Sanna M G, Swanson R V, Bourret R B, Simon M I
Division of Biology 147-75, California Institute of Technology, Pasadena 91125, USA.
Mol Microbiol. 1995 Mar;15(6):1069-79. doi: 10.1111/j.1365-2958.1995.tb02282.x.
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.
CheY是一种小的细胞质应答调节因子，在趋化途径中起关键作用。磷酸化CheY的浓度被认为决定细胞的游动行为：高水平的磷酸化CheY使细菌的鞭毛顺时针旋转并翻滚，而低水平的该蛋白磷酸化形式则允许鞭毛逆时针旋转并平稳游动。体内CheY的磷酸化状态由磷酸供体CheA的活性以及磷酸化CheY的去磷酸化拮抗作用决定。去磷酸化速率由磷酸化CheY的固有自水解活性和CheZ蛋白控制，CheZ蛋白可加速去磷酸化。我们分析了CheZ对几种突变型CheY蛋白去磷酸化速率的影响。鉴定出两个点突变，它们对CheZ活性的敏感性分别比野生型蛋白低50倍和5倍。尽管如此，在没有CheZ的情况下，这些突变体CheY23ND和CheY26KE的磷酸化和自动去磷酸化速率与野生型CheY相同。这些是cheY突变的首个例子，其降低了对CheZ磷酸酶活性的敏感性，但其固有磷酸化和自动去磷酸化速率未改变。有趣的是，Asn-23和Lys-26残基位于CheY远离磷酸化位点（Asp-57）的一侧，与先前描述的与组氨酸激酶CheA相互作用的位点不同，并且部分与涉及与鞭毛开关相互作用的区域重叠。
