GENETIC ANALYSIS OF THE SECRETORY APPARATUS OF E COLI

大肠杆菌分泌器官中的突变体，阻断了

Mutants in the secretory apparatus of E. coli, which block the synthesis of exported proteins, have been isolated. In eukaryotes, it has been shown that signal sequence mutations can recognize a component of the secretory apparatus; this recognition results in an arrest in the translation of exported proteins. Synthesis is not resumed until the arrested translation complex comes in contact with the endoplasmic reticulum (ER) membrane. The E. coli secretory mutant, secC, was isolated as a pseudorevertant of another secretion mutant, secA. At 23 degrees C, the secC mutant blocks the synthesis of transported proteins. In this mutant, the synthesis block of an exported protein can be reversed by mutating the signal sequence of that protein. This proposal has several aims. First, mutations in an exported protein, which specifically reverse the synthesis block of that protein in the secC mutant, will be isolated and sequenced. This work should define the domains of an exported protein that are involved in its synthesis. Second, the isolation of pseudorevertants of the secC mutant should identify genes whose protein products interact with the secC gene product. Third, the isolation and phenotypic analysis of a knock-out mutant in secC should ultimately unravel the role of the secC protein. This knock-out mutant will be constructed by using Tn5 mutagenesis. Further characterization of the secC mutant will also be done. Antibody directed against the SecA protein will be used to determine whether the SecC and SecA proteins interact. Immunoprecitation and temperature shift experiments, in the presence of a drug that prevents the initiation of protein synthesis, should determine if the block in synthesis is site-specific. The effect that the secC mutation has on the SecC proteins will also be evaluated. Protein secretion is an essential cellular process. Studies on the mechanism of secretion in E. coli may strengthen our understanding of this process in all organisms.