Purified Escherichia coli preprotein translocase catalyzes multiple cycles of precursor protein translocation

• Published in: Biochemistry (Easton) Vol. 32; no. 10; pp. 2626 - 2630
• Main Authors: Bassilana, Martine, Wickner, William
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 16-03-1993
• Subjects: Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphatases - metabolism; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacteriology; Binding Sites; Biological and medical sciences; Cell Membrane - enzymology; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Kinetics; Liposomes; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Membrane Transport Proteins; membrane vesicles; Microbiology; Permeability, membrane transport, intracellular transport; preprotein translocase; proOmpA protein; Proteolipids - metabolism; purification; reconstitution; SEC Translocation Channels; translocation; Membrane protein; Proteoliposome; Escherichia coli; Bacteria; Biosynthesis precursor; Kinetics; Membrane translocation; Comparative study; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00061a021

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.