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运输通过分离的载体进行，这些载体从一层膜萌发，然后选择性地与另一层膜融合。本文将重点研究酿酒酵母菌内质网和高尔基复合体之间的囊泡运输催化分子及其保守机制。遗传方法已经确定了这种运输途径所需的基本成分。然而，这一过程背后的许多分子机制仍然不清楚。我们的研究结合了分子遗传学方法和体外测定从内质网到早期高尔基区室的蛋白质运输。这种转运反应通过copii依赖性货物选择和囊泡出芽、usolp依赖性囊泡系结和SNARE蛋白依赖性膜融合等生化可分解阶段进行。我们用分离的膜和纯化的可溶性分子重现了这些阶段。我的研究计划的长期目标是阐明这些事件的催化机制，通过分析阶段特异性分析和重构实验与定义的蛋白质和脂质组分。本提案的目的是：剖析分选接头Erv26p与COPII外壳一起作用的机制，将分泌蛋白分选到er衍生的囊泡中；确定整体膜Yip1/Yif1/Yos1复合物在内质网囊泡出芽中的作用；研究特定脂类在COPII囊泡形成中的作用；并利用新建立的SNARE蛋白半胱氨酸-二硫交联分析内质网衍生囊泡在同型和异型膜融合反应中的命运。据估计，20-30%的细胞蛋白进入分泌途径。我们的实验目的旨在解决有关分泌蛋白如何选择性地从内质网输出并定向到高尔基复合体的基本问题。这个细胞内运输步骤对几乎所有细胞的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.