PROTEIN TRANSLOCATION ACROSS ESCHERICHIA COLI MEMBRANES

跨大肠杆菌膜的蛋白质易位

• 批准号: 3286301
• 负责人: PHANG C. TAI
• 金额: $ 20.44万
• 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/3286301
• 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; nucleic acid sequence; 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.

跨膜的蛋白质分泌是最重要，最复杂的蛋白质 生长细胞的生理过程。 该提议的主要目的是 在体外分析该过程的分子细节，特别是 将脂蛋白以外的蛋白质易位到细菌膜 涉及信号肽酶的囊泡I。 阐明蛋白质易位机制将结合 良好的体外遗传操作和生化研究 大肠杆菌碱性磷酸酶的易位系统和 OMPA进入倒的细胞质膜囊泡，以分析 ATP水解，膜蛋白和可溶性细胞质因子 蛋白质易位。 建立在最近的演示中 蛋白质中的SECY/PRLA，SECA，SECB和其他蛋白质因子参与 易位，纯化的因素和抗体将用于定义其 在此过程中的角色。 探索最近的生理含义 观察到SECA可以被磷酸化和去磷酸化，并且 蛋白质因子SECI抑制蛋白质易位，我们将测试 假设ATP要求可能是由于瞬时磷酸化引起的 和蛋白质转运过程所需的Seca的去磷酸化 通过膜，并调节蛋白质的流动 易位。 为了确定SECA在膜中的功能， 参与磷酸化和去磷酸化的成分将是 确定和表征。 化学与双功能交联 试剂，然后进行免疫沉淀，并与 膜囊泡和脂质体将用于识别成分 与Seca和Secy/PRLA有关蛋白质易位。 蛋白质 蛋白质易位涉及的因素将被纯化， 部分氨基酸序列将确定与 已知的蛋白质，如果新颖，这些基因将被克隆。 这些角色 前体分子易位能力的因素将是 检查。 对细菌中发现的机制的理解可能具有 人类细胞对蛋白质分泌的显着意义， 应该对医学上重要的重要含义 蛋白质在细菌中使用重组DNA。 此外， 了解微生物毒素的分泌对于 医学细菌学。