Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli.
The ATP-hydrolytic activity of SecA protein is stimulated up to 100-fold by the translocation-competent precursor to outer membrane protein A (pro-OmpA) in conjunction with inner-membrane vesicles bearing active SecY [Lill, R., Cunningham, K., Brundage, L., Ito, K., Oliver, D. & Wickner, W. (1989) EMBO J. 8, 961-966]. This reaction is saturable, with Michaelis-Menten kinetics for an enzyme with two substrates, ATP and pro-OmpA, and is defined as translocation ATPase. Another precursor protein, pre-PhoE, is also a substrate for this translocation ATPase. Neither OmpA nor its synthetic leader peptide are effective substrates for translocation ATPase, suggesting that both domains of the complete precursor are necessary for the reaction. The leader peptide is a potent inhibitor and apparently competes with pro-OmpA for necessary binding sites on translocation ATPase. After a brief preincubation, the activity of translocation ATPase becomes resistant to inhibition by leader peptide, suggesting that the leader peptide is recognized at an early step in the protein translocation pathway. Our enzymological studies show that translocation ATPase recognizes and functionally binds the leader region of precursor proteins.
Cunningham
K
K
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
Wickner
W
W
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D021382:Protein Sorting Signals / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D009842:Oligopeptides; D021382:Protein Sorting Signals; D006180:Proton-Translocating ATPases