INTRACELLULAR VESICLE FUSION IN YEAST

在真核细胞中，几个膜结合细胞器之间的运输是由囊泡介导的，囊泡从一个膜中萌发并选择性地与另一个膜融合。本研究的重点是在酿酒酵母中催化来自内质网（ER）的运输囊泡与高尔基复合体位点特异性融合的分子。这种融合事件的许多基本组成部分已经通过遗传方法确定，但是位点特异性囊泡融合的分子细节仍然不清楚。我研究的长期目标是用确定的蛋白质和脂质组分来重建这个反应，以阐明催化机制。囊泡融合的潜在机制似乎基本上是保守的。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实验来测量ER来源的运输囊泡与高尔基复合物的融合。我们用分离的膜和纯化的可溶性分子重现了这一事件。该反应分两个不同的生化步骤进行；首先，囊泡被“拴”在受体上，其次，一组独特的蛋白质催化稳定的对接和双分子层融合。本研究的目的如下：首先，开发清洁剂溶解内质网衍生囊泡的方法，并重建融合能力的蛋白脂质体，从而促进膜结合蛋白的酶学分析。其次，确定将囊泡功能性地系在高尔基腔室上的蛋白质-蛋白质相互作用。第三，用纯化的融合因子和分离的膜建立再关联实验。第四，鉴定内质网衍生转运囊泡所含的蛋白质，并确定它们在内质网到高尔基转运中的作用。将这些生化方法与模型遗传生物相结合，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。

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.