Mechanisms of COPII-Dependent Transport

描述(申请人提供)：在真核细胞中，不同的膜结合的隔间之间的细胞内运输通过分离的载体进行，这些载体从一个膜上发芽，然后选择性地与另一个膜融合。这项建议将集中在酿酒酵母中催化内质网(ER)和高尔基复合体之间的囊泡运输的分子和保守机制。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实验，测量了蛋白质从内质网到早期高尔基体的运输。这种转运反应经历了COPII依赖的货物选择和囊泡萌发、Usolp依赖的囊泡捆绑和SNARE蛋白依赖的膜融合等生化可分解阶段。我们已经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通过分析特定阶段的分析和特定蛋白质和脂肪组分的重建实验来阐明这些事件背后的催化机制。这项建议的目标是：剖析分选接头Erv26p与COPII外壳一起将分泌蛋白分拣到内质网衍生小泡中的机制；确定整合膜Yip1/Yif1/Yos1复合体在内质网小泡萌发中的作用；研究特定脂类在COPII小泡形成中的作用；以及利用新建立的SNARE蛋白半胱氨酸-二硫交联分析在同型和异型膜融合反应中内质网衍生小泡的命运。据估计，20-30%的细胞蛋白进入分泌途径。我们的实验目的是解决有关分泌蛋白如何有选择地从内质网输出并定向到高尔基复合体的基本问题。这一细胞内转运步骤对几乎所有细胞的生长和功能都是必不可少的。因此，拟议的研究对于理解许多与健康相关的问题是基本的，并且与理解胆固醇调节、阿尔茨海默病和囊性纤维化特别相关。

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.