GTPASE REGULATION OF ER EXPORT

我们的长期目标是从机制上了解膜结合蛋白和胞质蛋白的作用，这些蛋白指导哺乳动物细胞内质网（ER）的货物出口。一个关键步骤是将货物分选到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是未知的。为了实现这一目标，我们提出了一套补充的方法来剖析货物选择的机制，专注于中心作用的Sar1 GTcirs。具体目标I：哺乳动物Sar1 GT3的分子和结构分析。我们将研究的假设，特定的Sar1效应域直接的活性Sar1耦合货物选择下游靶蛋白参与核苷酸交换和水解，出口网站的形成和浓度到COPII囊泡。这些研究将涉及定点诱变和使用X射线晶体学和NMR阐明Sar1的结构。具体目标II：基于SAR 1的货物选择机制的新组件的功能。我们将探讨这一假设，目前未知的组件的预出芽复合物的一部分，协调货物选择与ER功能的其他方面集中货物到COPII囊泡的蛋白质机器。我们将阐明预出芽复合物的组成，使用质谱和表征的功能，在ER输出的新组件在体内和体外。具体目标三。信号指导Sar1依赖的货物选择。我们将测试的假设，即生物合成和内源性蛋白质，ER和ER后室之间的循环含有信号，作为配体，以促进ER出口。我们将利用成熟的体外货物招募和出芽测定来建立促进有效货物输出所需的最小信号。具体目标四。Sar1是一种形态发生的GT3。我们将研究这一假设，即Sar1是一个“形态发生”的GTdR参与的ER亚结构域专门在COPII囊泡形成的一代。这些研究将探索新的可能性，即膜相关受体，微管马达和微管是正常的包装和出口部位选择在体内的关键组成部分。这四个具体目标中的每一个都涉及限制Sar1 GTTRO在ER出口中的功能的关键问题。它们将使我们能够测试当前的模型，以首次对正常和疾病状态下ER功能的细胞生物学中的关键事件进行机械理解。

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.