Regulation of Rab activation at the Golgi complex

高尔基复合体 Rab 激活的调节

• 批准号: 9005350
• 负责人: J Christopher Fromme
• 金额: $ 29.08万
• 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/9005350
• 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; Lead; Logic; Membrane; Membrane Protein Traffic; Membrane Proteins; Modeling; Molecular; Mutate; Mutation; Organelles; Pathway interactions; Physiological; Play; Positioning Attribute; Proteins; Recruitment Activity; Regulation; Research; Role; Saccharomycetales; Site; Sorting - Cell Movement; Structure; Substrate Specificity; Sum; Time; Transport Vesicles; Uncertainty; Vesicle; Yeast Model System; base; bone; human disease; in vitro Assay; in vivo; particle; 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(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这个细胞器的物质流动仍不清楚。进出高尔基体的蛋白质和膜的运输由Rab GTP酶控制，Rab GTP酶通过招募效应器来产生、运输和系留运输囊泡。这些基本的RAB途径的主要调节者是RAB-环境基金蛋白，它必须“决定”是否激活它们的底物RAB。TRapp复合体是激活高尔基体RAB的GEF，但尚不清楚TRapp复合体活性的时间和大小是如何调节的。在发芽酵母模型中，有两个控制高尔基体交通的基本RAB：Ypt1(Rab1)和Ypt31/32(Rab11)。TRAPPI复合体是早期高尔基体中已建立的Ypt1激活剂。Ypt31/32的全球环境基金是未知的，也是有争议的主题。TRAPPII复合体被提议作为Ypt31/32的全球环境基金，尽管缺乏这一假设的确凿证据。围绕全球环境基金这一关键贩运途径的不确定性阻碍了这一领域的进展。使用生化方法，我们现在已经获得了将解决这一争议的数据。通过纯化TRAPPII复合体和进行生理性Rab激活实验，我们发现TRAPPII对Ypt1和Ypt31/32都是一个骨结合物。重要的是，Ypt31/32的强劲全球环境基金活性严格依赖于膜的存在，从而解释了为什么以前没有被其他组观察到，并意味着存在一种自动抑制机制。我们的长期目标是发现高尔基体功能的调控机制。本项目的目标是确定TRapp复合体激活Ypt1和Ypt31/32的调节机制。为了实现我们的研究目标，我们提出了以下三个目标：1)剖析两种不同Rab GTP酶激活TRAPPII的机制基础。体外试验将被用来研究TRAPPII如何激活两种不同的底物，并询问为什么激活Ypt31/32而不是Ypt1需要膜。然后，我们将使用酵母遗传学和细胞生物学来表征干扰体内TRAPPII活性的功能后果，以便了解TRAPPII复合体在高尔基体运输的背景下是如何调节的。2)确定TRAPPIII在高尔基体中的作用机制和功能意义。新的证据表明，TRAPPIII是Ypt1在高尔基体早期的生理环境。我们将研究TRAPPIII全球环境基金的活性是如何调节的，以及它在细胞中扮演什么角色。3)研究TRAPPII和TRAPIII与高尔基体结合的动力学特征。我们将确定已知的相互作用伙伴是否直接调节TRapp复合体的活性和动力学。我们还将确定TRapp复合体是否受与其他高尔基定位的GTP酶的串扰调节。总而言之，这些研究将导致一个全面的模式，以管制基本的高尔基羊贩运路径。