GTPase Regulation of the Golgi Complex

高尔基复合体的 GTP 酶调节

• 批准号: 10597649
• 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/10597649
• 关键词: Biochemical; Biological; Biological Assay; Cell Survival; Cell membrane; Cells; Communication; Defect; Destinations; Endoplasmic Reticulum; Endosomes; Eukaryotic Cell; Experimental Genetics; Family; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; In Vitro; Logic; Lysosomes; Membrane; Membrane Protein Traffic; Molecular; Nucleotides; Organelles; Pathway interactions; Physiological; Process; Protein Sortings; Proteins; Proteomics; Reaction; Regulation; Research; Role; Signal Transduction; Site; Sorting; Vesicle; Visualization; Work; experimental study; genetic approach; glycosylation; human disease; 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.

项目概要/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，但是 细胞如何调节通过该细胞器的物质流动仍然未知。 通过高尔基体进行完全糖基化和蛋白水解加工 高尔基体必须成功运输。 数百种膜和腔蛋白到达几个不同的亚细胞目的地，包括血浆 膜、内体、溶酶体和内质网蛋白质和膜的进出。 高尔基体的功能由 Arf 和 Rab 家族的 GTPases 控制，这些 GTPase 通过招募效应蛋白来发挥作用 产生、运输和束缚膜囊泡和小管这些必需 GTP 酶的主要调节因子。 通路是 GEF（鸟嘌呤核苷酸交换因子）蛋白，必须“决定”何时何地 对于大多数 GEF，我们不知道其定位、时间和激活方式。 因此，我们并不完全了解高尔基体的分子逻辑。 我们的实验室致力于破译高尔基体 GEF 正在监听哪些细胞信号以及如何监听。 GEF 解释这些信号，我们发现其中一些 GTP 酶转运途径。 通过蛋白质-蛋白质相互作用相互沟通，其中激活的 GTP 酶积极地 调节另一种 GTP 酶的 GEF，这一发现的一个重要意义是不同的 GTP 酶的激活。 尽管我们已经发现了一些高尔基体 GEF 的调节机制， 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 我们提出的研究计划试图回答的主要问题是：高尔基体是如何工作的。 GEF 做出分子决定？我们将通过研究每个高尔基体 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、体内功能测定、 我们将使用生化方法来确定精确的作用。 我们将使用结构方法来可视化每个 GEF“陷入困境”。 根据假设产生的 GTP 酶进行核苷酸交换的行为。 然后将在体内进行生化和结构实验以确定其后果 我们将使用公正的蛋白质组学和遗传学方法来揭示特定的相互作用和领域。 我们将使用已确定的 GEF 的身份并发现高尔基 GEF 的未知调节器。 采取细胞生物学方法来确定调节机制的生理重要性。 体内和体外实验的综合结果将使我们能够确定其机制 GEF 用于定位其作用位点、识别其底物并调节其活性。 将定义高尔基体 GEF 如何感知和整合信号，我们将全面了解它们如何工作 一起，我们将揭示支撑高尔基体功能的分子逻辑。