Molecular mechanisms of cargo capture into ER-derived transport vesicles

内质网衍生运输囊泡中货物捕获的分子机制

• 批准号: 7507898
• 负责人: Elizabeth A. Miller
• 金额: $ 30.04万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7507898
• 关键词: Animal Model; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biochemistry; Biogenesis; Capsid Proteins; Cells; Client; Communication; Complex; Cystic Fibrosis; Cytoplasmic Vesicles; Defect; Destinations; Development; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Ensure; Environment; Eukaryotic Cell; Event; Face; Genetic; Genetic Screening; Heart; Heart Diseases; Human Development; Intracellular Transport; Life; Link; Lipids; Lung diseases; Membrane; Molecular; Molecular Chaperones; Molecular Conformation; Nature; Numbers; Organelles; Osteogenesis Imperfecta; Pathway interactions; Personal Satisfaction; Play; Pliability; Population; Process; Protein Binding; Protein Secretion; Proteins; Proteome; Proteomics; Public Health; Quality Control; Regulation; Research Proposals; Role; Route; Saccharomyces cerevisiae; Saccharomycetales; Staging; Structure; Transport Vesicles; Vesicle; Work; absorption; base; cell growth; human disease; insight; mutant; novel; protein folding; protein misfolding; secretory protein; trafficking

DESCRIPTION (provided by applicant): Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the life of all eukaryotic cells. Fully one-third of the eukaryotic proteome is targeted to the membrane compartments that comprise the secretory pathway. These proteins must be faithfully delivered to these organelles after synthesis and folding in the endoplasmic reticulum (ER). The first step in protein delivery to downstream compartments is the selective capture of proteins into ER-derived transport vesicles, known as COPII vesicles for the cytoplasmic coat proteins that sculpt these transport carriers from the donor membrane. Cargo proteins are enriched in nascent vesicles through the action of the COPII coat subunit, Sec24p, which serves as a cargo- binding platform by providing multiple interfaces for cargo-coat interaction. Although we have gained enormous insight into this function of Sec24p through biochemical, genetic and structural analyses, we are currently lacking a full description of the diversity and flexibility of this process, which must accommodate a vast array of client proteins with distinct structures, functions, and ultimate destinations. We aim to fully understand the molecular interactions that drive export out of the ER, and are working to determine the full spectrum of cargo proteins that bind to Sec24p to ensure efficient ER egress. We use the model organism, Saccharomyces cerevisiae, to study this fundamental process using a combination of genetic, proteomic and biochemical approaches. This research proposal consists of two specific aims. (1) To determine the full repertoire of secretory proteins that interacts with the three known cargo-binding sites on Sec24p and to define the mode of interaction of novel client cargoes. (2) To determine the nature of cargo capture or coat assembly defects associated with three novel Sec24p mutants that were isolated from a structure-based genetic screen. Using these approaches, we seek to gain detailed insight into the molecular mechanisms that underlie the diversity and flexibility of cargo capture. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease and development that are impacted by early stages of protein biogenesis and deployment within the secretory pathway. We study the detailed molecular interactions that drive the selective capture of newly synthesized secretory proteins into transport vesicles that bud from the endoplasmic reticulum. Public Health Relevance: This process represents a critical quality control decision in the cell, and has important implications for the growing number of diseases that are caused by aberrant folding, trafficking and deployment of secretory proteins, including cystic fibrosis, familial heart disease and lung disease.

描述（由申请人提供）：有效而准确的蛋白质分泌是一个基本过程，在所有真核细胞的生活中起着关键作用。完全三分之一的真核蛋白质组针对构成分泌途径的膜室。这些蛋白质必须忠实地递送到内质网（ER）中的合成和折叠后。蛋白质向下游区室的第一步是将蛋白质的选择性捕获到ER衍生的传输囊泡中，被称为细胞质涂层蛋白，这些蛋白质被称为copii囊泡，这些蛋白质是从供体膜上雕刻这些传输载体的细胞质涂层蛋白。通过COPII涂层亚基Sec24p的作用将货物蛋白富含新生囊泡，该囊泡通过为货物涂层交互作用提供多个接口来充当货物结合平台。尽管我们通过生化，遗传和结构分析对SEC24P的这一功能有了深入的了解，但目前我们缺乏对此过程的多样性和灵活性的完整描述，该过程必须适应具有不同结构，功能，功能和最终目的地的大量客户蛋白。我们旨在充分了解将导出出口出口的分子相互作用，并正在努力确定与SEC24P结合的完整货物蛋白，以确保有效的EGERS。我们使用模型生物酿酒酵母使用遗传，蛋白质组学和生化方法的组合来研究这一基本过程。该研究建议由两个具体目标组成。 （1）确定与SEC24P上三个已知的货物结合位点相互作用的分泌蛋白的完整曲目，并定义了新型客户货物的相互作用方式。 （2）确定与从基于结构的遗传筛查中分离出的三个新型SEC24P突变体相关的货物捕获或外套组件缺陷的性质。使用这些方法，我们寻求详细了解载有货物捕获的多样性和灵活性的分子机制。最终，对这一基本真核过程的更详细的理解将对人类疾病和发育的许多方面具有重要意义，这些方面受到蛋白质生物发生和分泌途径内蛋白质生物发生和部署的影响。我们研究了详细的分子相互作用，这些相互作用将新合成的分泌蛋白选择性捕获到来自内质网的传输囊泡中。公共卫生相关性：此过程代表了细胞中的关键质量控制决策，并且对由于异常折叠，贩运和部署分泌蛋白（包括囊性纤维化，家族性心脏病和肺部病）引起的疾病的数量越来越重要。