Role of Ypt GTPases in Intracellular Trafficking

描述(申请人提供)：细胞内转运对于所有真核细胞的正常运作至关重要。在顺行和逆行通路的多个步骤中，蛋白质和膜在细胞内的隔室之间向前和向后运输。虽然对个别贩运步骤的管制进行了广泛的研究，但对这些步骤的协调知之甚少。YPT/Rab GTP酶已成为管制个别贩运步骤的关键角色。我们的长期目标是阐明YPT/Rab GTP酶如何将单个运输步骤整合到完整的途径中。关于细胞内运输的机制和机制的里程碑式的发现是在酵母中完成的，并被证明与人类有关。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，因为它允许利用复杂的遗传方法与分子和细胞方法相结合。 此外，相对较少的玩家数量(例如，酵母中11个Ypt，而人类为~70个RAB)和由此产生的简化的相互作用网络使酵母成为研究完整途径协调的极佳模型，正如这里所计划的那样。 拟议的研究将侧重于协调激活高尔基Ypt。高尔基体是细胞中的主要分选室，由顺式、内侧和反式三个功能池组成。ER和高尔基人之间的双向贩运发生在独联体一侧。在反式方面，双向运输途径将高尔基体与两个细胞室连接起来：外细胞膜和内小体。在酵母中，四个Ypt起高尔基体守门人的作用。YPT/Rab GTP酶被鸟嘌呤核苷酸交换因子(GEF)激活。根据我们的结果，我们认为四个高尔基Ypt的激活是通过一个被称为TRapp的模块化的全球环境基金复合体来实现的，从而协调所有进出高尔基体的运输。将调查以下具体问题：1)TRapp如何作为多个Ypt的环境基金？2)在顺行贩运中，TRapp对高尔基体出入境是否协调激活？3)在逆行贩运中，TRapp对高尔基体出入境是否激活Ypt？为了解决这些问题，我们将研究TAPP亚基与高尔基体Ypt的相互作用，它们在体外对YPT核苷酸转换的影响，以及体内的蛋白质运输和细胞内定位。最后，我们将用活细胞显微镜研究TAPPP复合体的调控动力学。 这项研究与人类健康有关，因为多个基本过程依赖于细胞内的运输：例如，蛋白质和多肽的分泌；受体、离子通道和离子泵在外细胞膜上的呈递；以及配体和受体的内化。由于细胞与环境的相互作用依赖于细胞内的运输，这一过程的损害影响到人体的每个系统，包括大脑、心脏和免疫系统的发育和功能。 公共卫生评论：这项拟议的研究旨在了解一个基本的细胞过程，即细胞内运输，在这个过程中，蛋白质和膜在细胞细胞器之间转移。这一过程是所有细胞正常运作所必需的，因此也是人体每个系统所必需的。阐明调节细胞内转运的机制与多种疾病有关，这些疾病是由物质运输受损引起的，这些物质要么是必不可少的，如糖尿病中的胰岛素、癌症中的生长因子受体，以及囊性纤维化中的CFTR，要么是有害的，如阿尔茨海默病中的淀粉样蛋白。

DESCRIPTION (provided by applicant): Intracellular trafficking is vital for proper functioning of all eukaryotic cells. In the multiple steps of the anterograde and retrograde pathways, proteins and membranes are transported forward and backward between intracellular compartments. Whereas the regulation of individual trafficking steps has been studied extensively, very little is known about the coordination of these steps. Ypt/Rab GTPases have emerged as key players in the regulation of individual trafficking steps. Our long-term goal is to elucidate how Ypt/Rab GTPases integrate individual transport steps into complete 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 using yeast as a model to address these complicated issues, because it allows utilizing 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 complete pathways, as planned here. The proposed research will focus on the coordinated activation of the Golgi Ypts. Golgi, the major sorting compartment in the cell, consists of three functional cisternae: cis, medial and trans. Bi-directional trafficking between the ER and the Golgi occurs on the cis side. On the trans side, bi-directional transport pathways connect the Golgi with two cellular compartments: the outer-cell membrane and endosomes. In yeast, four Ypts function as Golgi gatekeepers. Ypt/Rab GTPases are activated by guanine-nucleotide exchange factors (GEFs). Based on our results, we propose that activation of the four Golgi Ypts is achieved by one modular GEF complex termed TRAPP, which thereby coordinates all trafficking into and out of the Golgi. The following specific questions will be investigated: 1) How Does TRAPP function as a GEF for multiple Ypts? 2) Does coordinated activation of the Golgi Ypts by TRAPP couple Golgi entry and exit in anterograde trafficking? 3) Does activation of Ypts by TRAPP couple Golgi entry and exit in retrograde trafficking? To address these questions, TRAPP subunits will be examined for their interaction with the Golgi Ypts, their effect on Ypt nucleotide switching in vitro, and protein transport and intracellular localization in vivo. Finally, the dynamics of TRAPP complex modulation will be studied by live-cell microscopy. This study is relevant to human health because multiple essential processes depend on intracellular trafficking: e.g., secretion of proteins and peptides; presentation of receptors, ion channels and ion pumps on the outer-cell membrane; and internalization of ligands and receptors. Because the interaction of cells with their environment is dependent on intracellular trafficking, impairment of this process affects every system in the human body, including the development and functioning of the brain, heart, and immune system. PUBLIC HEALTH REVELANCE: The proposed research is aimed at understanding a basic cellular process, intracellular trafficking, in which proteins and membranes are transferred between cellular organelles. This process is required for proper functioning of all cells, and therefore for every system of the human body. Elucidation of the mechanisms that regulate intracellular trafficking is relevant to a variety of diseases caused by impaired transport of substances that are either essential, such as insulin in diabetes, growth-factor receptors in cancer, and CFTR in cystic fibrosis, or detrimental, such as ¿-amyloid in Alzheimer's disease.