Coordination of intracellular trafficking pathways by Ypt/Rab GTPases and their GEFs

摘要 YPT/Rab GTP酶及其激活剂--鸟嘌呤核苷酸交换因子(GEF)已经出现 作为多种细胞内转运途径的关键调节因子。在这些途径中，蛋白质和 膜在细胞内的隔室之间运输，对所有生物的正常功能至关重要 真核细胞。虽然对单个运输步骤的调节已被广泛研究，但较少有人研究 知道他们之间的协调。我们的长期目标是阐明YPT/Rab GTP酶及其GEF是如何 协调同一路径和不同路径中的各个运输步骤。具有里程碑意义的发现 关于细胞内运输的机制和机制是在酵母中制造的，并显示出 与人类有关。因此，我们将继续使用酵母作为模型来解决这些复杂的问题， 因为它允许使用复杂的遗传方法，结合分子和 细胞方法。此外，相对较少的球员数量(例如，酵母中的11个Ypt与~70个RAB 以及由此产生的简化的相互作用网络使酵母菌成为研究 按照这里的计划，协调运输步骤。 在这项提案中，我们将解决这个主要问题：不同的全球环境基金复合体如何协调 Ypt1在分泌和自噬途径中的多种功能？在分泌途径中，货物是 通过高尔基体从内质网输送到质膜。在 结构性和应激诱导的自噬途径，过量或受损的细胞成分被穿梭 通过自噬小体在溶酶体中降解。使用共享(Ypt1)或重叠(TRapp GEFS)监管机构引导我们提出，交通结果(目的地)的特殊性是通过使用 而途径之间的协调是通过使用共同的GTP酶Ypt1实现的。 这里将讨论以下具体问题：1)两个不同的TAPP复合体如何 在分泌途径中通过高尔基体协调YPT介导的进程？2)多个 TAPP复合体协调Ypt1在分泌途径中的功能，并具有保守的选择性 自噬途径？基于我们以前的研究，我们开发了YPT/RAB的工作假设 协调，并将使用遗传、细胞和生化方法相结合的方法来测试它们。 这项研究与人类健康高度相关，因为多个必要的过程，如分泌 激素，受体在外细胞膜上的呈现，配体和受体的内化， 以及对压力的反应，取决于有效和协调良好的细胞内贩运。因此， 人口贩运的损害影响到人体的每个系统，包括发育和功能。 大脑、心脏和免疫系统。酵母调节剂的同源物，我们打算在这里研究， 与多种人类疾病有直接关联，包括癌症和神经退行性疾病。

Abstract Ypt/Rab GTPases together with their activators, guanine-nucleotide exchange factors (GEFs), have emerged as key regulators of the multiple intracellular trafficking pathways. These pathways, in which proteins and membranes are transported between intracellular compartments, are vital for the proper functioning of all eukaryotic cells. Whereas the regulation of individual transport steps has been studied extensively, less is known about their coordination. Our long-term goal is to elucidate how Ypt/Rab GTPases and their GEFs coordinate individual transport steps in the same pathway and in different pathways. Landmark discoveries about the mechanisms and machinery that underlie intracellular trafficking were made in yeast and shown to pertain to humans. Therefore, we will continue to use yeast as a model to address these complicated issues, because it allows easy utilization of sophisticated genetic approaches in combination with molecular and cellular methods. Furthermore, the relatively small number of players (e.g., 11 Ypts in yeast versus ~70 Rabs in humans) and the resultant simplified interaction networks make yeast an excellent model for studying the coordination of transport steps, as planned here. In this proposal, we will address this major question: How do different GEF complexes coordinate the multiple functions of Ypt1 in the secretory and autophagy pathways? In the secretory pathway, cargo is delivered from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus. In the constitutive and stress-induced autophagy pathways, excess or damaged cellular components are shuttled for degradation in the lysosome through autophagosomes. The use of shared (Ypt1) or overlapping (TRAPP GEFs) regulators leads us to propose that specificity in traffic outcomes (destinations) is achieved via the use of specific TRAPPs, while coordination between pathways is achieved via use of a common GTPase, Ypt1. The following specific questions will be addressed here: 1) How do two different TRAPP complexes coordinate Ypt-mediated progression through the Golgi in the secretory pathway? 2) How do multiple TRAPP complexes coordinate the function of Ypt1 in the secretory pathway and conserved selective autophagy pathways? Based on our previous studies, we developed working hypotheses for Ypt/Rab coordination, and will employ a combination of genetic, cellular and biochemical approaches to test them. This study is highly relevant to human health because multiple essential processes, such as secretion of hormones, presentation of receptors on the outer-cell membrane, internalization of ligands and receptors, and response to stress, depend on efficient and well-coordinated intracellular trafficking. Therefore, impairment of trafficking affects every system in the human body, including the development and functioning of the brain, heart, and immune system. Homologs of the yeast regulators, which we propose to study here, were implicated directly in multiple human illnesses, including cancer and neurodegenerative diseases.