The role of the mature domain of proOmpA in the translocation ATPase reaction

• Published in: The Journal of biological chemistry Vol. 267; no. 35; pp. 25246 - 25250
• Main Authors: BASSILANA, M, ARKOWITZ, R. A, WICKNER, W
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-12-1992
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; adenosinetriphosphatase; Bacterial Outer Membrane Proteins - isolation & purification; Bacterial Outer Membrane Proteins - metabolism; Biological and medical sciences; Cell physiology; Electrophoresis, Polyacrylamide Gel; Endopeptidases - metabolism; Escherichia coli; Escherichia coli - metabolism; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Kinetics; Membrane and intracellular transports; Membrane Proteins; Molecular and cellular biology; Molecular Weight; OmpA protein; Peptide Fragments - isolation & purification; precursors; Protein Precursors - isolation & purification; Protein Precursors - metabolism; reaction; role; Serine Endopeptidases; translocation; C terminal peptide; Enzyme; ATPase; Escherichia coli; Reaction mechanism; Bacteria; Precursor; Membrane translocation; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)74032-0

The export of proOmpA, the precursor of outer membrane protein A from Escherichia coli, requires preprotein translocase, which
is comprised of SecA, SecY/E, and acidic phospholipids. Previous studies of proOmpA translocation intermediates (Schiebel,
E., Driessen, A. J. M., Hartl, F.-U., and Wickner, W. (1991) Cell 64, 927-939) suggested that the "slippage" of the translocating
polypeptide chain and the high level of ATP hydrolysis, characteristic of the "translocation ATPase," were part of a futile
cycle. To examine the role of the mature domain of proOmpA in its translocation-dependent ATP hydrolysis, we used chemical
cleavage to generate NH2-terminal fragments of this preprotein. Each fragment contained the 21-residue leader region and either
53 or 228 residues of the mature domain (preproteins P74 and P249, respectively). As observed with full-length proOmpA, the
translocation of each fragment requires ATP and both the SecA and SecY/E domains of translocase and is stimulated by the transmembrane
proton electrochemical gradient. The apparent maximal velocities of P74 and proOmpA translocation are similar. While the translocation
of P74 and of proOmpA show the same apparent Km for ATP, far less ATP is hydrolyzed during the translocation of P74. Thus,
the mature carboxyl-terminal domain of proOmpA has a major role in supporting the translocation ATPase.