Molecular Analysis of the CCC185 Golgin

描述(申请人提供)：这项研究的长期目标是了解蛋白质在人类细胞膜结合室之间运输的分子基础。蛋白质运输包括将货物收集到囊泡中、囊泡发芽、运动、系留和对接在靶膜上，以及随后的融合。系留和对接是膜流量中最不为人所知的步骤。本申请的具体目的是研究GCC185蛋白的分子功能，GCC185是GRIP结构域家族中185K的反式高尔基网络(TGN)定位蛋白。我们试图测试这一假设，即GCC185作为到达TGN的运输小泡的系留蛋白。关于GRIP结构域家族高尔基体，需要解决的最重要的问题是：1.这些蛋白实际上是拴系的吗？2.这些蛋白是与什么分子合作来实现系链的？3.这些蛋白是作为囊泡相关的系链还是靶相关的系链？4.这些蛋白是否与TGN特异性的SNARE蛋白结合，以及它们是如何在囊泡融合过程中或之后从高尔基体释放出来的？为了开始解决这些问题，我们建议：1.使用生化方法来确定GCC185高尔基复合体定位的分子基础；2.通过在SNARE耗竭的细胞中积累运输小泡来测试GCC185作为囊泡结合系链的模型；3.表征GCC185与涉及内体到高尔基转运的SNARE蛋白的结合，并测试GCC185是否催化TGN处SNARE复合体的形成；4.使用纯化的固定化GCC185建立膜系留实验，以探索其功能。这一点很重要，因为它将确定GCC185是一个真正的系留蛋白，并可能帮助我们首次纯化这些运输载体。这些实验将为GCC185定位到跨高尔基体网络的机制提供重要线索，以及这种蛋白质在那里起什么作用，以促进从晚期内体进入的运输小泡的对接和融合。这项工作对于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。

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.