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: 9234934
• 负责人: J Christopher Fromme
• 金额: $ 31.16万
• 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/9234934
• 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; Recruitment Activity; 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; 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 细胞如何调节这个细胞器中复杂的物质流动，在很大程度上仍是未知的。蛋白质和 高尔基体的膜交通是由Arf GTP酶控制的，Arf GTP酶通过招募效应器来产生 排出小泡并系住进入的小泡。激活Arf GTP酶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Arf-GEF是如何被调节的，以做出分子决定 何时何地激活它们的底物Arf蛋白。我们发现了监管机制， 管理跨高尔基体网络(TGN)本地化的Arf-全环基金的功能，第7节。我们发现第七节可以 在自动禁止和激活状态之间切换，并通过与激活状态的直接交互进行调节 四种不同高尔基GTP酶的形式：Ypt31/32(Rab11)、Ypt1(Rab1)、ARL1和Arf1。这一系列的 相互作用代表了这些著名的高尔基GTP酶之间以前未被认识到的串扰水平 小路。关于sec7调控的生化基础和细胞生物学的关键问题仍然存在。 支撑着这些互动。此外，我们已经确定了Gea1和Gea2，即Arf-GEF 定位于早期高尔基体，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管理高尔基建筑群的人口贩运。为了让这两个 拓宽和深化我们对高尔基体Arf激活调节的机械性理解，我们 1)分子内和分子间的分子内和分子间研究 调节Sec7的相互作用。我们将使用我们建立的体外试验来表征一种新发现的 不同的Sec7调节域之间的功能联系。使用一种新的方法来测量SEC7活性 ，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 高尔基货物装载量的变化。我们将进行基因内抑制物筛选，以确定新的功能 Sec7受规域之间的连接。最后，我们将寻求更多的结构信息 关于Sec7C-末端调节域。2)确定本地化调控机制和 早期高尔基Arf-GEF Gea1/2的活动。为了了解早期如何管制贩运-- 高尔基，我们将对Gea1/2膜的调节机制进行全面的研究 本地化和活跃性。我们将利用嵌合体和温度敏感突变体来鉴定和 描述调节定位和活性的Gea1/2区域。我们将把体外和体内结合起来 表征Gea1/2结合伙伴作用的方法。最后，我们将寻求结构性信息，以 解释Gea1/2规则的基础。我们预计这些研究的结果将是新的和完善的 高尔基建筑群管制贩运的机械模型。