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， 作为多种细胞内运输途径的关键调节剂。这些途径，其中蛋白质和 细胞膜在细胞内隔室之间运输，对于所有细胞的正常功能至关重要。 真核细胞虽然个别运输步骤的调节已被广泛研究， 了解他们的协调。我们的长期目标是阐明Ypt/Rab GTP酶及其GEF 协调同一途径和不同途径中的各个运输步骤。标志性发现 关于细胞内运输的机制和机制，是在酵母中进行的， 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， 因为它允许容易地利用复杂的遗传方法结合分子和 细胞方法。此外，相对较少的玩家（例如，酵母中11个Ypts与约70个Rabs 在人类中）和由此产生的简化的相互作用网络使酵母成为研究 协调运输步骤，如计划中所示。 在本提案中，我们将解决这一主要问题：不同的全球环境基金综合体如何协调 Ypt 1在分泌和自噬途径中的多种功能？在分泌途径中，货物是 从内质网通过高尔基体传递到质膜。在 在组成性和应激诱导的自噬途径中，过量或受损的细胞组分穿梭于 通过自噬体在溶酶体中降解。使用共享（Ypt 1）或重叠（TRAPP 全球环境基金）监管机构引导我们提出，交通结果（目的地）的特异性是通过使用 的特定TRAPP，而途径之间的协调是通过使用一个共同的GTd 1，Ypt 1。 以下具体问题将在这里解决：1）如何两个不同的TRAPP复合物 协调Ypt介导的进展，通过高尔基体的分泌途径？2)如何使多个 TRAPP复合物协调Ypt 1在分泌途径中的功能， 自噬途径基于我们以前的研究，我们提出了Ypt/Rab的工作假设 协调，并将采用遗传，细胞和生物化学方法相结合，以测试他们。 这项研究与人类健康高度相关，因为多个基本过程，如分泌 激素，受体在细胞外膜上的呈递，配体和受体的内化， 和对压力的反应，依赖于有效和协调良好的细胞内运输。因此，我们认为， 贩运的损害影响到人体的每一个系统，包括发育和运作， 大脑，心脏和免疫系统我们打算在这里研究的酵母调节剂的同源物， 直接与多种人类疾病有关，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.