Molecular Analysis of the CCC185 Golgin

CCC185 Golgin 的分子分析

• 批准号: 7634541
• 负责人: Suzanne R Pfeffer
• 金额: $ 28.58万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7634541
• 关键词: Address; Binding; Biochemical; Biological Assay; Cells; Collection; Complex; Disease; Docking; Endosomes; Event; Family; Goals; Golgi Apparatus; Health; Human; Immunofluorescence Microscopy; Membrane; Membrane Protein Traffic; Modeling; Molecular; Molecular Analysis; Names; Pathway interactions; Protein Binding; Proteins; Research; Role; SNAP receptor; Testing; Time; Transport Vesicles; Vesicle; Work; base; cell motility; late endosome; mutant; protein transport; research study; trafficking; trans-Golgi Network

DESCRIPTION (provided by applicant): The long term goal of this research is to understand the molecular basis of protein trafficking between membrane-bound compartments in human cells. Protein transport involves cargo collection into vesicles, vesicle budding, motility, tethering and docking at the target membrane, and subsequent fusion. Tethering and docking are the least understood steps in membrane traffic. The specific goal of this application is to investigate the molecular function of a protein named GCC185, a 185K, trans Golgi network (TGN)-localized protein of the GRIP domain family. We seek to test the hypothesis that GCC185 functions as a tethering protein for transport vesicles arriving at the TGN. With regard to GRIP domain-family Golgins, the most important questions that need to be resolved are: 1. Are these proteins actually tethers? 2. What molecules do these proteins partner with to achieve tethering? 3. Do these proteins act as vesicle-associated tethers or target-associated tethers? 4. Do these proteins bind to TGN-specific SNARE proteins, and how are they released from the Golgi during or after vesicle fusion? To begin to address these questions, we propose to: 1. Use biochemical approaches to determine the molecular basis for GCC185 Golgi complex localization; 2. Test a model for GCC185 as a vesicle-bound tether by accumulating transport vesicles in SNARE- depleted cells; 3. Characterize the binding of GCC185 to SNARE proteins implicated in endosome to Golgi transport, and test whether GCC185 catalyzes SNARE complex formation at the TGN; 4. Establish a membrane tethering assay using purified, immobilized GCC185 to explore its function. This is important because it will establish that GCC185 is a bona fide tethering protein, and may help us to purify these transport carriers for the first time. These experiments will provide important clues to the mechanism by which GCC185 is localized to the trans Golgi network, and what this protein does there, to facilitate the docking and fusion of transport vesicles, inbound from late endosomes. This work has broad implications for our understanding of vesicle docking and fusion events within the secretory and endocytic pathways that are essential for normal human health and disease.

描述（由申请人提供）：本研究的长期目标是了解人类细胞中膜结合区室之间蛋白质运输的分子基础。蛋白质转运包括货物收集到囊泡中，囊泡出芽，运动，在靶膜上系住和对接，以及随后的融合。系绳和对接是膜交通中最不为人所知的步骤。该应用程序的具体目标是研究名为GCC185的蛋白质的分子功能，GCC185是GRIP结构域家族的185K，反高尔基网络（TGN）定位蛋白。我们试图验证GCC185作为运输囊泡到达TGN的栓系蛋白的假设。关于GRIP域家族Golgins，需要解决的最重要的问题是：1。这些蛋白质实际上是系链吗？2. 这些蛋白质与什么分子合作来实现捆绑？3. 这些蛋白是囊泡相关系链还是靶标相关系链？4. 这些蛋白是否与tgn特异性SNARE蛋白结合？它们是如何在囊泡融合期间或之后从高尔基体中释放出来的？为了解决这些问题，我们建议：利用生物化学方法确定GCC185高尔基复合物定位的分子基础；2. 通过在SNARE缺失的细胞中积累运输囊泡，测试GCC185作为囊泡结合系链的模型；3. 表征GCC185与参与内体高尔基转运的SNARE蛋白的结合，并测试GCC185是否在TGN催化SNARE复合物的形成；4. 使用纯化的固定化GCC185建立膜系固实验，以探索其功能。这很重要，因为它将确定GCC185是一种真正的捆绑蛋白，并可能帮助我们首次纯化这些运输载体。这些实验将为GCC185定位于反式高尔基网络的机制提供重要线索，以及该蛋白在那里促进转运囊泡的对接和融合，从晚期核内体进入。这项工作对我们理解对正常人类健康和疾病至关重要的分泌和内吞途径中的囊泡对接和融合事件具有广泛的意义。