GTPASE REGULATION OF ER EXPORT

ER 出口的 GTPASE 监管

• 批准号: 6385922
• 负责人: William Edward Balch
• 金额: $ 39.27万
• 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: 1990-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385922
• 关键词: Golgi apparatus; X ray crystallography; endoplasmic reticulum; gene mutation; guanosinetriphosphatases; intracellular transport; laboratory rabbit; laboratory rat; mass spectrometry; molecular cloning; nuclear magnetic resonance spectroscopy; protein structure function; site directed mutagenesis; vesicle /vacuole

Our long range goal is to understand mechanistically the roles of membrane-bound and cytosolic proteins that direct the export of cargo from the endoplasmic reticulum (ER) of mammalian cells. A key step is the sorting of cargo into COPII vesicles. The events that underpin the physiological basis for cargo selection and concentration in COPII vesicles are unknown. To address this goal, we propose a complementary set of approaches to dissect the mechanism of cargo selection by focusing on the central role of the Sar1 GTPase. Specific Aim I: Molecular and structural analyses of the mammalian Sar1 GTPase. We will examine the hypothesis that specific Sar1 effector domains direct the activity of Sar1 in coupling cargo selection to downstream target proteins involved in nucleotide exchange and hydrolysis, export site formation and concentration into COPII vesicles. These studies will involve site-directed mutagenesis and elucidation of the structure of Sar1 using x-ray crystallography and NMR. Specific Aim II: Function of novel components of the Sar1-based cargo selection machinery. We will explore the hypothesis that currently unknown components of pre-budding complexes are part of a protein machine that coordinates cargo selection with other aspects of ER function to concentrate cargo into COPII vesicles. We will elucidate the composition of pre-budding complexes using mass spectrometry and characterize the function of novel components in ER export in vivo and in vitro. Specific Aim III. Signals directing Sar1-dependent cargo selection. We will test the hypothesis that biosynthetic and endogenous proteins that recycle between the ER and post-ER compartments contain signals that serve as ligands to promote ER export. We will utilize well-developed in vitro cargo recruitment and budding assays to establish the minimal signal(s) necessary to promote efficient cargo export. Specific Aim IV. Sar1 is a morphogenetic GTPase. We will examine the hypothesis that Sar1 is a 'morphogenic' GTPase involved in the generation of ER subdomains specialized in COPII vesicle formation. These studies will explore the novel possibility that membrane-associated receptors, microtubule motors and microtubules are critical components for normal packaging and export site selection in vivo. Each of the four specific aims addresses key issues circumscribing the function of the Sar1 GTPase in ER export. They will allow us to test current models to develop for the first time a mechanistic understanding of the crucial events in the cell biology of ER function in the normal and disease state.

我们的长期目标是从机制上理解膜结合蛋白和细胞质蛋白在指导哺乳动物细胞内质网（ER）输出货物中的作用。关键的一步是将货物分拣到COPII囊泡中。支持COPII囊泡中货物选择和浓度的生理基础的事件尚不清楚。为了实现这一目标，我们提出了一套互补的方法，通过关注Sar1 GTPase的核心作用来剖析货物选择的机制。特定目标1：哺乳动物Sar1 GTPase的分子和结构分析。我们将检验特定Sar1效应域的假设，即Sar1的活性在将货物选择偶联到下游目标蛋白的过程中，涉及核苷酸交换和水解，出口位点的形成和浓度进入COPII囊泡。这些研究将包括定点诱变和利用x射线晶体学和核磁共振阐明Sar1的结构。具体目标II：基于sar1的货物选择机器的新组件的功能。我们将探索一种假设，即目前未知的出芽前复合物成分是蛋白质机器的一部分，该蛋白质机器与内质网功能的其他方面协调货物选择，将货物集中到COPII囊泡中。我们将利用质谱法阐明芽前复合物的组成，并表征新成分在体内和体外内质网输出中的功能。具体目标三。指示sar1依赖货物选择的信号。我们将验证在内质网和内质网后室之间循环的生物合成和内源性蛋白质含有作为配体促进内质网输出的信号的假设。我们将利用成熟的体外货物招募和出芽试验来建立促进有效货物出口所需的最小信号。Sar1是一种形态发生的GTPase。我们将检验Sar1是一种“形态发生”的GTPase的假设，Sar1参与了COPII囊泡形成的内质网亚域的产生。这些研究将探索一种新的可能性，即膜相关受体、微管马达和微管是正常包装和体内出口位点选择的关键成分。这四个具体目标中的每一个都解决了限制Sar1 GTPase在ER输出中的功能的关键问题。它们将使我们能够测试当前的模型，从而首次对正常和疾病状态下内质网功能细胞生物学中的关键事件进行机制理解。