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和最复杂的 细胞生长中的生理过程。这项建议的主要目的是 在体外分析这一过程的分子细节，特别是 脂蛋白以外的蛋白质转位到细菌膜上 含有信号肽I的囊泡。 对蛋白质转位机制的阐明将结合 一种成熟的体外遗传操作和生化研究 大肠埃希氏菌碱性磷酸酶转位系统 OmpA转化为倒置细胞质膜囊泡的作用分析 三磷酸腺苷水解酶、膜蛋白和可溶性细胞质因子 蛋白质易位。以最近的演示为基础 SecY/PrlA、SecA、SecB等蛋白质因子在蛋白质中的作用 易位、纯化因子和抗体将被用来确定它们的 在这一过程中的角色。探索新近的生理意义 观察到Seca可以被磷酸化和去磷化，并且 一种蛋白质因子SecI抑制蛋白质转位，我们将测试 假设ATP需求可能是由于短暂的磷酸化 和蛋白质转运过程中所需的SecA去磷酸化 通过细胞膜，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成分将是 确定和表征的。双官能团化学交联 试剂，然后免疫沉淀，部分重组与 膜囊泡和脂质体将被用于鉴定成分 SecA和SecY/PrlA参与蛋白质易位。这种蛋白质 参与蛋白质转位的因子将被提纯， 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，这些基因将被克隆。这些角色的作用 影响前体分子移位能力的因素将是 检查过了。 对细菌中发现的机制的理解可能会有 对人类细胞和蛋白质分泌的重要意义 应该具有重要的实际意义，对于医学上重要的 在细菌中使用重组DNA的蛋白质。此外， 了解微生物毒素的分泌对于 医学细菌学。