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: 8273265
• 负责人: RUTILIO A FRATTI
• 金额: $ 26.73万
• 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/8273265
• 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. PUBLIC HEALTH RELEVANCE: Dysfunctional membrane fusion can have severe consequences to human health, and defects are found in many genetic and infectious diseases that are too numerous to list here. However, suffice it to say that proper membrane fusion is essential for cell viability and cellular functions that include the release of neurotransmitters, hormones and antibodies. Therefore, understanding the fundamental mechanisms of membrane fusion is critical if we are to understand the diseases that target this machinery.

描述（由申请人提供）：真核细胞由膜结合的细胞器划分，这些细胞器通过载有膜融合中的货物囊泡的运输来相互通信。我们使用从酿酒酵母分离的液泡溶酶体检查膜融合。液泡融合需要一组调节脂质，包括磷酸肌醇，麦角固醇，磷脂酸和二酰基甘油。融合发生在膜微区域中，其中调节脂质与蛋白质相互依存，催化融合蛋白，包括鼻虫，Rab GTPase YPT7链接复合物啤酒花和肌动蛋白。由于脂质双层通过脂质修饰经历了恒定的重塑，因此我们假设脂质物种的代谢互转换与膜融合完全耦合。在此提案中，我们研究了液泡膜的脂质组成与网罗的功能以及RAB7/YPT7核苷酸交换因子MON1之间的关系。我们计划通过追求以下目的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，以及如何通过局部脂质环境控制这些结构域； 2-确定脂质组成对编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RAB7/YPT7核苷酸交换因子MON1的募集和激活之间的关系。公共卫生照：膜融合对于细胞活力，神经传递，抗体和激素的分泌，血液凝结以及微生物的破坏至关重要。脂质代谢/修饰的失调与许多疾病有关，包括肉食 - 玛丽齿病，肥胖，2型糖尿病和心脏病。在每种情况下，膜运输和融合都受到有害影响，但是将脂质修饰和融合机械调节的机制仍然未知。拟议的研究将定义液泡膜的组成如何调节SNARE和MON1功能，并将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。 公共卫生相关性：功能失调的膜融合可能会对人类健康造成严重后果，并且在许多遗传和传染病中发现缺陷，这些疾病太多了，在这里列出。但是，可以说适当的膜融合对于细胞活力和细胞功能至关重要，包括释放神经递质，激素和抗体。因此，如果我们要了解针对这种机械的疾病，了解膜融合的基本机制至关重要。