Comparison in vitro of a high- and a low-abundance chemoreceptor of Escherichia coli:: Similar kinase activation but different methyl-accepting activities
Comparison in vitro of a high- and a low-abundance chemoreceptor of Escherichia coli:: Similar kinase activation but different methyl-accepting activities
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DOI：
10.1128/jb.180.24.6713-6718.1998
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发表时间：
1998-12-01
影响因子：
3.2
通讯作者：
Hazelbauer, GL
中科院分区：
文献类型：
--
作者：
Barnakov, AN;Barnakova, LA;Hazelbauer, GL
在大肠杆菌中，高丰度化学感受器的细胞数量大约是低丰度化学感受器的 10 倍。仅含有低丰度受体的细胞表现出异常低的翻滚频率，并且不能在空间梯度中有效迁移。这些缺陷反映了两种受体类别之间固有的活性差异。我们使用体外测定来研究这种差异。低丰度受体 Trg 介导的激酶 CheA 激活类似于 100 倍，仅比高丰度受体 Tar 的激活低两倍。相比之下，Trg 的体外甲基化活性不到 Tar 的 1/20。正如对高丰度受体所观察到的，Trg 激活的激酶随着甲基接受位点修饰的程度而变化。低甲基化对应于低激酶激活。因此，在仅 Trg 的细胞中，低受体甲基化将导致低激酶激活，相应地磷酸化 CheY 含量低，以及引诱剂结合调节激酶活性的动态范围降低。这些特征可以解释仅 Trg 单元表现出的低翻滚频率和低效出租车。因此，受体类别之间关键的功能差异可能是甲基接受活性。我们通过在 Trg 的羧基末端引入 CheR 结合五肽（通常仅在高丰度受体的羧基末端发现）来研究这种功能差异的结构基础。这种添加使 Trg 的体外甲基接受活性增强了 10 倍。
In Escherichia coli, high-abundance chemoreceptors are present in cellular amounts approximately 10-fold greater than low-abundance chemoreceptors. Cells containing only low-abundance receptors exhibit abnormally low tumble frequencies and do not migrate effectively in spatial gradients. These defects reflect an inherent activity difference between the two receptor classes. We used in vitro assays to investigate this difference. The low-abundance receptor Trg mediated an similar to 100-fold activation of the kinase CheA, only twofold less than activation by the high-abundance receptor Tar. In contrast, Trg was less than 1/20 as active as Tar for in vitro methylation. As observed for high-abundance receptors, kinase activation by Trg varied with the extend of modification at methyl-accepting sites; low methylation corresponded to low kinase activation. Thus, in Trg-only cells, low receptor methylation would result in low kinase activation, correspondingly low content of phospho-CheY, and a decreased dynamic range over which attractant binding could modulate kinase activity. These features could account for the low tumble frequency and inefficient taxis exhibited by Trg-only cells. Thus, the crucial functional difference between the receptor classes is likely to be methyl-accepting activity. We investigated the structural basis for this functional difference by introducing onto the carboxy terminus of Trg a CheR-binding pentapeptide, usually found only at the carboxy termini of high-abundance receptors. This addition enhanced the in vitro methyl-accepting activity of Trg 10-fold.