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，但细胞如何调节物质通过该细胞器的流动仍然未知。蛋白质和膜进出高尔基体的运输由Rab GTP酶控制，其通过募集效应子来产生、运输和系链运输囊泡而发挥作用。这些必需的Rab途径的主要调节因子是Rab-GEF蛋白，它们必须“决定”是否激活其底物Rab。TRAPP复合物是激活高尔基体Rab的GEF，但TRAPP复合物活性的时间和幅度如何调节尚不清楚。在芽殖酵母模型中，有两个重要的Rab控制高尔基体的交通：Ypt 1（Rab 1）和Ypt 31/32（Rab 11）。TRAPPI复合物是Ypt 1在早期高尔基体隔室的既定激活剂。Ypt 31/32的GEF未知，并且是有争议的主题。TRAPPII复合体已被提议作为Ypt 31/32的GEF，尽管这一假设缺乏确凿的证据。全球环境基金在这一关键贩运途径方面的不确定性阻碍了这一领域的进展。使用生物化学方法，我们现在已经获得了解决这一争议的数据。通过纯化TRAPPII复合物和进行生理Rab激活试验，我们发现TRAPPII作为Ypt 1和Ypt 31/32的真正GEF发挥作用。重要的是，Ypt 31/32的强GEF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，并暗示存在自抑制机制。我们的长期目标是揭示高尔基体功能调节的机制。本项目的目标是确定TRAPP复合物调节Ypt 1和Ypt 31/32激活的机制。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TRAPPII激活两种不同的Rab GTP酶的机制基础。体外试验将用于研究TRAPPII如何激活两种不同的底物，并询问为什么需要膜激活Ypt 31/32而不是Ypt 1。然后，我们将使用酵母遗传学和细胞生物学来表征在体内干扰TRAPPII活性的功能后果，以了解TRAPPII复合物是如何在高尔基体运输的背景下进行调节的。2)确定TRAPPIII在高尔基体功能的机制和功能意义。新出现的证据表明TRAPPIII是Ypt 1在早期高尔基体的生理GEF。我们将研究TRAPPIII GEF活性是如何调节的，以及它在细胞中起什么作用。3)表征TRAPPII和TRAPIII与高尔基体的动态关联。我们将确定是否已知的相互作用的合作伙伴直接调节TRAPP复合物的活性和动力学。我们还将确定TRAPP复合物是否受到与其他高尔基体定位的GTP酶的串扰的调节。总之，这些研究将导致一个全面的模型，为必要的高尔基体Rab贩运途径的监管。