Role of Ypt GTPases in Intracellular Trafficking

描述（由申请人提供）：细胞内运输对于所有真核细胞的正常功能至关重要。在顺行和逆行途径的多个步骤中，蛋白质和膜在细胞内区室之间向前和向后运输。尽管对个人贩运步骤的监管已进行了广泛研究，但对这些步骤的协调知之甚少。Ypt/Rab全球贸易点已成为管制贩运活动各个步骤的关键角色。我们的长期目标是阐明Ypt/Rab GTP酶如何将单个转运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发现是在酵母中发现的，并表明与人类有关。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（例如，酵母中的11个Ypts与人类中的约70个Rabs）以及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绝佳模型。 拟议的研究将集中在协调激活的高尔基体Ypts。高尔基体是细胞中的主要分选室，由三个功能池组成：顺式池、内侧池和反式池。ER和高尔基体之间的双向运输发生在顺式侧。在反式侧，双向运输途径将高尔基体与两个细胞隔室连接：细胞外膜和内体。在酵母中，四个Ypts作为高尔基体的看门人。Ypt/Rab GTP酶由鸟嘌呤核苷酸交换因子（GEF）激活。基于我们的研究结果，我们建议，激活的四个高尔基体Ypts是由一个模块化的全球环境基金复杂的称为TRAPP，从而协调所有贩运进出高尔基体。将研究以下具体问题：1）TRAPP如何作为多个YPTS的GEF？2)TRAPP对高尔基体Ypts的协同激活在顺行运输中是否与高尔基体的进出有关？3)TRAPP激活Ypts是否与高尔基体的进入和退出有关？为了解决这些问题，TRAPP亚基将检查它们与高尔基体Ypt的相互作用，它们在体外对Ypt核苷酸转换的影响，以及体内蛋白质转运和细胞内定位。最后，TRAPP复合物调制的动力学将通过活细胞显微镜进行研究。 这项研究与人类健康有关，因为多种基本过程取决于细胞内运输：例如，蛋白质和肽的分泌;受体、离子通道和离子泵在细胞外膜上的呈递;以及配体和受体的内化。由于细胞与其环境的相互作用依赖于细胞内运输，因此这一过程的损害会影响人体的每个系统，包括大脑，心脏和免疫系统的发育和功能。 公共卫生部门：拟议的研究旨在了解一个基本的细胞过程，细胞内运输，其中蛋白质和膜在细胞器之间转移。这个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的正常运作所必需的。阐明调节细胞内运输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，这些物质要么是必需的，如糖尿病中的胰岛素，癌症中的生长因子受体和囊性纤维化中的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.