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

描述(申请人提供)：这项工作旨在加深我们对真核细胞膜运输的理解。细胞内膜运输对细胞和生物体的发育至关重要。此外，囊泡连接因子中的各种故障，如保守的寡聚体高尔基复合体(COG)，会对蛋白质分选、糖基化和细胞器完整性等过程产生重大影响。我们提出了一个详细的结构-功能分析的机制，决定高保真双向蛋白质和脂肪在高尔基体运输。将研究SNARS、RABS和系留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序列。我们的长期目标是建立高尔基体内膜转运的分子机制。COG复合体是一个8亚基的外周高尔基膜异寡聚体蛋白复合体，被组织成A叶(Cog2-4)和B叶(Cog5-7)，Cog1p和Cog8p连接这两个叶。COG复合体被认为在囊泡拴系过程中发挥关键作用，该过程涉及负责糖链(多糖)生物合成的驻留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复合体功能的模型，在该模型中，COG复合体特异性地捆绑小泡，从跨高尔基体中回收回收的顺式/中位高尔基蛋白。我们假设COG复合体通过一系列蛋白质-蛋白质相互作用来协调囊泡拴系和融合。这些相互作用包括与线圈-线圈系留因子以及特定的RAB和SNARE分子的瞬时接触。这项建议的目标是定义人类COG复合体与高尔基体内对接和融合机械的核心组件相互作用的序列和分子机制。我们将通过(1)研究COG复合体与高尔基体内SNARE蛋白相互作用的分子细节，(2)表征高尔基体Rab蛋白与COG复合体相互作用的分子基础，(3)表征COG复合体的自组装和膜附着过程，以及(4)表征COG复合体、线圈-线圈拴系因子(S)、RABS和SNARS之间的功能相互作用，从而实现这一目标。这一建议在方法上是综合的，因为我们将应用各种细胞生物学、生化和显微工具来解决这个问题。我们预计，拟议的实验将测试我们的COG复合体功能模型，并解释在COG复合体活性急性和永久性耗尽后细胞中发生的细胞缺陷。 与公共卫生相关：在人类中，COG复合体亚单位的突变改变高尔基体组织和细胞膜运输(Ungar等人，2002年；Zolov和Lupashin，2005年；Shestakova等人，2006年；Ungar等人，2006年)，导致多个糖基化途径中断，最终导致精神运动迟缓和低眼压(COG1突变(Foulquier等人，2006年)和COG8突变(Foulquier等人，2007年)或死于心功能不全和严重肝病(COG7突变患者(Wu等人，2004年))。我们预计，拟议的实验将测试我们的COG复合体功能模型，为COG复合体活性改变患者发生的细胞缺陷提供解释，并为开发COG复合体相关疾病的治疗提供分子基础。

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.