Role of Ypt GTPases in Vesicular transport

描述（申请人摘要）：分泌途径是 细胞与其环境的相互作用，因为它是 蛋白质和肽被释放到周围的环境中， 通道和离子泵存在于质膜上，配体和 受体被内化。阐明调控这一过程的机制 这条通路与多种疾病有关， 一种物质的运输，这种物质在糖尿病中是必不可少的胰岛素， 囊性纤维化中的CFTR，或阿尔茨海默病中的β-淀粉样蛋白。 在分泌途径中，蛋白质在细胞内 隔室通过膜囊泡。小GTP酶在 调节这种囊泡运输。这些机制和机器， 囊泡形成、靶向和融合的基础是高度保守的， 酵母和人。我们将使用酵母作为模型系统来解决这些复杂的 问题，因为这种生物体允许使用强大的遗传方法， 除了分子和细胞。我们的长期目标是了解 Ypt/Rab GTP酶如何调节和协调分泌的离散步骤， 通路拟议的研究将重点关注具有以下功能的蛋白质 Ypt GTP酶的调节子或效应子。它们将用于解决三个 重要的问题1)Ypt核苷酸的调节子在 Ypt介导的蛋白质转运中的循环作用？在GTP和 在辅助因素的推动下，GDP绑定形式被认为对 Ypt/Rab函数。然而，很少有人知道这些因素如何定位 它们的Ypt/Rab靶点，促进核苷酸交换或水解，并影响 Ypt GTdiff函数。为了解决这些问题，监管机构的本地化， 它们的功能及其与Ypt GTP酶的相互作用将在 体内和体外。2)Ypt蛋白在GT3级联中起作用吗？这 现有的假说表明，Ypt蛋白影响核苷酸循环， 其他GTP酶协调分泌途径的离散步骤。的 存在和性质的GT3级联将首先调查， 体外，稍后将在体内测试它们的作用。3)效应器在 Ypt GTP酶在蛋白质转运中的作用Ypt/Rab蛋白，像其他GTP酶一样， 被认为是通过募集下游效应物发挥作用， 其将信号从GTP结合的GTP酶传递到膜对接和/或 聚变装置为了确定Ypt效应器发挥作用的机制， 我们将研究Ypt效应基因突变对囊泡出芽的影响， 靶向或融合，以及它们与Ypt GTP酶相互作用的性质。

DESCRIPTION (APPLICANT'S ABSTRACT): The secretory pathway is central to the interaction of cells with their environment as it is the route by which proteins and peptides are released into the surrounding melieu, receptors, ion channels and ion pumps are presented on the plasma membrane, and ligands and receptors are internalized. Elucidation of the mechanisms that regulate this pathway is relevant for a variety of diseases that are caused by impaired transport of a substance that is either essential as insulin in diabetes and CFTR in cystic fibrosis, or detrimental as beta-amyloid in Alzheimer disease. In the secretory pathway proteins are transported between intracellular compartments via membranous vesicles. Small GTPases play a key role in the regulation of this vesicular trafficking. The mechanisms and machinery that underlie vesicle formation, targeting and fusion are highly conserved between yeast and man. We will use yeast as a model system to address these complicated issues, since this organism allows the use of powerful genetic approaches in addition to molecular and cellular ones. Our long term goal is to understand how Ypt/Rab GTPases regulate and coordinate the discrete steps of the secretory pathway. The proposed research will focus on proteins that function as regulators or effectors for Ypt GTPases. They will be used to address three important questions. 1) What is the role that regulators of Ypt nucleotide cycling play in Ypt-mediated protein transport? Cycling between the GTP- and GDP-bound forms, facilitated by accessory factors, is considered crucial for Ypt/Rab function. However, little is known regarding how these factors locate their Ypt/Rab targets, facilitate nucleotide exchange or hydrolysis, and affect Ypt GTPase function. To resolve these issues, the localization of regulators, their function and their interaction with Ypt GTPases will be examined both in vivo and in vitro. 2) Do Ypt proteins function in GTPase cascades? This existing hypothesis suggests that Ypt proteins affect nucleotide cycling of other GTPases to coordinate discrete steps of the secretory pathway. The existence and the nature of GTPase cascades will be first investigated in vitro, and later their role will be tested in vivo. 3) What role do effectors of Ypt GTPases play in protein transport? Ypt/Rab proteins, like other GTPases, are believed to function through the recruitment of down-stream effectors, which transmit signals from the GTP-bound GTPase to the membrane docking and/or fusion apparatus. To determine the mechanism by which Ypt effectors function, we will study the effect of mutations in Ypt effector genes on vesicle budding, targeting, or fusion, and the nature of their interaction with Ypt GTPases.