Ito Jun, Taylor Nicolas L, Castleden Ian, Weckwerth Wolfram, Millar A Harvey, Heazlewood Joshua L
Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Western Australia, Australia.
Proteomics. 2009 Sep;9(17):4229-40. doi: 10.1002/pmic.200900064.
Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2-DE separations of Arabidopsis mitochondrial proteins and affinity-enriched phosphoproteins using the Pro-Q Diamond phospho-specific in-gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.
植物线粒体在细胞能量产生、新陈代谢和应激反应中发挥着核心作用。最近在哺乳动物和酵母线粒体中进行的磷酸化蛋白质组学研究表明，蛋白质磷酸化可能是广泛的重要线粒体过程中的一种调节机制。本研究使用亲和富集和蛋白质组学工具，对模式植物拟南芥细胞悬浮液中纯化的线粒体中的蛋白质磷酸化进行了研究。在拟南芥线粒体蛋白质和使用Pro-Q Diamond磷酸特异性凝胶内染料亲和富集的磷酸化蛋白质的二维电泳分离中，检测到18种推定的磷酸化蛋白质，包括线粒体代谢酶、热休克蛋白、一种蛋白酶和几种功能未知的蛋白质。与酵母和小鼠的线粒体磷酸化蛋白质组比较表明，这三个物种几乎没有经过验证的共同磷酸化蛋白质。通过二氧化钛富集和串联质谱对18种线粒体蛋白质中的7种的磷酸化位点进行了表征。在此过程中，还鉴定出了71种来自拟南芥蛋白质的磷酸肽，这些蛋白质不存在于线粒体中，但在各种类型的线粒体制剂中作为污染物被发现，这表明与拟南芥中的其他细胞成分相比，线粒体成分的磷酸化水平较低。从这项研究中获得的信息有助于更好地理解拟南芥在亚细胞和细胞水平上的蛋白质磷酸化。
