In Vivo Acetylation of CheY, a Response Regulator in Chemotaxis of Escherichia coli Jianshe Yan 1 †, Rina Barak 1 †, Orna Liarzi 1 , Alla Shainskaya 2 and Michael Eisenbach 1 ⁎ 1 Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel 2 Biological Mass Spectrometry Facility, Department of Biological Services, The Weizmann Institute of Science, 76100 Rehovot, Israel Received 12 October 2007; received in revised form 26 December 2007; accepted 27 December 2007 Available online 5 January 2008 CheY, the excitatory response regulator in the chemotaxis system of Escherichia coli, can be modulated by two covalent modifications: phosphorylation and acetylation. Both modifications have been detected in vitro only. The role of CheY acetylation is still obscure, although it is known to be involved in chemotaxis and to occur in vitro by two mechanisms—acetyl-CoA synthetase-catalyzed transfer of acetyl groups from acetate to CheY and autocatalyzed transfer from AcCoA. Here, we succeeded in detecting CheY acetylation in vivo by three means—Western blotting with a specific anti-acetyl-lysine antibody, mass spectrometry, and radiolabeling with [ 14 C]acetate in the presence of protein-synthesis inhibitor. Unexpectedly, the level and rate of CheY acetylation in vivo were much higher than that in vitro. Thus, before any treatment, 9–13% of the lysine residues were found acetylated, depending on the growth phase, meaning that, on average, essentially every CheY molecule was acetylated in vivo. This high level was mainly the outcome of autoacetylation. Addition of acetate caused an incremental increase in the acetylation level, in which acetyl-CoA synthetase was involved too. These findings may have far- reaching implications for the structure–function relationship of CheY. © 2007 Elsevier Ltd. All rights reserved. Edited by I. B. Holland Keywords: acetylation; bacterial chemotaxis; chemotaxis; CheY; response regulator Introduction Chemotactic signals in bacteria such as Escherichia coli are transduced from the receptor supramolecu- lar complex to the flagellar–motor supramolecular complex by the response regulator CheY. This re- sponse regulator undergoes a dual covalent modi- fication: phosphorylation and acetylation. Both covalent modifications shift the flagellar rotation from the default direction, counterclockwise, to a higher probability of clockwise rotation. Phosphor- ylation, which is modulated by the histidine kinase CheA and the phosphatase CheZ according to changes in the receptor's occupancy and methyla- tion level, shifts the rotational direction by increas- ing the binding of CheY to the switch protein FliM at the base of the flagellar–motor supramolecular complex (for reviews, see Refs. 1–5). Acetylation is achieved both by acetyl-CoA synthetase (Acs; with acetate as the acetyl donor) 6,7 and by autocatalysis (with acetyl-CoA as the acetyl donor), 8 and evidence for its involvement in chemotaxis has been pro- vided. 9 Both covalent modifications are linked: the kinase CheA inhibits CheY acetylation, whereas the phosphatase CheZ enhances it; conversely, Acs increases the phosphorylation level of CheY. 10 Thus, CheY phosphorylation and acetylation appear to be coregulated, similarly to some mammalian transcription factors. 11–16 When CheY is isolated from E. coli and purified, it is already acetylated to a low extent, suggesting that CheY maintains in vivo a basal level of acetylation. 7 *Corresponding author. E-mail address: m.eisenbach@weizmann.ac.il. † J.Y. and R.B. contributed equally to this work. Abbreviations used: Acs, acetyl-CoA synthetase; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; LC, liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; KLH, keyhole limpet hemocyanin; TFA, trifluoroacetic acid. doi:10.1016/j.jmb.2007.12.070 J. Mol. Biol. (2008) 376, 1260–1271 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.