GTPAse Regulation of ER Export

ER 出口的 GTPA 酶监管

• 批准号: 6784109
• 负责人: William Edward Balch
• 金额: $ 42.23万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6784109
• 关键词: Vesiculovirus; X ray crystallography; endoplasmic reticulum; enzyme activity; enzyme mechanism; genetic models; genetically modified animals; guanine nucleotide exchange factors; guanosinetriphosphatases; intracellular transport; laboratory mouse; laboratory rabbit; laboratory rat; protein protein interaction; protein structure function; site directed mutagenesis; vesicle /vacuole; virus protein

DESCRIPTION (provided by applicant): Our long-range goal is to understand the roles of membrane-bound and cytosolic proteins that direct the sorting of cargo into COPII vesicles budding from endoplasmic reticulum (ER) of mammalian cells. The mechanism by which cargo is selected and concentrated during export is unknown. To address this goal, we will focus on the central role of the Sar1 GTPase and its upstream and downstream effectors in cargo selection. Aim I. Molecular basis for Sar1 activation leading to transitional element formation. We will examine the mechanism by which Sarl directs the assembly of export sites through interaction with the mammalian Sec12 guanine nucleotide exchange factor (GEF) to generate tubular elements that form using a KIF-dependent process. Specific Aim II. Biochemical basis for cargo selection. We will explore the hypothesis that novel components of pre budding complexes are part of a protein machine that coordinates cargo selection with other aspects of ER function to select cargo into COPII vesicles. We will address the molecular basis for cargo recognition, Sec23/24 recruitment and coat polymerization by Sar1, and generate transgenic mouse models to pursue the unique functions of mammalian Sec24 (A-D) isoforms. Specific Aim Ill. Vesicle maturation. We will explore the basis for coordination of cargo recruitment with the recruitment of molecular tethers and targeting/fusion determinants. We will determine the structural organization of the tether p115 using x-ray crystallography. We will explore the contribution of Sec13/31 to the completion of budding and fission of COPII vesicles from the ER through biochemical, molecular and structural analyses, allowing us to test different models for COPII coat polymerization. The proposed studies should allow us to develop significant mechanistic insight into the crucial early events in the secretory pathway that control the flow of nearly one-third of genome into the cell.

描述（由申请人提供）：我们的长期目标是了解膜结合蛋白和胞质蛋白的作用，这些蛋白指导将货物分选到从哺乳动物细胞的内质网 (ER) 出芽的 COPII 囊泡中。出口过程中选择和集中货物的机制尚不清楚。为了实现这一目标，我们将重点关注 Sar1 GTPase 及其上游和下游效应器在货物选择中的核心作用。目标 I. Sar1 激活导致过渡元素形成的分子基础。我们将研究 Sarl 通过与哺乳动物 Sec12 鸟嘌呤核苷酸交换因子 (GEF) 相互作用指导输出位点组装的机制，以生成使用 KIF 依赖性过程形成的管状元件。具体目标二。货物选择的生化基础。我们将探讨这样的假设：出芽前复合物的新成分是蛋白质机器的一部分，该蛋白质机器协调货物选择与 ER 功能的其他方面，以将货物选择到 COPII 囊泡中。我们将解决 Sar1 的货物识别、Sec23/24 招募和外壳聚合的分子基础，并生成转基因小鼠模型以追求哺乳动物 Sec24 (A-D) 同工型的独特功能。具体目标 Ill. 囊泡成熟。我们将探索货物招募与分子系链招募和靶向/融合决定因素协调的基础。我们将使用 X 射线晶体学确定系链 p115 的结构组织。我们将通过生化、分子和结构分析探索 Sec13/31 对 COPII 囊泡从内质网完成出芽和裂变的贡献，使我们能够测试 COPII 涂层聚合的不同模型。拟议的研究应该使我们能够对控制近三分之一基因组流入细胞的分泌途径中的关键早期事件产生重要的机制洞察。