Martin Torsten, Sharma Rita, Sippel Claudia, Waegemann Karin, Soll Jürgen, Vothknecht Ute C
Department Biology I, Botany, Ludwig-Maximilians-University Munich, Menzingerstrasse 67, D-80638 Munich, Germany.
J Biol Chem. 2006 Dec 29;281(52):40216-23. doi: 10.1074/jbc.M606580200. Epub 2006 Nov 7.
A serine/threonine protein kinase that is able to phosphorylate chloroplast-destined precursor proteins was purified from leaf extract of Arabidopsis thaliana and was identified by mass spectrometry. The protein kinase, encoded by AT2G17700, belongs to a small protein family comprising in addition AT4G35780 and AT4G38470. All three proteins were expressed heterologously in Escherichia coli and characterized with regard to their properties in precursor protein phosphorylation. They were able to phosphorylate several chloroplast-destined precursor proteins within their cleavable presequences. In contrast, a mitochondria-destined precursor protein was not a substrate for these kinases. For all three enzymes, the phosphorylation reaction was specific for ATP with apparent K(m) values between 14 and 67 microM. They did not utilize other NTPs nor were those able to compete for ATP in the reaction. An excess of ADP was able to inhibit ATP-dependent phosphorylation. Furthermore, all three kinases exhibited autophosphorylation. The protein kinases described here could represent subunits of a regulatory network involved in the cytosolic events of chloroplast protein import.
从拟南芥叶提取物中纯化出一种能够磷酸化叶绿体靶向前体蛋白的丝氨酸/苏氨酸蛋白激酶，并通过质谱鉴定。该蛋白激酶由AT2G17700编码，属于一个小蛋白家族，该家族还包括AT4G35780和AT4G38470。这三种蛋白均在大肠杆菌中异源表达，并对其在前体蛋白磷酸化方面的特性进行了表征。它们能够在可裂解的前导序列内磷酸化几种叶绿体靶向前体蛋白。相比之下，线粒体靶向前体蛋白不是这些激酶的底物。对于所有三种酶，磷酸化反应对ATP具有特异性，表观K(m)值在14至67微摩尔之间。它们不利用其他NTP，也不能在反应中竞争ATP。过量的ADP能够抑制ATP依赖性磷酸化。此外，所有三种激酶都表现出自身磷酸化。这里描述的蛋白激酶可能代表参与叶绿体蛋白输入胞质事件的调控网络的亚基。
