Allosteric Regulation of SNAREs and the Rab GEF Mon1 by Phosphoinositides

描述(申请人提供)：真核细胞由膜结合的细胞器分隔，这些细胞器通过运输最终达到膜融合的货运小泡相互通信。我们使用从酿酒酵母中分离的空泡溶酶体来检查膜融合。空泡融合需要一组调节脂质，包括磷脂酰肌醇、麦角固醇、磷脂酸和二酰甘油。融合发生在膜微域，在那里调节脂与催化融合的蛋白质相互依赖地组装，包括SNARS、Rab GTP酶Ypt7、系留复合体HOP和肌动蛋白。由于脂质双层通过脂类修饰进行不断的重塑，我们假设脂类之间的代谢相互转化与膜融合是完整耦合的。在这项建议中，我们研究了液泡膜的脂质组成与SNARS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标来阐明脂质组成的作用和空泡融合的调节：1-检测融合过程中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的控制；2-确定脂质组成对SNARE复合体形成亲和力的作用；3-检测脂质磷脂酰肌醇3-磷酸与Rab7/Ypt7核苷酸交换因子Mon1的募集和激活的关系。公共卫生相关性：膜融合对于细胞存活、神经传递、抗体和激素的分泌、血液凝结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有关，包括夏科-玛丽-图斯病、肥胖、2型糖尿病和心脏病。在每一种情况下，膜运输和融合都受到有害的影响，但将脂质修饰和融合机制调节联系在一起的机制仍不清楚。这项拟议的研究将确定液泡膜的组成如何调节SNARE和Mon1的功能，并将为我们提供关于脂质修饰与人类健康和疾病之间的关系的线索。 与公共卫生相关：膜融合功能障碍可能会对人类健康造成严重后果，许多遗传和传染病中都发现了缺陷，这里无法列出这些缺陷。然而，可以说，适当的膜融合对于细胞存活和细胞功能是必不可少的，包括神经递质、激素和抗体的释放。因此，如果我们要了解以膜融合为目标的疾病，了解膜融合的基本机制是至关重要的。

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.