Bourret R B, Hess J F, Simon M I
Division of Biology, California Institute of Technology, Pasadena 91125.
Proc Natl Acad Sci U S A. 1990 Jan;87(1):41-5. doi: 10.1073/pnas.87.1.41.
The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior.
在大肠杆菌的趋化信号转导途径中，CheY蛋白由CheA磷酸化，并由CheZ去磷酸化。有人提出CheY的磷酸化发生在一个天冬氨酸残基上。利用定点诱变技术将CheY的八个天冬氨酸残基逐一替换。天冬氨酸-12、天冬氨酸-13或天冬氨酸-57的替换导致趋化性丧失。大多数突变型CheY蛋白仍能被CheA磷酸化，但表现出改变的生化特性，包括从CheA接受磷酸的能力降低、磷酸基团稳定性改变和/或对CheZ介导的去磷酸化具有抗性。在位置57处有替换的CheY蛋白的特性最为异常，这与天冬氨酸-57是酰基磷酸形成的正常位点这一假设一致。本文提供了天冬氨酸-57突变体中另一个磷酸化位点的证据。磷酸化的CheY被认为会导致翻滚行为。然而，一种在体外未被磷酸化的显性突变型CheY蛋白在体内CheA不存在的情况下会导致翻滚。这种表型表明，野生型CheY蛋白中磷酸化的作用是稳定一种能产生翻滚行为的瞬时构象变化。
