MOLECULAR GENETICS OF PEPTIDE EXPORT IN E. COLI

蛋白质在不同细胞内的正确定位 舱室对正常运作至关重要 活着的细胞。蛋白质出口的过程一直是 在过去十年中进行了深入的研究。对于革兰氏阴性 细菌，这个问题可以通过考虑有限的 一组隔室：a)细胞质，b)细胞膜， C)周质间隙，d)外膜和e) 胞外环境。对分子方面的了解很少。 细菌分泌生物活性多肽的机制 变成了媒介。拟议工作的长期目标是 在分子水平上表征通向 出口两种由大肠杆菌菌株产生的多肽。这些 多肽具有抗生素活性，似乎是从 该细胞通过新的出口途径。为了实现这些目标，我们 建议将多肽的成熟途径确定为 它们从细胞内前体到细胞外活性 表格。这将使用以下组合来实现 生物化学和遗传方法。免疫沉淀法 在各种突变体中积累的标记中间体 多肽出口存在缺陷将为此提供证据 路径。将产生蛋白质融合以确定 可能参与多肽输出的膜蛋白的拓扑结构。 四十三年单一氨基酸替代物的集合 将生成氨基酸多肽。生物化学分析 这些突变形式将提供与 多肽的结构使其具有各种功能。

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.