Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ.
CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow counter-clockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephosphorylation rates of these mutants. CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of cheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephosphorylation rates. Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.
Sanna
M G
MG
Division of Biology 147-75, California Institute of Technology, Pasadena 91125, USA.
Swanson
R V
RV
Bourret
R B
RB
Simon
M I
MI
D001426:Bacterial Proteins; D001665:Binding Sites; D002465:Cell Movement / Q000235:genetics; D002633:Chemotaxis / Q000235:genetics*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D000071677:Histidine Kinase; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation / Q000235:genetics*; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D010749:Phosphoprotein Phosphatases