MECHANISM OF PROTEIN SECRETION IN E COLI

这项建议的主要目标是分析E. 这是分泌蛋白质所必需的。 胞质膜。我们将继续用基因方法来 定义了细菌的基因，这些基因编码了 出口机械。我们将制定新的选择策略 突变体在分泌方面存在多向性缺陷。我们将继续 两种分泌物的体内遗传和生化特性 我们最近鉴定的基因，secD和secE。这 鉴定涉及，克隆，测序，分离 零突变，制备针对基因产物的抗体 以及这些产物的蜂窝位置的确定。 我们正在分析sec基因产物在体外培养中的作用。 蛋白质分泌系统。这项工作是通过合作完成的 与戴博士和他的同事们一起，他们已经建立了 Secy和SecA基因在机体功能中的重要作用 在这个系统中。我们将研究生态环境的调节机制。 SEC基因。我们已经证明了secA和secD基因是 由细胞的分泌需求调节。突变 阻断分泌导致去抑制这些物质的合成 蛋白质。我们将使用遗传方法来确定 这些基因的调控以及涉及的调控因素。我们 将进行研究以确定出口的属性是否 这一过程将使我们能够开发出解决问题的遗传方法 蛋白质折叠。B-半乳糖苷酶检测结果显示 蛋白质折叠的可能性，正常情况下 细胞质可能会干扰它们的跨膜转移。 如果是这样的话，对干扰基因突变的基因选择 折叠是可能的。至少有一种经过充分研究的蛋白质，即 色氨酸合成酶B亚基，将被用作模型系统 用于这些研究。

The major goal of this proposal is to analyze the components of E. coli which are required for secretion of proteins across the cytoplasmic membrane. We will continue genetic approaches to defining the genes of the bacteria which code for components of the export machinery. We will develop new strategies for selecting mutants pleiotropically defective in secretion. We will continue in vivo genetic and biochemical characterization of two secretion genes which we have recently identified, secD and secE. This characterization involves, cloning, sequencing, the isolation of null mutations, the preparation of antibodies to the gene products and the determination of the cellular location of these products. We are analyzing the role of the sec gene products in an in vitro protein secretion system. This work is being done in collaboration with Dr. P.-C. Tai and his colleagues, who have established the essential role of the secY and secA genes for the functioning of this system. We will study the mechanism of regulation of the sec genes. We have shown that the secA and secD genes are regulated by the secretion needs of the cell. Mutations which block secretion cause a derepression in the synthesis of these proteins. We will use genetic approaches to define the site of regulation of these genes and the regulatory factors involved. We will do studies to determine whether the properties of the export process will allow us to develop a genetic approach to the problem of protein folding. Results with B-galactosidase suggest the possibility that the folding of proteins which, are normally cytoplasmic may interfere with their transfer across the membrane. If this is so, a genetic selection for mutants that interfere with folding would be possible. At least one well-studied protein, the B subunit of tryptophan synthetase, will be used as a model system for these studies.