PROTEIN TRANSLOCATION & EXPORT IN GRAM-NEGATIVE BACTERIA

蛋白质的分泌是最重要也是最复杂的生理过程之一 生长细胞中的过程。这项建议的主要目的是分析 这一过程在体外的分子细节，即蛋白质易位 进入细菌膜囊泡。此外，我们还将进一步研究如何 在某些革兰氏阴性菌中，蛋白质通过双膜排泄 细菌。 对蛋白质移位机制的阐明是非常重要的。 在我们新开发的高效提取系统的帮助下， 碱性磷酸酶和OmpA蛋白的mRNA，以及内膜 囊泡，都来自大肠杆菌。该系统可以分离出不同阶段的 从翻译的移位，这使得生化 蛋白质易位的分析要简单得多。有了这个系统，我们将 寻找蛋白质转位需要ATP和 质子推动力，以及蛋白质的复合体是否可能是 真核信号识别颗粒的细菌等价物，以及 其他细胞质蛋白也参与其中。鉴定膜蛋白 在转位过程中，我们将使用部分重组、蛋白酶 分泌蛋白前体与膜的失活、交联 蛋白质、阻断功能的特异性抗体和有缺陷的突变体 分泌物。 革兰氏阴性菌双膜蛋白排泄的研究 细菌，我们将研究生物合成和排泄途径 铜绿假单胞菌溶血素和霍乱弧菌毒素。在基础上建设 毒素向外排泄需要不存在的基因的证据 在大肠杆菌细胞中，我们将从假单胞菌中引入额外的基因 大肠杆菌，并将寻找将毒素排泄到外部的转化子 而不是将其分泌到周质中。如果结果是肯定的， 将确定所需的基因(S)和蛋白质(S)。 对细菌中发现的机制的理解可能会有 对人类细胞分泌蛋白质的影响，并且应该有 对医疗工业应用的重要实际意义 细菌中的重组DNA。此外，微生物毒素的研究 与医学细菌学直接相关。

Protein secretion 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, i.e. protein translocation into bacterial membrane vesicles. In addition, we will examine further how proteins are excreted past a double membrane in certain Gram-negative bacteria. The elucidation of the mechanism of protein translocation is greatly facilitated by our newly developed, efficient system utilizing extracts, mRNA for alkaline phosphatase and OmpA protein, and inner membrane vesicles, all from E. coli. This system can separate the stage of translocation from that of translation, which makes the biochemical analysis of protein translocation much simpler. With this system we will pursue indications that protein translocation requires ATP as well as protonmotive force, and whether a complex of proteins that may be the bacterial equivalent of the eukaryotic signal recognition particle, and other cytoplasmic proteins, are involved. To identify membrane proteins involved in translocation we will use partial reconstitution, protease inactivation, cross-linking of precursors of secreted proteins to membrane proteins, specific antibodies to block function, and mutants defective in secretion. To study protein excretion past a double membrane in Gram-negative bacteria, we will examine the biosynthesis and excretion pathway of Pseudomonas aeruginosa hemolysin and Vibrio cholera toxin. Building on evidence that excretion of toxin to the exterior requires genes not present in E. coli cells, we will introduce additional genes from Pseudomonas into E. coli and will seek transformants that excrete toxin to the exterior instead of secreting it into the periplasm. If the results are positive, the required gene(s) and protein(s) will be identified. An understanding of mechanisms discovered in bacteria is likely to have implications for secretion of proteins by human cells, and should have important practical implications for the medical-industrial use of recombinant DNA in bacteria. Furthermore, the studies of microbial toxins are directly relevant to medical bacteriology.