Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit
Chloroplast acetyl-CoA carboxylase activity is 2-oxoglutarate-regulated by interaction of PII with the biotin carboxyl carrier subunit
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DOI：
10.1073/pnas.0910097107
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发表时间：
2010-01-05
影响因子：
11.1
通讯作者：
Hodges, Michael
中科院分区：
文献类型：
--
作者：
Bourrellier, Ana Belen Feria;Valot, Benoit;Hodges, Michael
PII蛋白是参与调节细菌和植物氮代谢的信号整合子。在感知细胞碳和能量可用性后，PII通过与靶蛋白相互作用传递信号，从而调节其生物活性。植物PII定位于质体；因此，为了鉴定新的PII靶蛋白，我们对拟南芥叶片叶绿体可溶性提取物进行了PII亲和层析。只有当Mg-ATP存在于结合介质中时，才会保留一些蛋白质，并且通过使用含有2-氧戊二酸酯的洗脱缓冲液特异性地从树脂中释放出来。SDS/ page分辨蛋白谱带的质谱鉴定出plastidial acetyl-CoA羧化酶（ACCase）的生物素羧基载体蛋白亚基和其他三个含有类似生物素/脂酰结合基的蛋白作为假定的PII靶点。ACCase是启动质体中脂肪酸合成的关键酶。在补充了外源ATP的体外重组实验中，重组拟南芥PII抑制了叶绿体ACCase的活性，而在2-氧戊二酸盐、丙酮酸盐或草酰乙酸盐的存在下，这种情况完全逆转。抑制作用是pii剂量依赖性的，并且似乎是pii特异性的，因为ACCase活性在其他测试蛋白存在下没有改变。PII降低了ACCase反应的V-max，但没有改变乙酰辅酶a的K-m。这些数据表明，PII功能已经在细菌和植物系统之间进化，以控制质体中脂肪酸合成的碳代谢途径。
The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotin/lipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the V-max of the ACCase reaction without altering the K-m for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids.