PROTEIN TRANSLOCATION ACROSS ESCHERICHIA COLI MEMBRANES

跨大肠杆菌膜的蛋白质易位

• 批准号: 3286303
• 负责人: PHANG C. TAI
• 金额: $ 18.33万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286303
• 关键词: Escherichia coli; adenosinetriphosphatase; alkaline phosphatase; antibacterial antibody; bacterial genetics; bacterial proteins; bacterial toxins; bioenergetics; crosslink; cytoplasm; gene expression; genetic recombination; hydrolysis; lipid bilayer membrane; liposomes; membrane permeability; membrane proteins; molecular cloning; peptidases; phosphorylation; protein biosynthesis; protein purification; protein transport; recombinant DNA; secretion; tissue /cell culture

Protein secretion across membranes is one of the most important and complex physiological processes in growing cells. The main aim of this proposal is to analyze the molecular details of this process in vitro, specifically translocation of proteins other than lipoproteins into bacterial membrane vesicles involving signal peptidase I. The elucidation of the mechanism of protein translocation will combine genetic manipulation and biochemical studies in a well-established in vitro system for the translocation of Escherichia coli alkaline phosphatase and OmpA into inverted cytoplasmic membrane vesicles to analyze the roles of ATP hydrolysis, membrane proteins and soluble cytoplasmic factors in protein translocation. Building on the recent demonstration of the involvement of SecY/PrlA, SecA, SecB and other protein factors in protein translocation, purified factors and antibodies will be used to define their roles in the process. Exploring the physiological implication of recent observations that SecA can be phosphorylated and dephosphorylated and that a protein factor SecI inhibits protein translocation, we will test the hypothesis that the ATP requirement may be due to transient phosphorylation and dephosphorylation of SecA required for the process of protein transit through the membranes, and that SecI regulates the flow of protein translocation. To determine the functions of SecA in the membranes, the components involved in the phosphorylation and dephosphorylation will be identified and characterized. Chemical crosslinking with bifunctional reagents, followed by immunoprecipitation, and partial reconstitution with membrane vesicles and liposomes will be used to identify the components involved with SecA and SecY/PrlA in protein translocation. The protein factors that are involved in protein translocation will be purified, the partial amino acid sequence will be determined to search for homology with known proteins, and if novel, the genes will be cloned. The roles of these factors on the translocation competency of precursor molecules will be examined. An understanding of mechanisms discovered in bacteria is likely to have significant implications for secretion of proteins by human cells and should have important practical implications for the medically important proteins with the use of recombinant DNA in bacteria. Furthermore, understanding the secretion of microbial toxins is also important to medical bacteriology.

蛋白质跨膜分泌是生物体内最重要和最复杂的 生长细胞的生理过程。 这项建议的主要目的是 在体外分析这个过程的分子细节， 脂蛋白以外的蛋白质向细菌膜的移位 涉及信号肽酶I的囊泡。 蛋白质转位机制的阐明将联合收割机 基因操作和生化研究在一个良好建立的体外 用于大肠杆菌碱性磷酸酶易位的系统， OmpA进入倒置的细胞质膜囊泡，以分析OmpA的作用。 ATP水解、膜蛋白和可溶性胞质因子在 蛋白质易位 在最近展示的 SecY/PrlA、SecA、SecB等蛋白因子参与蛋白质 易位，纯化的因子和抗体将用于定义它们的 过程中的角色。 探索最近的生理意义 观察到SecA可以被磷酸化和去磷酸化， 蛋白因子SecI抑制蛋白质易位，我们将测试 假设ATP需求可能是由于瞬时磷酸化 以及蛋白质转运过程所需的SecA去磷酸化 SecI调节蛋白质的流动 易位 为了确定SecA在膜中的功能， 参与磷酸化和去磷酸化的组分将被 识别和表征。 双官能化学交联 试剂，然后进行免疫沉淀，并用 膜囊泡和脂质体将用于鉴定组分 参与SecA和SecY/PrlA蛋白质转运。 蛋白质 参与蛋白质转运的因子将被纯化， 将确定部分氨基酸序列以搜索与 已知的蛋白质，如果是新的，基因将被克隆。 这些角色 影响前体分子易位能力的因素将是 考察 对细菌中发现的机制的理解可能会 对人类细胞分泌蛋白质的重要意义， 应该对医学上重要的 在细菌中使用重组DNA的蛋白质。 此外，委员会认为， 了解微生物毒素的分泌也很重要， 医学细菌学