Characterization of mammalian COG complex-interacting intra-Golgi trafficking mac

描述（由申请人提供）：这项工作旨在进一步了解真核细胞中的膜运输。细胞膜内运输对细胞和机体发育至关重要。此外，囊泡系结因子（如保守寡聚高尔基（COG）复合体）的各种功能障碍对蛋白质分选、糖基化和细胞器完整性等过程有重大影响。我们提出了一个详细的结构功能分析的机制，决定高尔基体的高保真双向蛋白质和脂质运输。将研究SNAREs、Rabs和系绳因子之间的功能相互作用；我们还希望确定导致囊泡对接和融合的事件顺序。我们的长期目标是建立高尔基膜内转运的分子机制。COG复合体是一种由8个亚基组成的外周高尔基膜异质寡聚蛋白复合体，由Cog1p和Cog8p连接成A叶（Cog2-4）和B叶（Cog5-7）。COG复合物被认为在囊泡系结过程中起关键作用，涉及负责糖链（聚糖）生物合成的常驻蛋白的逆行高尔基转运。基于我们和其他实验室的数据，我们提出了一个COG复合体功能模型，其中COG复合体特异性地系住囊泡，从反式高尔基区室中回收回收顺式/中位高尔基蛋白。我们假设COG复合体通过一系列蛋白质相互作用来协调囊泡的捆绑和融合。这些相互作用包括与线圈系绳因子和特定的Rab和SNARE分子的短暂接触。本研究的目标是确定人类COG复合体与高尔基对接融合机制核心组分相互作用的序列和分子机制。我们将通过(1)研究COG复合体与高尔基SNARE蛋白相互作用的分子细节，(2)表征高尔基Rab蛋白与COG复合体相互作用的分子基础，(3)表征COG复合体自组装和膜附着的过程，以及(4)表征COG复合体、线圈-线圈系缚因子、Rabs和SNAREs之间的功能相互作用来实现这一目标。这个建议在方法上是综合的，因为我们将应用各种细胞生物学，生物化学和微观工具来解决这个问题。我们预计提出的实验将测试我们的COG复合物功能模型，并解释在COG复合物活性急性和永久性耗尽后细胞中发生的细胞缺陷。

DESCRIPTION (provided by applicant): This work is designed to further our understanding of membrane trafficking in eukaryotic cells. Intracellular membrane trafficking is critically important to cellular and organismal development. Furthermore, various malfunctions in vesicle tethering factors, such as the Conserved Oligomeric Golgi (COG) complex, have a significant impact on processes such as protein sorting, glycosylation and organelle integrity. We propose a detailed structural-functional analysis of the machinery that determines high-fidelity bidirectional protein and lipid trafficking in the Golgi apparatus. The functional interplay between SNAREs, Rabs and tethering factors will be investigated; we also hope to identify the sequence of events that lead to vesicle docking and fusion. Our long-term goal is to establish the molecular mechanism through which intra-Golgi membrane trafficking is accomplished. The COG complex is an eight subunit peripheral Golgi membrane hetero-oligomeric protein complex that is organized into lobes A (Cog2-4) and B (Cog5-7) with Cog1p and Cog8p bridging these lobes. The COG complex is thought to play a critical role in vesicle tethering processes involving retrograde Golgi transport of resident proteins responsible for sugar chain (glycan) biosynthesis. Based on data from our and other laboratories, we proposed a model for COG complex function in which COG complex specifically tethers vesicles that retrieve recycling cis/medial-Golgi proteins from trans-Golgi compartments. We hypothesize that the COG complex orchestrates vesicle tethering and fusion through a cascade of protein-protein interactions. These interactions include transient contacts with coil-coil tethering factors and specific Rab and SNARE molecules. The goal of this proposal is to define both the sequence and the molecular mechanism of human COG complex interactions with core components of intra-Golgi docking and fusion machinery. We will accomplish this goal through (1) investigation the molecular details of interaction between the COG complex and intra-Golgi SNARE proteins, (2) characterization the molecular basis by which Golgi Rab proteins interact with the COG complex, (3) characterization the processes of COG complex self-assembly and membrane attachment and (4) characterization the functional interplay between the COG complex, coil-coil tethering factor(s), Rabs and SNAREs. This proposal is integrated methodologically because we will apply various cell biological, biochemical and microscopic tools to approach the problem. We anticipate that proposed experiments would test our model of COG complex function and explain cellular defects occurring in cells after both acute and permanent depletion of COG complex activity. Public Health Relevance: In humans, mutations in COG complex subunits alter the Golgi organization and intracellular membrane trafficking (Ungar et al., 2002; Zolov and Lupashin, 2005; Shestakova et al., 2006; Ungar et al., 2006), resulting in disruption of multiple glycosylation pathways, and, ultimately in suffering from psychomotor retardation and hypotonia (COG1-mutant (Foulquier et al., 2006), and COG8-mutant (Foulquier et al., 2007) patients) or in lethality from cardiac insufficiency and severe liver disease (COG7-mutant (Wu et al., 2004) patients). We anticipate that proposed experiments would test our model for the COG complex function, provide explanation for cellular defects occurring in patients with altered COG complex activity and provide molecular basis for development treatments for the COG complex-related deceases.