Mechanisms of COPII-Dependent Transport

描述（由申请人提供）：在真核细胞中，不同膜结合区室之间的细胞内转运通过解离的载体进行，这些载体从一个膜出芽，然后选择性地与另一个膜融合。本论文主要研究酿酒酵母内质网和高尔基复合体之间囊泡转运的催化分子和保守机制。遗传学方法已经确定了这一运输途径所需的基本组成部分。然而，这一过程背后的许多分子机制仍然不清楚。我们的研究将联合收割机分子遗传学方法与体外测定方法相结合，测定蛋白质从ER到早期高尔基体的转运。这种转运反应通过COPII依赖性货物选择和囊泡出芽、Usolp依赖性囊泡束缚和SNARE蛋白依赖性膜融合的生化可解析阶段进行。我们用分离的膜和纯化的可溶性分子再现了这些阶段。我的研究计划的长期目标是阐明这些事件背后的催化机制，通过分析特定阶段的测定和重建实验与定义的蛋白质和脂质馏分。本研究的主要目的是：分析Erv 26 p与COPII外壳共同作用将分泌蛋白分选到ER囊泡中的机制，确定整合膜Yip 1/Yif 1/Yos 1复合物在ER囊泡出芽中的作用，研究COPII囊泡形成中特定脂质种类的作用。并使用新建立的SNARE蛋白半胱氨酸-二硫键交联测定法分析ER衍生的囊泡在同型和异型膜融合反应中的命运。据估计，20-30%的细胞蛋白质进入分泌途径。我们的实验目的是解决分泌蛋白如何选择性地从内质网出口，并直接到高尔基复合体的基本问题。这种细胞内转运步骤对于几乎所有细胞生长和功能都是必不可少的。因此，拟议的研究是了解许多健康相关问题的基础，特别是与了解胆固醇调节，阿尔茨海默病和囊性纤维化有关。

DESCRIPTION (provided by applicant): In eukaryotic cells, intracellular transport between distinct membrane-bound compartments proceeds through dissociated carriers that bud from one membrane and then fuse selectively with another. This proposal will focus on the molecules and conserved mechanisms that catalyze vesicular transport between the endoplasmic reticulum (ER) and Golgi complex in Saccharomyces cerevisiae. Genetic approaches have identified essential components required for this transport pathway. However, many of the molecular mechanisms underlying this process remain obscure. Our studies combine molecular genetic approaches with in vitro assays that measure protein transport from the ER to early Golgi compartments. This transport reaction proceeds through the biochemically resolvable stages of COPII-dependent cargo selection and vesicle budding, Usolp-dependent vesicle tethering, and SNARE protein-dependent membrane fusion. We have reproduced these stages with isolated membranes and purified soluble molecules. The long-term goal of my research program is to elucidate catalytic mechanisms underlying these events though analysis of stage-specific assays and reconstitution experiments with defined protein and lipid fractions. The objectives of this proposal are to: dissect the mechanism by which the sorting adaptor Erv26p functions with the COPII coat to sort secretory proteins into ER-derived vesicles; determine the role of the integral membrane Yip1/Yif1/Yos1 complex in vesicle budding from the ER; investigate the role of specific lipid species in COPII vesicle formation; and analyze the fate of ER derived vesicles in homotypic and heterotypic membrane fusion reactions using newly established SNARE protein cysteine-disulfide cross-linking assays. It is estimated that 20-30% of cellular proteins enter the secretory pathway. Our experimental aims are designed to address fundamental questions on how secretory proteins are selectively exported from the ER and directed to the Golgi complex. This intracellular transport step is essential for virtually all cell growth and function. Therefore, the proposed studies are basic for understanding numerous health related issues and are specifically relevant to understanding cholesterol regulation, Alzheimer's disease and cystic fibrosis.