; Diglycerides; Disease; Environment; Equilibrium; Ergosterol; Eukaryota; Eukaryotic Cell; Event; Family; Guanine Nucleotide Exchange Factors; Health; Heart Diseases; Hereditary Disease; Homeostasis; Homologous Gene; Hormones; Human; Insulin Resistance; Kinetics; Laboratories; Lead; Lecithin; Length; Link; Lipase; Lipid Bilayers; Lipids; Lysosomes; Mammals; Mediating; Membrane; Membrane Fluidity; Membrane Fusion; Membrane Microdomains; Membrane Protein Traffic; Metabolic; Microbe; Modeling; Modification; Mutation; Neuropathy; Non-Insulin-Dependent Diabetes Mellitus; Nucleotides; Obesity; Organelles; Peripheral Nervous System; Phosphatidic Acid; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiology; Proteins; Regulation; Role; SNAP receptor; Saccharomyces cerevisiae; Signal Transduction; Sphingolipids; System; Testing; Thick; Transmembrane Domain; Vacuole; Vesicle; Yeasts; chemical property; disorder subtype; late endosome; lipid metabolism; lipid transport; loss of function mutation; neurotransmission; neurotransmitter release; phosphatidylinositol 3-phosphate; physical property; protein function; protein protein interaction; rab GTP-Binding Proteins; rho GTP-Binding Proteins; stoichiometry; trafficking

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.

描述（由申请人提供）：真核细胞由膜结合细胞器分隔，这些细胞器通过运输货物囊泡相互通信，最终导致膜融合。我们使用从酿酒酵母中分离的液泡溶酶体来检查膜融合。液泡融合需要一组调节脂质，包括磷酸肌醇、麦角甾醇、磷脂酸和二酰基甘油。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，包括 SNARE、Rab GTPase Ypt7、束缚复合物 HOPS 和肌动蛋白。由于脂质双层通过脂质修饰不断重塑，我们假设脂质种类的代谢相互转化与膜融合整体耦合。在本提案中，我们研究了液泡膜的脂质组成与 SNARE 以及 Rab7/Ypt7 核苷酸交换因子 Mon1 的功能之间的关系。我们计划通过以下目标来阐明脂质组成的作用和液泡融合的调节： 1 - 检查融合过程中 SNARE 跨膜结构域的功能以及这些结构域如何受局部脂质环境控制； 2 - 确定脂质成分对 SNARE 复合物形成亲和力动力学的作用； 3 - 检查脂质磷脂酰肌醇 3-磷酸与 Rab7/Ypt7 核苷酸交换因子 Mon1 的招募和激活之间的关系。公共健康相关性：膜融合对于细胞活力、神经传递、抗体和激素的分泌、血液凝固以及微生物的破坏至关重要。脂质代谢/修饰失调与许多疾病有关，包括腓骨肌萎缩症、肥胖、2 型糖尿病和心脏病。在每种情况下，膜运输和融合都会受到有害影响，但连接脂质修饰和融合机制调节的机制仍然未知。这项研究将明确液泡膜的组成如何调节 SNARE 和 Mon1 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。