INTRACELLULAR VESICLE FUSION IN YEAST

酵母细胞内囊泡融合

• 批准号: 2835565
• 负责人: CHARLES K BARLOWE
• 金额: $ 31.54万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2835565
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; cell free system; endoplasmic reticulum; guanosinetriphosphatases; intracellular transport; membrane fusion; membrane proteins; molecular cloning; protein protein interaction; protein sequence; protein structure function; vesicle /vacuole

In eukaryotic cells, transport between several membrane-bound organelles is mediated by vesicles that bud from one membrane and fuse selectively with another. This study focuses on molecules that catalyze site-specific fusion of transport vesicles derived from the endoplasmic reticulum (ER) with the Golgi complex in Saccharomyces cerevisiae. Many of the essential components of this fusion event have been identified through genetic approaches, however the molecular details of site-specific vesicle fusion remain obscure. The long-term goal of my investigation is to reconstitute this reaction with defined protein and lipid fractions for the elucidation of catalytic mechanisms. The underlying mechanisms of vesicle fusion appear to be fundamentally conserved. Therefore these studies are basic for illuminating endocrine and exocrine secretion as well as neurotransmission. Our studies use an in vitro assay that measures the fusion of ER- derived transport vesicles with the Golgi complex. We have reproduced this event with isolated membranes and purified soluble molecules. The reaction proceeds in two biochemically distinct steps; first, vesicles are "tethered" to the acceptor, and second, a distinct set of proteins catalyze stable docking and bilayer fusion. The objectives of this study are as follows: First, develop methods to detergent solubilize ER-derived vesicles and reconstitute fusion competent proteoliposomes, thereby facilitating an enzymological analysis of membrane bound proteins. Second, determine the protein-protein interactions that functionally tether vesicles to the Golgi compartment. Third, establish reassociation assays with purified fusion factors and isolated membranes. Fourth, identify proteins contained on ER-derived transport vesicles and determine their roles in ER to Golgi transport. Combining these biochemical approaches with a model genetic organism provides my laboratory a unique opportunity to dissect the underlying mechanisms of vesicle fusion.

在真核细胞中，几个膜结合的细胞器之间的运输是由从一个膜出芽并选择性地与另一个膜融合的囊泡介导的。 本研究的重点是分子催化位点特异性融合的运输囊泡来自内质网（ER）的高尔基体在酿酒酵母。 这种融合事件的许多重要组成部分已被确定通过遗传方法，但位点特异性囊泡融合的分子细节仍然模糊。 我研究的长期目标是用确定的蛋白质和脂质组分重建该反应，以阐明催化机制。 囊泡融合的基本机制似乎是基本保守的。 因此，这些研究是阐明内分泌和外分泌以及神经传递的基础。我们的研究使用了一种体外试验，测量ER衍生的转运囊泡与高尔基体复合体的融合。 我们用分离的膜和纯化的可溶性分子再现了这一事件。 该反应在两个生物化学上不同的步骤中进行;第一，囊泡被“拴”到受体，第二，一组不同的蛋白质催化稳定的对接和双层融合。 本研究的目的如下：首先，开发方法，洗涤剂溶解ER衍生的囊泡和重组融合感受态脂蛋白体，从而促进酶的分析膜结合蛋白。 第二，确定蛋白质-蛋白质相互作用，功能拴囊泡高尔基体区室。第三，用纯化的融合因子和分离的膜建立再结合试验。 第四，确定ER衍生的运输囊泡上所含的蛋白质，并确定它们在ER到高尔基体运输中的作用。 将这些生化方法与模型遗传有机体相结合，为我的实验室提供了一个独特的机会来剖析囊泡融合的潜在机制。