Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan.
Plant Cell Physiol. 2013 Jun;54(6):875-92. doi: 10.1093/pcp/pct037. Epub 2013 Feb 26.
The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system.
硫氧还蛋白（Trx）系统已知在细胞氧化还原调节中发挥关键作用，但植物线粒体中的其靶蛋白在很大程度上仍未被阐明。在这项研究中，我们系统地筛选了植物线粒体中的 Trx 靶候选物。从拟南芥芽、菠菜叶和土豆块茎中制备线粒体蛋白提取物，然后将其亚组分分为可溶性基质和不溶性膜部分。将蛋白提取物加载到固定有拟南芥线粒体定位 o 型 Trx 突变蛋白的亲和柱上，其中活性部位的两个内部半胱氨酸之一被丝氨酸取代。通过蛋白质组学方法鉴定与突变 Trx 形成混合二硫键中间体的蛋白质。该程序确定了 101 种参与广泛线粒体过程的 Trx 靶候选蛋白。此外，生化测定表明，潜在的 Trx 靶蛋白之一，交替氧化酶，实际上受 Trx 调控。这项研究为通过 Trx 系统介导的线粒体生物学中多种功能的调节机制提供了深入了解。
