Barak Rina, Yan Jianshe, Shainskaya Alla, Eisenbach Michael
Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel.
J Mol Biol. 2006 Jun 2;359(2):251-65. doi: 10.1016/j.jmb.2006.03.033. Epub 2006 Mar 31.
One of the processes by which CheY, the excitatory response regulator of chemotaxis in Escherichia coli, can be activated to generate clockwise flagellar rotation is by acetyl-CoA synthetase (Acs)-mediated acetylation. Deletion of Acs results in defective chemotaxis, indicating the involvement of Acs-mediated acetylation in chemotaxis. To investigate whether Acs is the sole acetylating agent of CheY, we purified the latter from a delta acs mutant. Mass spectrometry analysis revealed that this protein is partially acetylated in spite of the absence of Acs, suggesting that CheY can be post-translationally acetylated in vivo by additional means. Using [14C]AcCoA in the absence of Acs, we demonstrated that one of these means is autoacetylation, with AcCoA serving as an acetyl donor and with a rate similar to that of Acs-mediated acetylation. Biochemical characterization of autoacetylated CheY and mass spectrometry analysis of its tryptic digests revealed that its acetylated lysine residues are those found in CheY acetylated by Acs, but the acetylation-level distribution among the acetylation sites was different. Like CheY acetylated by Acs, autoacetylated CheY could be deacetylated by Acs. Also similarly to the case of Acs-mediated acetylation, the phosphodonors of CheY, CheA and acetyl phosphate, each inhibited the autoacetylation of CheY, whereas the phosphatase of CheY, CheZ, enhanced it. A reduced AcCoA level interfered with chemotaxis to repellents, suggesting that CheY autoacetylation may be involved in chemotaxis of E. coli. Interestingly, this interference was restricted to repellent addition and was not observed with attractant removal, thus endorsing our earlier suggestion that the signaling pathway triggered by repellent addition is not identical to that triggered by attractant removal.
大肠杆菌趋化性的兴奋性反应调节因子CheY可被激活以产生顺时针鞭毛旋转的过程之一是通过乙酰辅酶A合成酶（Acs）介导的乙酰化作用。Acs缺失会导致趋化性缺陷，这表明Acs介导的乙酰化作用参与了趋化性。为了研究Acs是否是CheY的唯一乙酰化剂，我们从Δacs突变体中纯化了CheY。质谱分析表明，尽管不存在Acs，该蛋白仍被部分乙酰化，这表明CheY可以通过其他方式在体内进行翻译后乙酰化。在没有Acs的情况下使用[14C]乙酰辅酶A，我们证明其中一种方式是自身乙酰化，乙酰辅酶A作为乙酰供体，其速率与Acs介导的乙酰化相似。对自身乙酰化的CheY进行生化表征及其胰蛋白酶消化产物的质谱分析表明，其乙酰化的赖氨酸残基与被Acs乙酰化的CheY中的相同，但乙酰化位点之间的乙酰化水平分布不同。与被Acs乙酰化的CheY一样，自身乙酰化的CheY可以被Acs去乙酰化。同样与Acs介导的乙酰化情况类似，CheY的磷酸供体CheA和乙酰磷酸均抑制CheY的自身乙酰化，而CheY的磷酸酶CheZ则增强其自身乙酰化。乙酰辅酶A水平降低会干扰对驱避剂的趋化性，这表明CheY自身乙酰化可能参与大肠杆菌的趋化性。有趣的是，这种干扰仅限于添加驱避剂时，在去除引诱剂时未观察到，因此支持了我们之前的观点，即添加驱避剂引发的信号通路与去除引诱剂引发的信号通路不同。
