Mechanisms of COPII-dependent Transport

COPII 依赖的运输机制

• 批准号: 6610150
• 负责人: CHARLES K BARLOWE
• 金额: $ 42.49万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610150
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; catalyst; cell free system; endocytosis; endoplasmic reticulum; intracellular transport; laboratory rabbit; mass spectrometry; membrane fusion; membrane proteins; molecular genetics; protein structure function; protein transport; vesicle /vacuole

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.

描述（由申请人提供）：在真核细胞中，几个膜结合细胞器之间的运输是由囊泡介导的，囊泡从一个膜中萌发并选择性地与另一个膜融合。本文主要研究了在酿酒酵母菌内质网和高尔基复合体之间催化囊泡运输的分子。这种运输途径的许多重要组成部分已经通过遗传和生化方法确定；然而，潜在的分子机制尚不清楚。我们的研究结合了分子遗传学方法和体外分析，测量了从内质网到高尔基体的蛋白质运输。这种转运反应通过copii依赖性囊泡出芽、us1p依赖性囊泡系结和SNARE蛋白依赖性膜融合的不同生化阶段进行。我们用分离的膜和纯化的可溶性分子重现了这些阶段。我研究的长期目标是重建内质网/高尔基转运中具有明确蛋白质和脂质组分的不同亚反应，以阐明催化机制。本提案的目的是研究COPII外壳将特定的分泌货物分类到囊泡中的机制，确定将囊泡与受体膜功能连接在一起的分子接触，并设计新的检测方法来监测膜融合阶段的亚反应。细胞内运输的许多基本机制在一个物种内以及从酵母到哺乳动物的多种运输途径中都是保守的。因此，这些研究是阐明内分泌和外分泌的基础，具有广泛的医学意义。