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.

项目摘要/摘要 高尔基体络合物是真核细胞中所有蛋白质近三分之一的中心分类站，但 细胞如何调节材料通过该细胞器的复杂流仍然很大程度上未知。蛋白质和 高尔基的膜流量由ARF GTPases控制，该ARF GTPase通过招募效应子来发挥作用 外向的囊泡和系绳传入囊泡。激活ARF GTPase途径的主调节器是 ARF-GEF（鸟嘌呤核苷酸交换因子）。为了了解高尔基的分子逻辑 贩运，我们必须了解如何调节高尔基Arf-gef，以做出分子决定 在何时何地激活其底物ARF蛋白。我们有发现的监管机制 控制反式高尔基网络（TGN）局部的ARF-GEF的功能，SEC7。我们发现Sec7可以 自抑制和激活状态之间的切换，并通过与激活的直接相互作用进行调节 四种不同的高尔基GTPase的形式：YPT31/32（RAB11），YPT1（RAB1），ARL1和ARF1。这个集合 相互作用代表了这些突出的高尔基GTPase之间先前未欣赏的串扰水平 途径。关于SEC7调节和细胞生物学的生化基础，仍然存在关键问题 支撑这些相互作用。此外，我们已经确定GEA1和GEA2，ARF-GEFS 本地化到早期高尔基体的隔室是通过与SEC7不同的机制来调节的。 我们的长期目标是确定细胞如何调节高尔基体复合物的运输。为了两者 扩大和加深我们对高尔基体ARF激活调节的机械理解，我们 提出以下目标的目的：1）研究分子内和分子间 调节SEC7的相互作用。我们将使用我们既定的体外测定法来表征新鉴定的 不同的SEC7调节域之间的功能联系。使用新的测定法来测量SEC7活动 Vivo，我们将探讨SEC7集成多个GTPase信号的可能性，并可以感知和回应 高尔基货物负载的变化。我们将执行基因内抑制器屏幕以识别新功能 SEC7监管域之间的连接。最后，我们将寻求其他结构信息 关于SEC7 C末端调节域。 2）确定调节本地化的机制和 早期Golgi ARF-GEFS GEA1/2的活性。为了了解在早期如何调节贩运的方式 高尔基，我们将对调节GEA1/2膜的机制进行全面研究 本地化和活动。我们将利用嵌合体和温度敏感的突击剂来识别和 表征调节定位和活性的GEA1/2区域。我们将在体外和体内结合 表征GEA1/2结合伙伴角色的方法。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是新的和精致的 调节高尔基综合体贩运的机械模型。