GTPase Regulation of the Golgi Complex (Diversity Supplement 2023)

高尔基复合体的 GTPase 调节（多样性补充资料 2023）

• 批准号: 10800329
• 负责人: J Christopher Fromme
• 金额: $ 5.79万
• 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/10800329
• 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.

项目摘要/摘要 高尔基体复合物是真核细胞中几乎所有蛋白质中几乎三分之一的中心分类站，但是 细胞如何调节材料通过该细胞器的流动仍然未知。秘书必须 穿过高尔基体以完全糖基化并进行蛋白水解处理。高尔基必须 成功地将数百个膜和腔蛋白运送到几个不同的亚细胞 包括质膜，内体，溶酶体和内质网中的目的地。 蛋白质和膜流入高尔基体的蛋白质和膜流量受ARF和RAB的GTPase控制 通过招募效应蛋白来生成，运输和绑扎膜的家庭 囊泡和块茎。这些基本GTPase途径的主调节器是GEF （鸟嘌呤核苷酸交换因子）必须“决定”何时何地激活其蛋白质 基板GTPase。对于大多数这些GEF，我们不知道本地化，时机和 他们的活动的大小受到调节。因此，我们不完全理解 高尔基人贩运。我们的实验室重点是解释Golgi GEF正在听的蜂窝信号 以及GEFS如何解释这些信号。我们发现其中几个GTPase 贩运途径通过蛋白质 - 蛋白质相互作用相互通信 激活的GTPase积极调节另一GTPase的GEF。一个重要的含义 发现不同途径的激活是协调的。虽然我们发现了 某些高尔基GEF的监管机制，其他机制仍然很众所周知，并且 这些途径的总体分子逻辑才刚刚开始焦点。主要问题 我们提出的研究计划试图回答：高尔基GEF如何做出分子决定？ 我们将通过使用广泛的工具来调查每个高尔基GEF，以解决这个问题 生化重构反应，结构生物学，体内功能测定，活细胞成像和 基因实验。我们将使用生化方法来确定GEF的确切作用 监管亚基和域。我们将使用结构方法可视化每个GEF“陷入困境 在其GTPase上进行核丁基交换的行为。根据假设产生的突变体 然后，由生化和结构实验引起的，将在体内研究以确定 扰动特定相互作用和域的后果。我们将使用公正的蛋白质组学和 揭示未知GEF的身份并发现未知调节剂的遗传方法 Golgi GEFS。我们将使用已建立的细胞生物学方法来确定生理 监管机制的重要性。综上所述，体内和体外的综合结果 实验将使我们能够确定GEF所用的机制将其定位到其作用部位， 确定其底物并调节其活动。因此，我们将定义Golgi GEFS的感觉 和综合信号，我们将对它们如何共同工作有整体看法，我们将揭示 分子逻辑基于高尔基体的功能。