ER export and the quality control checkpoint during membrane protein biogenesis

膜蛋白生物合成过程中的内质网输出和质量控制检查点

• 批准号: 7267734
• 负责人: Elizabeth A. Miller
• 金额: $ 29万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7267734
• 关键词: ATP-Binding Cassette Transporters; Animal Model; Binding; Biochemical Genetics; Biochemistry; Biogenesis; Capsid Proteins; Cell membrane; Cells; Client; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoplasm; Cytoplasmic Vesicles; Defect; Diabetes Mellitus; Disease; Employee Strikes; Endoplasmic Reticulum; Ensure; Equilibrium; Eukaryotic Cell; Event; Genetic; Heart Diseases; Homologous Gene; Human; Insulin Resistance; Intracellular Transport; Kinetics; Lead; Left; Lung diseases; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Chaperones; Mutation; Numbers; Osteogenesis Imperfecta; Pathology; Pathway interactions; Pharmaceutical Preparations; Platelet Factor 4; Play; Poison; Process; Proteins; Pump; Quality Control; Research Proposals; Resources; Role; Saccharomyces cerevisiae; Transport Vesicles; Ubiquitin; Vesicle; Yeasts; human disease; improved; multicatalytic endopeptidase complex; mutant; novel; novel therapeutics; protein folding; protein function; protein misfolding; response; uptake

DESCRIPTION (provided by applicant): Eukaryotic cells dedicate significant resources to the deployment of proteins to the membrane compartments that comprise the secretory pathway. Biogenesis of all membrane proteins starts with the folding and assembly of newly synthesized proteins within the endoplasmic reticulum (ER), often with the aid of cellular chaperones. Cells must strike a precise balance between ensuring that only fully folded proteins are allowed to leave the ER and avoiding the accumulation of misfolded proteins within the ER lumen. Uptake of newly synthesized cargo molecules into ER-derived transport vesicles only occurs once proteins are fully folded. These transport vesicles, known as COPII vesicles for the cytoplasmic coat proteins that drive membrane curvature and select cargo, thus play a critical role in regulating forward transport of new proteins. We study the close relationship between protein folding and packaging into ER- derived COPII vesicles in the model organism, Saccharomyces cerevisiae. Using a combination of genetics and biochemistry, we aim to define the cellular machinery that acts at the interface between protein folding and ER export. A model for examining this process is the yeast ABC transporter, Yor1, a plasma membrane protein that acts as a drug pump to clear toxic substances from the yeast cytoplasm. Yor1 is a homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR), defects in which cause cystic fibrosis. Deletion of a Phe residue in Yor1, equivalent to the major disease-related mutation in human cystic fibrosis, causes Yor1, like mutant CFTR, to be ER-retained and degraded by the cytoplasmic ubiquitin/proteasome pathway. Thus Yor1 is a useful model that allows the direct comparison of the intracellular itineraries of native and aberrant forms of a single protein. This research proposal consists of four specific aims. (1) To define the molecular mechanisms that drive uptake of Yor1 into COPII vesicles and assess how protein folding influences this event. (2) To determine how cellular chaperones contribute to Yor1 biogenesis and assess how the kinetics of chaperone/client interactions may influence COPII binding and thereby regulate ER export. (3) To identify and characterize novel factors that may facilitate Yor1 biogenesis, including specific membrane chaperones and more general folding factors. (4) To determine the mechanisms by which the unfolded protein response improves the folding and/or transport of misfolded proteins. Ultimately, a better understanding of cellular machinery that acts to regulate protein folding and forward transport may lead to novel therapeutic approaches to treat the many diseases associated with aberrant protein folding within the secretory pathway.

描述（由申请人提供）：真核细胞将大量资源专用于蛋白质的部署到构成分泌途径的膜室。所有膜蛋白的生物发生始于内质网（ER）内新合成蛋白（ER）的折叠和组装，通常借助细胞伴侣。细胞必须在确保仅允许完全折叠的蛋白质离开ER并避免ER腔内错误折叠蛋白的积累之间取得精确的平衡。仅一旦蛋白质完全折叠，才能将新合成的货物分子吸收到ER衍生的传输囊泡中。这些转运囊泡，称为驱动膜曲率并选择货物的细胞质涂层蛋白的复合囊泡，因此在调节新蛋白的​​正向转运方面起着至关重要的作用。我们研究了模型生物体酿酒酵母中的蛋白质折叠和包装中蛋白质折叠和包装之间的关系。使用遗传学和生物化学的组合，我们旨在定义对蛋白质折叠和ER导出之间界面作用的细胞机制。检查此过程的模型是酵母ABC转运蛋白YOR1，一种质膜蛋白，可作为药物泵清除酵母细胞质的有毒物质。 YOR1是人囊性纤维化跨膜电导调节剂（CFTR）的同源物，在其中引起囊性纤维化。 YOR1中PHE残基的删除相当于人囊性纤维化中与疾病相关的主要突变，会导致YOR1（例如突变CFTR）被胞质泛质泛素/蛋白酶体途径递增和降解。因此，YOR1是一个有用的模型，可以直接比较单个蛋白质天然和异常形式的细胞内行程。该研究建议由四个具体目标组成。 （1）定义将YOR1吸收到Copii囊泡中的分子机制，并评估蛋白质折叠如何影响这一事件。 （2）确定细胞伴侣如何促进YOR1生物发生，并评估伴侣/客户相互作用的动力学如何影响COPII结合，从而调节ER导出。 （3）识别和表征可能有助于YOR1生物发生的新因素，包括特定的膜伴侣和更一般的折叠因子。 （4）确定展开的蛋白质反应的机制可改善错误折叠蛋白的折叠和/或运输。最终，对调节蛋白质折叠和前进转运的细胞机械的更好理解可能会导致新型的治疗方法，以治疗与分泌途径中异常蛋白质折叠相关的许多疾病。