Regulation of Rab activation at the Golgi complex

高尔基复合体 Rab 激活的调节

• 批准号: 9197321
• 负责人: J Christopher Fromme
• 金额: $ 29.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197321
• 关键词: Autophagocytosis; Binding; Biochemical; Biological Assay; Carrier Proteins; Cell Survival; Cells; Cellular Membrane; Cellular biology; Complex; Conflict (Psychology); Data; Defect; Dissection; Eukaryotic Cell; Genes; Goals; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Health; Human Biology; Logic; Membrane; Membrane Protein Traffic; Modeling; Molecular; Mutate; Mutation; Organelles; Pathway interactions; Physiological; Play; Positioning Attribute; Protein Sortings; Proteins; Recruitment Activity; Regulation; Research; Role; Saccharomycetales; Site; Sorting - Cell Movement; Structure; Substrate Specificity; Sum; Transport Vesicles; Uncertainty; Vesicle; Vesicle Transport Pathway; Yeast Model System; bone; human disease; in vitro Assay; in vivo; particle; protein transport; public health relevance; rab GTP-Binding Proteins; reconstitution; trafficking; yeast genetics

 DESCRIPTION (provided by applicant): 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. Protein and membrane traffic into and out of the Golgi is controlled by Rab GTPases that function by recruiting effectors to generate, transport, and tether transport vesicles. The master regulators of these essential Rab pathways are the Rab-GEF proteins that must "decide" whether to activate their substrate Rabs. The TRAPP complexes are GEFs that activate Rabs at the Golgi, but it is unknown how the timing and magnitude of TRAPP complex activity is regulated. In the budding yeast model, there are two essential Rabs that control Golgi traffic: Ypt1 (Rab1), and Ypt31/32 (Rab11). The TRAPPI complex is the established activator of Ypt1 at early Golgi compartments. The GEF for Ypt31/32 is unknown, and is the subject of controversy. The TRAPPII complex has been proposed to serve as the GEF for Ypt31/32, although firm evidence for this hypothesis is lacking. The uncertainty surrounding the GEF for this critical trafficking pathway is hindering progress in the field. Using a biochemical approach, we have now obtained data that will resolve this controversy. By purifying the TRAPPII complex and performing physiological Rab activation assays, we have discovered that TRAPPII functions as a bone fide GEF for both Ypt1 and Ypt31/32. Importantly, robust GEF activity for Ypt31/32 is strictly dependent upon the presence of membranes, thus explaining why it was not observed previously by other groups, and implying the existence of an autoinhibitory mechanism. Our long-term goal is to uncover mechanisms governing the regulation of Golgi function. The goal of this project is to determine the mechanisms regulating the activation of Ypt1 and Ypt31/32 by the TRAPP complexes. In order to achieve our research goals, we propose the following three aims: 1) Dissect the mechanistic basis for TRAPPII activation of two different Rab GTPases. In vitro assays will be used to investigate how TRAPPII can activate two different substrates, and to ask why membranes are required for activation of Ypt31/32 but not Ypt1. We will then use yeast genetics and cell biology to characterize the functional consequences of perturbing TRAPPII activity in vivo, in order to understand how the TRAPPII complex is regulated in the context of Golgi trafficking. 2) Determine the mechanism and functional significance of TRAPPIII function at the Golgi. Emerging evidence points to TRAPPIII as the physiological GEF for Ypt1 at the early Golgi. We will investigate how TRAPPIII GEF activity is regulated and what role it plays in the cell. 3) Characterize the dynamics of TRAPPII and TRAPIII association with the Golgi. We will determine whether known interacting partners directly regulate the activity and dynamics of TRAPP complexes. We will also determine whether the TRAPP complexes are regulated by crosstalk with other Golgi-localized GTPases. In sum, these studies will lead to a comprehensive model for the regulation of the essential Golgi Rab trafficking pathways.

 描述（由申请人提供）：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，但细胞如何调节通过该细胞器的物质流动仍然未知。蛋白质和膜运输进出高尔基体。由 Rab GTP 酶控制，Rab GTP 酶通过招募效应子来生成、运输和束缚运输囊泡。这些重要 Rab 途径的主要调节因子是 Rab-GEF 蛋白。 TRAPP 复合物是在高尔基体上激活 Rab 的 GEF，但目前尚不清楚在芽殖酵母模型中，TRAPP 复合物活性的时间和强度是如何调节的。控制高尔基体交通：Ypt1 (Rab1) 和 Ypt31/32 (Rab11) TRAPPI 复合体是早期高尔基体中 Ypt1 的既定激活剂。 Ypt31/32 的 GEF 未知，并且已提出将 TRAPPII 复合体用作 Ypt31/32 的 GEF，尽管缺乏支持这一假设的确凿证据。围绕这一关键贩运途径的 GEF 的不确定性。使用生化方法，我们现在获得了可以解决这一争议的数据，通过纯化 TRAPPII 复合物并进行生理性 Rab 激活测定，我们发现 TRAPPII 具有真正的功能。 Ypt1 和 Ypt31/32 的 GEF 重要的是，Ypt31/32 的强大 GEF 活性严格依赖于膜的存在，从而解释了为什么其他组以前没有观察到它，并暗示了我们长期存在的自身抑制机制。长期目标是揭示调节高尔基体功能的机制 该项目的目标是确定 TRAPP 复合物调节 Ypt1 和 Ypt31/32 激活的机制。为了实现我们的研究目标，我们提出以下三个目标： 1) 剖析 TRAPPII 激活两种不同 Rab GTP 酶的机制基础 将使用体外测定来研究 TRAPPII 如何激活两种不同的底物，并探究为什么膜是这样的。然后，我们将使用酵母遗传学和细胞生物学来表征扰乱 TRAPPII 体内活性的功能后果，以便了解 TRAPPII 是如何激活的。 2) 确定 TRAPPIII 在高尔基体中的功能的机制和功能意义 新的证据表明 TRAPPIII 作为早期高尔基体中 Ypt1 的生理 GEF。 3) 描述 TRAPPII 和 TRAPIIII 与高尔基体关联的动态特征 我们将确定已知的相互作用伙伴是否直接调节高尔基体的活性和动态。我们还将确定 TRAPP 复合物是否受到与其他高尔基体本地化 GTP 酶的串扰调节。总之，这些研究将产生一个调节基本高尔基体 Rab 运输途径的综合模型。