Regulation of GlnK activity:: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum
Regulation of GlnK activity:: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum
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DOI：
10.1111/j.1365-2958.2004.04247.x
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发表时间：
2004-10-01
影响因子：
3.6
通讯作者：
Burkovski, A
中科院分区：
文献类型：
--
作者：
Strösser, J;Lüdke, A;Burkovski, A
P（II）型信号转导蛋白在许多细菌的氮调节中起着重要作用。响应于细胞内氮状态，这些蛋白质通过修饰/去修饰事件（例如通过磷酸化或尿苷酰化）而发挥其功能并与其他蛋白质相互作用。在这项研究中，我们表明，GlnK，唯一的P（II）型蛋白在谷氨酸棒杆菌，腺苷酸化响应氮饥饿和deadenylated当氮供应再次提高。这两个过程都依赖于GlnD蛋白。如突变体分析所示，该酶的修饰活性位于酶的N-末端部分，而去修饰依赖于其C-末端结构域。除了其修饰状态外，GlnK蛋白还响应于细胞氮供应的变化而改变其细胞内定位。虽然在氮饥饿期间存在于细胞质中，但GlnK蛋白在氮饥饿期后响应于铵脉冲而被隔离到细胞质膜。加入铵后，约2-5%的GlnK库位于细胞质膜上。GlnK与细胞质膜的结合依赖于铵转运蛋白AmtB，其在与GlnK和GlnD相同的转录单位中编码，即amtB-glnK-glnD操纵子。相反，结构上相关的甲基铵/铵通透酶AmtA不结合GlnK。膜结合的GlnK蛋白是稳定的，最有可能阻止AmtB依赖的铵转运，以防止在饥饿后富氨条件下的有害的无效循环，而大多数GlnK在2-4分钟内被降解。氮代谢的其它蛋白质如谷氨酰胺合成酶或与铵同化无关的蛋白质如烯醇化酶和ATP合成酶亚基F（1）β在这些条件下是稳定的。我们对不同突变菌株的分析表明，至少有三种不同的蛋白酶影响GlnK的降解，即FtsH、ClpCP和ClpXP蛋白酶复合物。
P(II)-type signal transduction proteins play a central role in nitrogen regulation in many bacteria. In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g. by phosphorylation or uridylylation. In this study, we show that GlnK, the only P(II)-type protein in Corynebacterium glutamicum, is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again. Both processes depend on the GlnD protein. As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain. Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply. While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period. About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition. GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnK-glnD operon. In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK. The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min. Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F(1)beta, are stable under these conditions. Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.