Molecular Analysis of the CCC185 Golgin

描述（由申请人提供）：这项研究的长期目标是了解人类细胞膜结合区室之间蛋白质运输的分子基础。蛋白质运输涉及将货物收集到囊泡中、囊泡出芽、运动、束缚和停靠在目标膜上以及随后的融合。束缚和对接是膜交通中最不为人所知的步骤。此应用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、反式高尔基体网络 (TGN) 定位蛋白质。我们试图检验 GCC185 作为到达 TGN 的运输囊泡的束缚蛋白的假设。对于GRIP结构域家族Golgins来说，需要解决的最重要的问题是： 1.这些蛋白质实际上是束缚蛋白吗？ 2. 这些蛋白质与哪些分子配合以实现束缚？ 3. 这些蛋白质是否充当囊泡相关系链或靶标相关系链？ 4. 这些蛋白是否与 TGN 特异性 SNARE 蛋白结合，它们在囊泡融合期间或之后如何从高尔基体释放？为了开始解决这些问题，我们建议： 1. 使用生化方法确定 GCC185 高尔基复合体定位的分子基础； 2. 通过在 SNARE 耗尽的细胞中积累转运囊泡，测试 GCC185 作为囊泡结合系链的模型； 3.表征GCC185与涉及内体至高尔基体转运的SNARE蛋白的结合，并测试GCC185是否催化TGN处SNARE复合物的形成； 4. 使用纯化的固定化 GCC185 建立膜束缚测定，以探索其功能。这很重要，因为它将确定 GCC185 是一种真正的束缚蛋白，并可能帮助我们首次纯化这些运输载体。这些实验将为 GCC185 定位于反高尔基体网络的机制提供重要线索，以及该蛋白在那里的作用，以促进从晚期内涵体入站的运输囊泡的对接和融合。这项工作对于我们理解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，这对于正常人类健康和疾病至关重要。

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.