Peltier J B, Ytterberg J, Liberles D A, Roepstorff P, van Wijk K J
Department of Biochemistry, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden.
J Biol Chem. 2001 May 11;276(19):16318-27. doi: 10.1074/jbc.M010503200. Epub 2001 Jan 26.
A 350-kDa ClpP protease complex with 10 different subunits was identified in chloroplast of Arabidopsis thaliana, using Blue-Native gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight and nano-electrospray tandem mass spectrometry. The complex was copurified with the thylakoid membranes, and all identified Clp subunits show chloroplast targeting signals, supporting that this complex is indeed localized in the chloroplast. The complex contains chloroplast-encoded pClpP and six nuclear-encoded proteins nCpP1-6, as well as two unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein was identified in this complex; it does not belong to any of the known Clp genes families and is here assigned ClpS1. Expression and accumulation of several of these Clp proteins have never been shown earlier. Sequence and phylogenetic tree analysis suggests that nClpP5, nClpP2, and nClpP8 are not catalytically active and form a new group of Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1 is a regulatory subunit positioned at the axial opening of the ClpP/R core. Several truncations and errors in intron and exon prediction of the annotated Clp genes were corrected using mass spectrometry data and by matching genomic sequences with cDNA sequences. This strategy will be widely applicable for the much needed verification of protein prediction from genomic sequence. The extreme complexity of the chloroplast Clp complex is discussed.
利用蓝色非变性凝胶电泳，随后进行基质辅助激光解吸电离飞行时间质谱和纳米电喷雾串联质谱，在拟南芥叶绿体中鉴定出了一种具有10种不同亚基的350 kDa ClpP蛋白酶复合体。该复合体与类囊体膜共纯化，所有鉴定出的Clp亚基均显示出叶绿体靶向信号，这支持该复合体确实定位于叶绿体中。该复合体包含叶绿体编码的pClpP和6种核编码蛋白nCpP1 - 6，以及两种未确定的Clp同源物（nClpP7、nClpP8）。在该复合体中还鉴定出一种额外的Clp蛋白；它不属于任何已知的Clp基因家族，在此被命名为ClpS1。此前从未有过这些Clp蛋白中几种的表达和积累情况的报道。序列和系统发育树分析表明，nClpP5、nClpP2和nClpP8没有催化活性，形成了一组新的高等植物Clp蛋白，与蓝藻ClpR蛋白直系同源，分别重新命名为ClpR1、-2和-3。我们推测ClpR1、-2和-3是七聚体环的一部分，而ClpS1是位于ClpP/R核心轴向开口处的调节亚基。利用质谱数据并通过将基因组序列与cDNA序列匹配，校正了注释的Clp基因在内含子和外显子预测中的几个截断和错误。该策略将广泛适用于对基因组序列蛋白质预测进行急需的验证。文中还讨论了叶绿体Clp复合体的极端复杂性。
