GTPase Regulation of the Golgi Complex

高尔基复合体的 GTP 酶调节

• 批准号: 10379948
• 负责人: J Christopher Fromme
• 金额: $ 57.38万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10379948
• 关键词: Address; Biochemical; Biological; Biological Assay; Cell Survival; Cell membrane; Cells; Defect; Destinations; Endoplasmic Reticulum; Endosomes; Eukaryotic Cell; Family; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; In Vitro; Logic; Lysosomes; Membrane; Membrane Protein Traffic; Membrane Proteins; Molecular; Nucleotides; Organelles; Pathway interactions; Physiological; Process; Protein Sortings; Proteins; Proteomics; Reaction; Regulation; Research; Role; Signal Transduction; Site; Sorting - Cell Movement; Vesicle; Work; base; experimental study; genetic approach; human disease; imaging genetics; in vivo; live cell imaging; mutant; programs; protein protein interaction; reconstitution; recruit; structural biology; tool; trafficking

Project Summary/Abstract The Golgi complex is the central sorting station for nearly a third of all proteins in eukaryotic cells, but how cells regulate the flow of material through this organelle remains unknown. Secretory traffic must pass through the Golgi to be fully glycosylated and proteolytically processed. The Golgi must successfully traffic hundreds of membrane and lumenal proteins to several different sub-cellular destinations including the plasma membrane, endosomes, lysosomes, and the endoplasmic reticulum. Protein and membrane traffic into and out of the Golgi is controlled by GTPases of the Arf and Rab families that function by recruiting effector proteins to generate, transport, and tether membrane vesicles and tubules. The master regulators of these essential GTPase pathways are the GEF (guanine-nucleotide exchange factor) proteins that must “decide” where and when to activate their substrate GTPases. For most of these GEFs, we do not know how the localization, timing, and magnitude of their activity is regulated. Therefore, we do not fully understand the molecular logic of Golgi trafficking. Our lab has focused on deciphering what cellular signals the Golgi GEFs are listening to, and how the GEFs interpret these signals. We have discovered that several of these GTPase trafficking pathways communicate with each other through protein-protein interactions in which an activated GTPase positively regulates the GEF of another GTPase. One important implication of this finding is that activation of the distinct pathways are coordinated. Although we have uncovered regulatory mechanisms for some of the Golgi GEFs, the others remain poorly understood, and the overall molecular logic of these pathways is only beginning to come into focus. The primary question our proposed research program seeks to answer is: How do the Golgi GEFs make molecular decisions? We will address this question by investigating each of the Golgi GEFs with a broad set of tools, utilizing biochemical reconstitution reactions, structural biology, in vivo functional assays, live-cell imaging, and genetic experiments. We will use biochemical approaches to determine the precise roles of GEF regulatory subunits and domains. We will use structural approaches to visualize each GEF “caught in the act” of performing nucleotide exchange on its GTPase. Mutants generated based on hypotheses arising from biochemical and structural experiments will then be investigated in vivo to determine the consequences of perturbing specific interactions and domains. We will use unbiased proteomic and genetic approaches to reveal the identity of unknown GEFs and to discover unknown regulators of the Golgi GEFs. We will use established cell biological approaches to determine the physiological importance of the regulatory mechanisms. Taken together, the combined results of in vivo and in vitro experiments will enable us to determine the mechanisms the GEFs use to localize to their site of action, identify their substrate, and regulate their activity. Therefore, we will define how the Golgi GEFs sense and integrate signals, we will obtain a holistic view of how they work together, and we will uncover the molecular logic underpinning how the Golgi functions.

项目摘要/摘要 高尔基复合体是真核细胞中近三分之一蛋白质的中央分选站，但 细胞如何调节物质通过这个细胞器的流动尚不清楚。秘密流量必须通过 通过高尔基体进行完全糖基化和蛋白水解性加工。高尔基必须成功地通过 成百上千的膜和腔蛋白到达几个不同的亚细胞目的地，包括血浆 膜、内质体、溶酶体和内质网。蛋白质和膜的进出流量 由Arf和Rab家族的GTP酶控制，它们的功能是通过招募效应蛋白来 产生、运输和系留膜泡和小管。这些必需的GTP酶的主要调节者 途径是必须“决定”何时何地发生的全球环境基金蛋白。 激活它们的底物GTP酶。对于大多数这样的GEF，我们不知道本地化、时机和 它们的活动规模是受监管的。因此，我们并不完全了解高尔基体的分子逻辑。 贩卖人口。我们的实验室专注于破译高尔基GEF正在监听的细胞信号，以及如何监听 全球环境基金解释了这些信号。我们发现其中几条GTP酶的运输途径 通过蛋白质-蛋白质相互作用相互作用，其中GTP酶被积极激活 调节另一种GTP酶的全环基金。这一发现的一个重要含义是激活了不同的 路径是协调的。尽管我们已经发现了一些高尔基GEF的监管机制， 其他途径仍然知之甚少，这些途径的整体分子逻辑才刚刚开始 把焦点对准。我们提出的研究计划试图回答的主要问题是：高尔基人是如何 GEF做分子决定吗？我们将通过调查每个高尔基GEF来解决这个问题 广泛的工具，利用生化重建反应，结构生物学，体内功能分析， 活细胞成像和基因实验。我们将使用生化方法来确定确切的角色 全球环境基金监管亚单位和领域。我们将使用结构化方法来可视化每个全球环境基金 在其GTP酶上进行核苷酸交换的行为。基于假设产生的突变体 生物化学和结构实验将在体内进行研究，以确定 扰乱特定的相互作用和领域。我们将使用无偏见的蛋白质组和遗传学方法来揭示 以确定未知GEF的身份，并发现高尔基GEF的未知调节者。我们将使用已建立的 用细胞生物学的方法确定生理重要性的调节机制。已被占用 总之，体内和体外实验的综合结果将使我们能够确定这种机制 GEF用来定位它们的作用部位，识别它们的底物，并调节它们的活动。因此，我们 将定义Golgi GEF如何感知和集成信号，我们将获得它们如何工作的整体视图 一起，我们将揭示支持高尔基体功能的分子逻辑。