A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.
A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.
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DOI：
10.1104/pp.119.1.219
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发表时间：
1999
期刊：
影响因子：
7.4
通讯作者：
Emmanuelle Pigaglio;N. Durand;Christian Meyer
中科院分区：
文献类型：
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作者：
Emmanuelle Pigaglio;N. Durand;Christian Meyer
先前已经表明，烟草（Nicotiana tabacum）硝酸还原酶（NR）的N-末端结构域参与了在黑暗中发生的磷酸化导致的酶失活（L. Nussaiti，M.阿森茨角Meyer，J.P. Boutin，and M. Caboche [1995] Plant Cell 7：611-621）。缺乏该N-末端结构域的突变NR蛋白的活性不再受光暗转换的调节。在这项研究中，在烟草NR的N-末端结构域进行了较小的缺失，去除了高等植物NR中保守的蛋白质基序。然后将得到的截短的NR编码序列融合到花椰菜花叶病毒35 S RNA启动子上，并引入到密切相关的烟草种的NR缺陷突变体中。我们发现，删除一个保守的延伸的酸性残基导致一个活跃的NR蛋白，这是比野生型酶更热敏感，但它是相对不敏感的失活磷酸化在黑暗中。因此，去除该酸性片段似乎对NR激活状态具有与缺失N末端结构域相同的影响。对这些观察结果的一个假设性解释是，阻碍失活的特定因子仍然与截短的酶结合。还发现来源于该酸性蛋白基序的合成肽是酪蛋白激酶II的良好底物。
It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.