PURIFIED ESCHERICHIA-COLI PREPROTEIN TRANSLOCASE CATALYZES MULTIPLE CYCLES OF PRECURSOR PROTEIN TRANSLOCATION

Escherichia coli preprotein translocase, composed of the peripheral membrane protein SecA bound at the integral membrane domain SecY/E, has been isolated and functionally reconstituted [Brundage, L., Hendrick, J.P., Schiebel, E., Driessen, A.J.M., & Wickner, W. (1990) Cell 62, 649-657]. It is not known whether this purified enzyme supports multiple turnover cycles and how its kinetics compare with translocase in inverted membrane vesicles. We now report a quantitative comparison of the translocation of the outer membrane protein A precursor (proOmpA) by purified preprotein translocase and by inner membrane vesicles. ProOmpA cross-linked to bovine pancreatic trypsin inhibitor was used for quantitative titration of the functional translocation sites. The rate of proOmpA translocation per active site in this purified system is 25% of that observed in inverted membrane vesicles. Each functional site can catalyze multiple cycles of precursor translocation. These results indicate that the purified preprotein translocase properly reconstitutes translocation.