IN-VIVO STUDIES OF THE ROLE OF SECA DURING PROTEIN EXPORT IN ESCHERICHIA-COLI

SecA is found in Escherichia coli both tightly associated with the cyt oplasmic membrane where it functions as a translocation ATPase during protein export and free in the cytosol (R. J. Cabelli, K. M. Dolan, L. Qian, and D. B. Oliver, J. Biol. Chem. 266:24420-24427, 1991; D. B. O liver and J. Beckwith, Cell 30:311-319, 1982; W. Wickner, A. J. M. Dri essen, and F.-U. Hartl, Annu. Rev. Biochem. 60:101-124, 1991). Here we show that SecA can be immunoprecipitated from the cytosol in complex with both fully elongated and nascent species of the precursor of malt ose-binding protein, an exported, periplasmic protein. In addition, un der conditions in which the distribution of SecA between the cytosolic and membrane-bound states changes from that normally observed, the di stribution of precursor maltose-binding protein changes in parallel. T hese results support the idea that cytosolic SecA plays a role in expo rt. With the aim of determining the roles of the multiple binding site s for ATP on SecA, we compared the export defect in a culture off. col i expressing a temperature-sensitive allele of secA with the defect in a culture treated with sodium azide. The results indicate that the mu tational change and treatment with sodium azide inhibit export by affe cting different steps in the cycle of ATP binding and hydrolysis by Se cA.