MOLECULAR GENETICS OF PEPTIDE EXPORT IN E. COLI

蛋白质在各种细胞内的正确定位 隔室对正常运作至关重要 活的细胞。 蛋白质出口的过程一直是 在过去的十年里，我们进行了深入的研究。 对革兰氏阴性 细菌，这个问题可以通过考虑有限的 一组隔室：a）细胞质，B）细胞质膜， c）周质空间，d）外膜和e）细胞膜。 细胞外环境 对于分子结构知之甚少 细菌分泌生物活性肽的机制 进入介质。 拟议工作的长期目标是 在分子水平上表征导致 E.杆菌 这些 肽具有抗生素活性，似乎是从 通过新的输出途径来控制细胞。 为了实现这些目标，我们 建议确定肽的成熟途径， 它们从细胞内前体变成细胞外活性物质， forms. 这将通过以下组合来实现： 生物化学和遗传学方法。 的免疫沉淀 在各种突变体中积累的标记中间体 肽输出的缺陷将为此提供证据 通路 将产生蛋白质融合物以确定 参与肽输出的假定膜蛋白的拓扑结构。 一组43个氨基酸序列中的单个氨基酸替换， 将产生氨基酸肽。 的生化分析 这些突变形式将提供信息， 肽的结构与其各种功能。

The correct localization of proteins within the various cellular compartments is of critical importance to the normal functioning of the living cell. The process of protein export has been intensively studied during the last decade. For gram-negative bacteria, the problem can be simplified by considering a limited set of compartments: a) the cytoplasm, b) the cytoplasmic membrane, c) the periplasmic space, d) the outer membrane and e) the extracellular milieu. Little is known as to the molecular mechanisms by which bacteria secrete biologically active peptides into the medium. The long term objectives of the proposed work are to characterize at the molecular level the steps leading to the export of two peptides produced by strains of E. coli. These peptides possess antibiotic activity and appear to be exported from the cell via novel export pathways. To achieve these goals, we propose to determine the pathway of maturation of the peptides as they go from intracellular precursors to extracellular active forms. This will be accomplished using a combination of biochemical and genetic approaches. Immunoprecipitation of labelled intermediates which accumulate in a variety of mutants defective in peptide export will provide the evidence for this pathway. Protein fusions will be generated to determine the topology of a putative membrane protein involved in peptide export. A collection of single amino acid replacements in the forty-three amino acid peptide will be generated. Biochemical analysis of these mutant forms will provide information that will relate the structure of the peptide to its various functions.