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.
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