PROTEIN TRANSLOCATION & EXPORT IN GRAM-NEGATIVE BACTERIA

蛋白质分泌是机体最重要、最复杂的生理过程之一， 在生长的细胞中。 本提案的主要目的是分析 体外这一过程的分子细节，即蛋白质易位 转化成细菌膜囊泡。 此外，我们将进一步研究如何 在某些革兰氏阴性菌中， 细菌 蛋白质转位机制的阐明， 通过我们新开发的利用提取物的高效系统， 碱性磷酸酶和OmpA蛋白的mRNA，以及内膜 囊泡，均来自E.杆菌 该系统可以将 从翻译中转移，这使得生物化学 蛋白质易位分析简单得多。 有了这个系统， 追求的迹象表明，蛋白质易位需要ATP以及 质子动力，以及蛋白质复合物是否可能是 所述真核信号识别颗粒的细菌等价物，和 其他细胞质蛋白也参与其中。 来鉴定膜蛋白 涉及易位，我们将使用部分重建，蛋白酶 失活，分泌蛋白质前体与膜的交联 蛋白质，阻断功能的特异性抗体，以及 分泌物 研究革兰氏阴性杆菌的蛋白质跨膜排泄， 细菌，我们将研究生物合成和排泄途径， 绿脓杆菌溶血素和霍乱弧菌毒素。 基础上 证据表明，毒素排泄到外部需要基因不存在 在大肠大肠杆菌细胞，我们将引入额外的基因从假单胞菌， E.大肠杆菌，并将寻求转化分泌毒素到外部 而不是分泌到周质中 如果结果是肯定的， 将鉴定所需的基因和蛋白质。 对细菌中发现的机制的理解可能会 对人类细胞分泌蛋白质的影响，应该有 重要的实际意义的医疗工业使用的 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.