Bren A, Welch M, Blat Y, Eisenbach M
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10090-3. doi: 10.1073/pnas.93.19.10090.
Chemotaxis in bacteria is controlled by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein CheY. When phosphorylated, CheY binds to FliM, which is one of the proteins that constitute the "gear box" (or "switch") of the flagellar motor. Consequently, the motor shifts from the default direction of rotation, counterclockwise, to clockwise rotation. This biased rotation is terminated when CheY is dephosphorylated either spontaneously or, faster, by a specific phosphatase, CheZ. Logically, one might expect CheZ to act directly on FliM-bound CheY. However, here we provide direct biochemical evidence that, in contrast to this expectation, phosphorylated CheY (CheY approximately P), bound to FliM, is protected from dephosphorylation by CheZ. The complex between CheY approximately P and FliM was trapped by cross-linking with dimethylsuberimidate, and its susceptibility to CheZ was measured. CheY approximately P complexed with FliM, unlike free CheY approximately P, was not dephosphorylated by CheZ. However, it did undergo spontaneous dephosphorylation. Nonspecific cross-linked CheY dimers, measured as a control, were dephosphorylated by CheZ. No significant binding between CheZ and any of the switch proteins was detected. It is concluded that, in the termination mechanism of signal transduction in bacterial chemotaxis, CheZ acts only on free CheY approximately P. We suggest that CheZ affects switch-bound CheY approximately P by shifting the equilibrium between bound and free CheY approximately P.
细菌中的趋化作用是通过调节鞭毛旋转方向来控制的。这种调节由趋化蛋白CheY执行。磷酸化时，CheY与FliM结合，FliM是构成鞭毛马达“齿轮箱”（或“开关”）的蛋白质之一。因此，马达从默认的逆时针旋转方向转变为顺时针旋转。当CheY自发去磷酸化或更快地被特定磷酸酶CheZ去磷酸化时，这种偏向旋转就会终止。从逻辑上讲，人们可能期望CheZ直接作用于与FliM结合的CheY。然而，在这里我们提供了直接的生化证据，与这种预期相反，与FliM结合的磷酸化CheY（CheYP）受到CheZ的保护而不被去磷酸化。通过与亚氨二甲酸二甲酯交联捕获CheYP与FliM之间的复合物，并测量其对CheZ的敏感性。与游离的CheYP不同，与FliM复合的CheYP不会被CheZ去磷酸化。然而，它确实会发生自发去磷酸化。作为对照测量的非特异性交联CheY二聚体被CheZ去磷酸化。未检测到CheZ与任何开关蛋白之间有明显的结合。得出的结论是，在细菌趋化作用信号转导的终止机制中，CheZ仅作用于游离的CheYP。我们认为，CheZ通过改变结合态和游离态CheYP之间的平衡来影响与开关结合的CheY~P。
