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.
Bassilana
M
M
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
Wickner
W
W
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D026901:Membrane Transport Proteins; D011510:Proteolipids / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011510:Proteolipids; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins