INTRACELLULAR VESICLE FUSION IN YEAST

酵母细胞内囊泡融合

• 批准号: 2701682
• 负责人: CHARLES K BARLOWE
• 金额: $ 22.01万
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701682
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; cell free system; endoplasmic reticulum; intracellular transport; membrane fusion; membrane proteins; molecular cloning; protein sequence; vesicle /vacuole

In eukaryotic cell, intracellular transport between membrane-bound compartments is mediated by vesicles that bud from one membrane and fuse selectively with another. This proposal focuses on site-specific fusion of ER-derived transport vesicles with the Golgi complex in Saccharomyces cerevisiae. Although some of the essential components of this vesicle fusion event have been identified, the molecular details associated with this process remain obscure. Thus, the long-term goal of this investigation is to use a combined genetic and biochemical approach to identify and characterized all of the factors required for this vesicle fusion reaction/ A more complete understanding of the regulation and mechanisms that underlie intracellular vesicle fusion could assist in the prevention and treatment of several human diseases. In vitro synthesis of ER-derived transport vesicles has been reconstituted with yeast membranes and three purified protein fractions. Vesicles formed under these conditions are competent for fusion with the Golgi apparatus and are distinct from the donor membrane fraction. A novel procedure has been devised to obtain highly purified ER-derived transport vesicles in sufficient quantities to examine their molecular composition. The targeting and fusion of these purified vesicles requires both a crude cytosol and an acceptor membrane fraction. The availability of purified vesicles and of a functional fusion assay in a model genetic organism provides a unique opportunity to investigate intracellular membrane fusion. The specific aims of this proposal are to characterize the vesicle proteins and soluble factors required for site-specific fusion of ER-derived vesicles with the Golgi apparatus. These purified vesicles contain a distinct set of polypeptides that are tightly associated with the membrane. Tom determine the roles of these membrane-associated proteins in specific steps of ER to Golgi transport, affinity-purified antibodies that neutralize the function of individual vesicle proteins will be used in the cell-free assay. Vesicle proteins implicated in targeting or fusion will be isolated, their polypeptide sequences determined and the encoding genes cloned. The isolation and characterization of soluble factors required for vesicle fusion with the Golgi complex will be achieved through the fractionation of the crude cytosol required to drive this cell-free fusion reaction. Initial efforts will focus on Ypt1p and Slyp, two proteins that are required for vesicle fusion and for which mutant alleles are available. Biochemical complementation assays that employ membranes and cytosol from ypt1 and sly1 mutant strains may provide a means of purification of these activities from a wild-type cytosol.

在真核细胞中，膜结合的胞内转运 隔室是由从一层膜萌发并融合的小泡调节的。 有选择地和另一个人一起。这项建议侧重于特定地点的融合 酵母菌内含高尔基复合体的内质网运输囊泡 酿酒。虽然这种水泡的一些基本成分 核聚变事件已经确定，与之相关的分子细节 这一过程仍然模糊不清。因此，这一计划的长期目标是 研究是使用基因和生物化学相结合的方法来 确定并描述这种水泡所需的所有因素 融合反应/更全面地了解调节和 细胞内囊泡融合的基础机制可能有助于 预防和治疗几种人类疾病。 重组内质网转运囊泡的体外合成 用酵母膜和三种纯化的蛋白质组分。水泡 在这些条件下形成的能够与高尔基体融合 与供体膜组分不同。一本小说 已经设计了获得高度纯化的内质网衍生转运的程序 足够数量的囊泡来检查它们的分子组成。 这些纯化小泡的靶向和融合需要一种粗制的 胞浆和受体膜组分。纯品的可获得性 模型遗传有机体中的囊泡和功能融合试验 为研究细胞内膜融合提供了一个独特的机会。 这项建议的具体目的是描述囊泡蛋白的特征。 以及内质网来源的位点特异性融合所需的可溶性因子 高尔基体的囊泡。这些纯化的囊泡含有一种 与膜紧密相关的一组不同的多肽。 TOM确定这些膜相关蛋白在特定情况下的作用 ER向高尔基体运输的步骤，亲和纯化的抗体 中和单个囊泡蛋白的功能将用于 无细胞检测。与靶向或融合有关的囊泡蛋白将 分离、测定其多肽序列及编码基因 克隆的。所需可溶性因子的分离和鉴定 囊泡与高尔基复合体的融合将通过 推动这种无细胞融合所需的粗胞浆的分级 反应。最初的努力将集中在Ypt1p和Slyp这两种蛋白质上 是囊泡融合所必需的，并且突变的等位基因是可用的。 利用细胞膜和胞浆进行生化互补分析 Ypt1和sly1突变株可能提供一种提纯这些病毒的方法。 野生型胞浆的活性。