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

哺乳动物 COG 复合体相互作用的高尔基体内运输机制的表征

• 批准号: 8272541
• 负责人: VLADIMIR V LUPASHIN
• 金额: $ 35.8万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8272541
• 关键词: Acute; Affect; Anabolism; Binding; Biochemical; Biological; Cardiac; Cells; Complex; Data; Defect; Development; Distal; Docking; Enzyme Interaction; Eukaryotic Cell; Event; GTP Binding; Goals; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Human; Intracellular Membranes; Investigation; Laboratories; Lead; Liver diseases; Lobe; Medial Golgi; Membrane; Membrane Protein Traffic; Microscopic; Modeling; Molecular; Muscle hypotonia; Mutation; Organelles; Pathway interactions; Patients; Peripheral; Play; Polysaccharides; Process; Proteins; Recruitment Activity; Recycling; Role; SNAP receptor; Site; Sorting - Cell Movement; Testing; Vesicle; Work; base; design; glycosylation; lipid transport; mutant; protein complex; protein protein interaction; protein transport; public health relevance; receptor; research study; self assembly; sugar; therapy development; tool; trafficking

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.

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。细胞内膜运输对细胞和生物体的发育至关重要。此外，囊泡拴系因子中的各种故障，例如保守寡聚体高尔基体（COG）复合物，对蛋白质分选、糖基化和细胞器完整性等过程具有显著影响。我们提出了一个详细的结构功能分析的机器，确定高保真双向蛋白质和脂质贩运的高尔基体。SNARE，Rabs和拴系因子之间的功能相互作用将被调查，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膜内运输的分子机制。COG复合物是八个亚基的外周高尔基体膜异源寡聚蛋白复合物，其被组织成叶A（Cog 2 -4）和B（Cog 5 -7），Cog 1 p和Cog 8 p桥接这些叶。COG复合物被认为在涉及负责糖链（聚糖）生物合成的驻留蛋白质的逆行高尔基体运输的囊泡拴系过程中起关键作用。基于我们和其他实验室的数据，我们提出了一个模型，COG复杂的功能，其中COG复合物专门拴囊泡检索回收顺式/中间高尔基体蛋白从trans-Golgi隔间。我们假设COG复合物通过蛋白质-蛋白质相互作用的级联来协调囊泡的束缚和融合。这些相互作用包括与螺旋-螺旋栓系因子和特异性Rab和SNARE分子的瞬时接触。这个提议的目标是定义人类COG复合物与高尔基体内对接和融合机制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复合物和高尔基体内陷阱蛋白之间相互作用的分子细节，（2）表征高尔基体Rab蛋白与COG复合物相互作用的分子基础，（3）表征COG复合物自组装和膜附着的过程，以及（4）表征COG复合物之间的功能相互作用，线圈-线圈系留因子、Rabs和SNARE。这个建议是集成的方法，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，拟议的实验将测试我们的模型的COG复杂的功能，并解释细胞缺陷后发生在细胞中的急性和永久性消耗的COG复杂的活动。 公共卫生相关性：在人类中，COG复合物亚基的突变改变高尔基体组织和细胞内膜运输（Ungar et al.，2002; Zolov和Lupashin，2005; Shestakova等人，2006; Ungar等人，2006），导致多种糖基化途径的破坏，并最终患有精神发育迟滞和张力减退（COG 1-突变体（Foulquier et al.，2006）和COG 8-突变体（Foulquier等人，2007）患者）或死于心功能不全和严重肝病（COG 7突变体（Wu等人，2004）患者）。我们预计，拟议的实验将测试我们的模型的COG复杂的功能，提供解释的细胞缺陷发生在患者与改变COG复杂的活动，并提供分子基础上开发治疗的COG复杂相关的疾病。