Role of Ypt/Rab GTPases in intra-cellular Trafficking

描述（由申请人提供）：Ypt/Rab GTP酶及其激活剂鸟嘌呤-核苷酸交换因子（GEF）已成为细胞内运输的关键调节因子。蛋白质和膜在细胞内区室之间运输的这个过程对于所有真核细胞的正常功能至关重要。虽然个别运输步骤的监管已被广泛研究，很少有人知道他们的协调。我们的长期目标是阐明Ypt/Rab GTP酶及其GEF如何协调多个运输步骤。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，因为它允许结合分子和细胞方法容易地利用复杂的遗传方法。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运输步骤协调的绝佳模型。拟议的研究重点是协调激活的胞外Ypts。在胞吐途径中，蛋白质从内质网（ER）通过高尔基体运输到质膜（PM）。从ER到高尔基体的运输需要蛋白质的正确折叠，并且错误折叠的蛋白质从ER穿梭以进行降解。自噬是错误折叠膜蛋白降解的主要途径。在酵母中，Ypt 1和Ypt 31/32对调节所有这些转运步骤。基于我们的研究结果，我们提出这些Ypts的激活是由一个模块化的GEF复合物TRAPP实现的，TRAPP由此协调胞外途径的步骤以及其与自噬的交叉。将研究以下具体问题：1）TRAPP I和TRAPP II在体内的定位和GEF活性是什么？2)TRAPP II复合物的动态组装如何影响其Ypt-GEF特异性？3)Ypt 1在内质网-高尔基体和内质网-自噬转运中的功能是如何协调的？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荧光显微镜进行研究。这项研究与人类健康高度相关，因为多个基本过程取决于有效和协调良好的细胞内运输：例如，蛋白质的分泌， 肽;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。

DESCRIPTION (provided by applicant): Ypt/Rab GTPases together with their activators, guanine-nucleotide exchange factors (GEFs), have emerged as key regulators of intracellular trafficking. This process, in which proteins and membranes are transported between intracellular compartments, is 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 multiple transport steps. 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. The proposed research focuses on coordinated activation of exocytic Ypts. In the exocytic pathway, proteins are transported from the endoplasmic reticulum (ER), through the Golgi, to the plasma membrane (PM). Transport from the ER to Golgi requires proper folding of proteins, and misfolded proteins are shuttled from the ER for degradation. Autophagy is a major degradation pathway of misfolded membrane proteins. In yeast, Ypt1 and the Ypt31/32 pair regulate all these transport steps. Based on our results, we propose that activation of these Ypts is achieved by one modular GEF complex, TRAPP, which thereby coordinates steps of the exocytic pathway as well as its intersection with autophagy. The following specific questions will be investigated: 1) what are the localization and GEF activity of TRAPP I and TRAPP II in vivo? 2) How does dynamic assembly of the TRAPP II complex affect its Ypt-GEF specificity? 3) How is the function of Ypt1 in ER-to-Golgi and ER-to-autophagy transport coordinated? 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 i vivo. Finally, the dynamics of TRAPP complex modulation will be studied by fluorescence microscopy. This study is highly relevant to human health because multiple essential processes depend on efficient and well-coordinated 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.