Regulation of Arf GTPase activation at the Golgi complex

高尔基复合体 Arf GTP 酶激活的调节

• 批准号: 9415454
• 负责人: J Christopher Fromme
• 金额: $ 31.1万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9415454
• 关键词: AGFG1 gene; Binding; Biochemical; Biochemistry; Biological Assay; C-terminal; Cell Survival; Cells; Cellular Membrane; Cellular biology; Chimera organism; Collection; Complex; Data; Defect; Eukaryotic Cell; Feedback; Funding; Genetic; Goals; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Income; Investigation; Link; Logic; Measures; Membrane; Membrane Protein Traffic; Modeling; Molecular; Organelles; Outcome Study; Pathway interactions; Process; Protein Sortings; Proteins; Regulation; Role; SNAP receptor; Saccharomycetales; Signal Transduction; Sorting - Cell Movement; Vesicle; Yeast Model System; Yeasts; human disease; in vitro Assay; in vivo; intermolecular interaction; novel; recruit; structural biology; temperature sensitive mutant; trafficking; trans-Golgi Network; yeast genetics

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 complex flow of material through this organelle remains largely unknown. Protein and membrane traffic at the Golgi is controlled by Arf GTPases that function by recruiting effectors to make outgoing vesicles and tether incoming vesicles. The master regulators that activate Arf GTPase pathways are Arf-GEFs (guanine nucleotide exchange factors). In order to understand the molecular logic of Golgi trafficking, we must understand how the Golgi Arf-GEFs are regulated to make the molecular decision of where and when to activate their substrate Arf proteins. We have uncovered regulatory mechanisms that govern the function of the trans-Golgi network (TGN)-localized Arf-GEF, Sec7. We discovered that Sec7 can switch between autoinhibited and activated states, and is regulated by direct interactions with the activated forms of four different Golgi GTPases: Ypt31/32 (Rab11), Ypt1 (Rab1), Arl1, and Arf1. This collection of interactions represents a previously unappreciated level of crosstalk between these prominent Golgi GTPase pathways. Key questions remain regarding the biochemical basis for Sec7 regulation and the cell biology underpinning these interactions. Furthermore, we have determined that Gea1 and Gea2, the Arf-GEFs that localize to early Golgi compartments, are regulated through mechanisms that are distinct from those of Sec7. Our long-term goal is to determine how cells regulate trafficking at the Golgi complex. In order to both broaden and deepen our mechanistic understanding of the regulation of Arf activation at the Golgi, we propose the following Aims for this project: 1) Investigate the intra-molecular and inter-molecular interactions that regulate Sec7. We will use our established in vitro assays to characterize a newly identified functional link between distinct Sec7 regulatory domains. Using a new assay for measuring Sec7 activity in vivo, we will explore the possibilities that Sec7 integrates multiple GTPase signals and can sense and respond to changes in Golgi cargo load. We will perform an intragenic suppressor screen to identify new functional connections between Sec7 regulatory domains. Finally, we will seek additional structural information regarding the Sec7 C-terminal regulatory domains. 2) Determine the mechanisms regulating localization and activity of the early-Golgi Arf-GEFs Gea1/2. In order to understand how trafficking is regulated at the early- Golgi, we will perform a comprehensive investigation of the mechanisms regulating Gea1/2 membrane localization and activity. We will utilize chimeras and temperature sensitive-mutants to identify and characterize the regions of Gea1/2 regulating localization and activity. We will combine in vitro and in vivo approaches to characterize the roles of Gea1/2 binding partners. Finally, we will seek structural information to explain the basis for Gea1/2 regulation. We expect the outcome of these studies will be new and refined mechanistic models for regulation of trafficking at the Golgi complex.

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复杂物质流仍然很大程度上未知。蛋白质和 高尔基体的膜交通由 Arf GTPases 控制，Arf GTPases 通过招募效应器来发挥作用 输出囊泡和束缚传入囊泡。激活 Arf GTPase 途径的主要调节因子是 Arf-GEF（鸟嘌呤核苷酸交换因子）。为了理解高尔基体的分子逻辑 贩运，我们必须了解高尔基体 Arf-GEF 是如何调节的，以做出分子决定 何时何地激活其底物 Arf 蛋白。我们已经发现了监管机制 管理跨高尔基体网络（TGN）定位的 Arf-GEF，Sec7 的功能。我们发现 Sec7 可以 在自动抑制和激活状态之间切换，并通过与激活状态的直接相互作用来调节 四种不同高尔基体 GTP 酶的形式：Ypt31/32 (Rab11)、Ypt1 (Rab1)、Arl1 和 Arf1。这本合集 相互作用代表了这些突出的高尔基体 GTP 酶之间以前未被认识到的串扰水平 途径。关于 Sec7 调节的生化基础和细胞生物学的关键问题仍然存在 支撑这些相互作用。此外，我们还确定了 Gea1 和 Gea2，即 Arf-GEF 定位于早期高尔基体区室，通过与 Sec7 不同的机制进行调节。 我们的长期目标是确定细胞如何调节高尔基复合体的运输。为了两者 为了扩大和加深我们对高尔基体 Arf 激活调节机制的理解，我们 为该项目提出以下目标： 1）研究分子内和分子间 调节 Sec7 的相互作用。我们将使用我们已建立的体外测定来表征新鉴定的 不同 Sec7 监管域之间的功能联系。使用新的检测方法测量 Sec7 活性 vivo，我们将探索Sec7整合多个GTPase信号并能够感知和响应的可能性 高尔基体货物负载的变化。我们将进行基因内抑制子筛选，以确定新的功能 Sec7 监管域之间的连接。最后，我们将寻求额外的结构信息 关于 Sec7 C 末端监管域。 2) 确定本地化调控机制 早期高尔基体 Arf-GEF Gea1/2 的活性。为了尽早了解贩运活动是如何受到监管的 高尔基体，我们将对Gea1/2膜的调节机制进行全面的研究 本地化和活动。我们将利用嵌合体和温度敏感突变体来识别和 描述 Gea1/2 调节定位和活动的区域。我们将体外和体内结合起来 表征 Gea1/2 结合伴侣作用的方法。最后，我们将寻求结构信息 解释 Gea1/2 调节的基础。我们预计这些研究的结果将会是新的和完善的 高尔基复合体贩运调节的机械模型。