Mechanisms of COPII-dependent Transport

描述（由申请人提供）：在真核细胞中，几种膜结合细胞器之间的转运由从一个膜出芽并选择性地与另一个膜融合的囊泡介导。该建议的重点是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。通过遗传学和生物化学方法已经鉴定了该转运途径的许多重要组分;然而，潜在的分子机制仍不清楚。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到高尔基复合体的转运。这种转运反应通过COPII依赖性囊泡出芽、Us 1 p依赖性囊泡束缚和SNARE蛋白依赖性膜融合的生化不同阶段进行。我们用分离的膜和纯化的可溶性分子再现了这些阶段。我的研究的长期目标是重建不同的子反应ER/高尔基体运输与确定的蛋白质和脂质馏分的催化机制的阐明。该提案的目的是调查COPII涂层将特定分泌货物分类到囊泡中的机制，确定功能性地将囊泡拴系到受体膜的分子接触，并设计新的检测方法来监测膜融合阶段的子反应。许多细胞内运输的这些基本机制是保守的多种运输途径在一个物种和从酵母到哺乳动物。因此，这些研究是阐明内分泌和外分泌的基础，对医学具有广泛的重要意义。

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.