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（由适用提供）：高尔基复合物是真核细胞中几乎所有蛋白质中几乎三分之一的中心分类站，但是细胞如何调节材料通过该细胞器的流动仍然未知。蛋白质和膜流入高尔基体受到Rab GTPases的控制，这些Rab GTPase通过招募生效者来生成，运输和绳索运输蔬菜。这些基本RAB途径的主要调节剂是Rab-GEF蛋白，必须“决定”是否激活其底物Rabs。 Trapp复合物是激活高尔基体Rabs的GEF，但未知如何调节Trapp复合活动的时间和幅度。在萌芽的酵母模型中，有两个控制高尔基体流量的必需RAB：YPT1（RAB1）和YPT31/32（RAB11）。 Trappi复合物是早期高尔基体室内YPT1的已建立活化剂。 YPT31/32的GEF尚不清楚，并且是争议的主题。尽管缺乏牢固的证据，但已提出Trappii综合体作为YPT31/32的GEF。 GEF围绕这一关键贩运途径的不确定性正在阻碍该领域的进步。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复合物并进行物理RAB激活评估，我们发现Trappii在YPT1和YPT31和YPT31/32方面均充当骨骼GEF。重要的是，YPT31/32的鲁棒GEF活性严格取决于机制的存在，从而解释了为什么其他群体先前未观察到它，并暗示存在自抑制机制。我们的长期目标是发现管理高尔基功能调节的机制。该项目的目的是确定TRAPP复合物对YPT1和YPT31/32的机理调节。为了实现我们的研究目标，我们提出以下三个目的：1）剖析两种不同RAB GTPase的trappii激活机械基础。体外测定将用于研究Trappii如何激活两个不同的底物，并询问为什么激活YPT31/32而不是YPT1需要机制。然后，我们将使用酵母遗传学和细胞生物学来表征体内Trappii活性的功能后果，以了解如何在高尔基运输的背景下调节Trappii复合物。 2）确定trappiii功能在高尔基体上的机制和功能意义。新兴的证据指出TrappIII是YPT1的物理GEF，我们将研究如何调节Trappiii GEF活性以及它在细胞中的作用。 3）表征Trappii和Trapiii与高尔基体关联的动力学。我们将确定已知的相互作用伙伴是否直接调节TRAPP复合物的活性和动力学。我们还将确定trapp复合物是否通过与其他高尔基体定位的GTPases串扰调节。总而言之，这些研究将导致一个综合模型，以调节基本的高尔基rab贩运途径。