Conserved Aspartate Residues and Phosphorylation in Signal Transduction by the Chemotaxis Protein CheY
The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior.
Additional Information© 1990 by National Academy of Sciences Contributed by Melvin I. Simon, October 11, 1989 We thank P. Matsumura for plasmid pRL22 and K. Borkovich and T. Wilkie for critical reading of the manuscript. This work was supported by Grant AI1926 from the National Institutes of Health (to M.I.S.), by National Research Service Award Fellowship A107798 (to R.B.B.); and by Damon Runyon-Walter Winchell Cancer Fund Fellowship DRG-915 (to J.F.H.). R.B.B. and J.F.H. contributed equally to this work. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Published - BOUpnas90.pdf