Phosphorylation-dependent binding of the chemotaxis signal molecule CheY to its phosphatase, CheZ.
Bacterial chemotaxis is accomplished by regulating the direction of flagellar rotation. The primary target of the control appears to be CheY, a diffusible clockwise-signal molecule which interacts with the switch at the base of the flagellar motor and causes clockwise rotation. The regulatory mechanism appears to be phosphorylation/dephosphorylation of CheY. Here we demonstrate that CheZ, which accelerates the dephosphorylation of CheY, binds to CheY (immobilized on CNBr-activated Sepharose beads), that the binding to phosphorylated CheY is higher by over 2 orders of magnitude than the binding to nonphosphorylated CheY, and that the binding to both the phosphorylated and nonphosphorylated forms of CheY is significantly higher in the presence of Mg2+. We also show that the mutant proteins CheY13DK, CheY57DE, and CheY109KR bind CheZ to the same extent as wild-type CheY. The extent of the binding of these mutant proteins was not, however, increased in the presence of acetyl phosphate, the phosphorylating agent. The results indicate that neither a conformation which has a clockwise-causing activity in vivo nor phosphorylation is sufficient, alone, for maximal binding of CheZ to CheY and that Mg2+ is required for the binding of these proteins as well as for the phosphorylation and dephosphorylation of CheY.
Blat
Y
Y
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Eisenbach
M
M
D001426:Bacterial Proteins; D001665:Binding Sites; D002633:Chemotaxis; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D029968:Escherichia coli Proteins; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium