Mechanisms of COPII-dependent Transport

描述(申请人提供)：在真核细胞中，几个膜结合的细胞器之间的运输是由从一个膜上发芽并选择性地与另一个膜融合的小泡介导的。这项建议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。通过遗传和生化方法已经确定了这种运输途径的许多基本成分；然而，潜在的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试验，测量蛋白质从内质网到高尔基复合体的运输。这种转运反应经历了COPII依赖的囊泡萌发、Us1p依赖的囊泡拴系和SNARE蛋白依赖的膜融合这三个不同的生化阶段。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。我研究的长期目标是用确定的蛋白质和脂肪组分重建内质网/高尔基体运输中不同的子反应，以阐明催化机制。这项建议的目的是研究COPII涂层将特定的分泌货物分类为小泡的机制，确定将小泡功能性地拴在受体膜上的分子接触，并设计新的分析方法来监测膜融合阶段的次级反应。许多这些细胞内运输的基本机制在物种内和从酵母到哺乳动物的多种运输途径中是保守的。因此，这些研究是阐明内分泌和外分泌分泌的基础，对医学具有广泛的意义。

DESCRIPTION (provided by applicant): In eukaryotic cells, transport between several membrane-bound organelles is mediated by vesicles that bud from one membrane and fuse selectively with another. This proposal focuses on the molecules that catalyze vesicular transport between the endoplasmic reticulum (ER) and Golgi complex in Saccharomyces cerevisiae. Many essential components of this transport pathway have been identified though genetic and biochemical approaches; however the underlying molecular mechanisms remain unclear. Our studies combine molecular genetic approaches with in vitro assays that measure protein transport from the ER to the Golgi complex. This transport reaction proceeds through the biochemically distinct stages of COPII-dependent vesicle budding, Us1p-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 investigation is to reconstitute distinct sub-reactions in ER/Golgi transport with defined protein and lipid fractions for the elucidation of catalytic mechanisms. The objectives of this proposal are to investigate the mechanisms by which the COPII coat sorts specific secretory cargo into vesicles, determine the molecular contacts that functionally tether vesicles to acceptor membranes and devise new assays that monitor sub-reactions in the membrane fusion stage. Many of these fundamental mechanisms in intracellular transport are conserved for multiple trafficking pathways within a species and from yeast to mammals. Therefore, these studies are basic for illuminating endocrine and exocrine secretion and will be of broad importance to medicine.