Thermosensing properties of mutant aspartate chemoreceptors with methyl-accepting sites replaced singly or multiply by alanine
Thermosensing properties of mutant aspartate chemoreceptors with methyl-accepting sites replaced singly or multiply by alanine
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DOI：
10.1128/jb.179.21.6573-6580.1997
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发表时间：
1997-11-01
影响因子：
3.2
通讯作者：
Kawagishi, I
中科院分区：
文献类型：
--
作者：
Nishiyama, SI;Nara, T;Kawagishi, I
天冬氨酸化学受体Tar具有温度敏感功能，其四个甲基化位点(Gln295、Glu302、Gln309和Glu491)的共价修饰调节了该功能。如果没有翻译后脱酰亚胺，焦油就没有热敏能力。当Gln295和Gln309被脱胺为Glu时，未甲基化和高度甲基化的形式分别起到热传感器和冷传感器的作用。在本研究中，我们对甲基化位点进行了丙氨酸扫描突变，虽然丙氨酸替换影响了信号偏向和甲基化水平，但所有突变体都保留了天冬氨酸敏感功能。具有单一替换的那些具有几乎正常的温度敏感特性，这表明在任何特定甲基化位置的替换不会严重损害温度敏感功能。在翻译后修饰缺陷的背景下，一些丙氨酸取代恢复了温度敏感能力。在保留两个谷氨酸残基的突变体中发现了温敏感受器，在含有一个谷氨酸残基或没有谷氨酸残基的突变体中发现了冷感应器。这一结果表明，甲基化部位的负电荷是决定温度传感器表型的一个因素，尽管侧链的大小和形状也可能是重要的。热的、冷的和零的温度传感器表型有明显的区别，没有发现中间表型。因此，甲基化位点的共价修饰导致的不同温度敏感表型可能反映不同的结构状态。还讨论了对热敏机制的更广泛的影响。
The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modification of its four methylation sites (Gln295, Glu302, Gln309, and Glu491). Without posttranslational deamidation, Tar has no thermosensing ability. When Gln295 and Gln309 are deamidated to Glu, the unmethylated and heavily methylated forms function as warm and cold sensors, respectively. In this study, we carried out alanine-scanning mutagenesis of the methylation sites, Although alanine substitutions influenced the signaling bias and the methylation level, all of the mutants retained aspartate-sensing function. Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function. In the posttranslational modification-defective background, some of the alanine substitutions restored thermosensing ability. Warm sensors were found among mutants retaining two glutamate residues, and cold sensors were found among those with one or no glutamate residue. This result suggests that the negative charge at the methylation sites is one factor that determines thermosensor phenotypes, although the size and shape of the side chain may also be important. The warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes were found. Thus, the different thermosensing phenotypes that result from covalent modification of the methylation sites may reflect distinct structural states. Broader implications for the thermosensing mechanism are also discussed.