Systematic exploration of thioredoxin target proteins in plant mitochondria.
Systematic exploration of thioredoxin target proteins in plant mitochondria.
复制标题
DOI：
10.1093/pcp/pct037
复制
发表时间：
2013-06
影响因子：
4.9
通讯作者：
Keisuke Yoshida;K. Noguchi;K. Motohashi;T. Hisabori
中科院分区：
文献类型：
--
作者：
Keisuke Yoshida;K. Noguchi;K. Motohashi;T. Hisabori
众所周知，硫氧还蛋白（Trx）系统在细胞氧化还原调节中发挥着关键作用，但其在植物线粒体中的靶蛋白在很大程度上仍然未知。在这项研究中，我们系统地筛选了植物线粒体中的 Trx 候选靶点。从拟南芥芽、菠菜叶和马铃薯块茎中制备线粒体蛋白提取物，然后细分为可溶性基质和不溶性膜部分。将蛋白质提取物加载到固定拟南芥线粒体定位的 o 型 Trx 突变蛋白的亲和柱上，其中活性位点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了与突变的 Trx 形成混合二硫键中间体的蛋白质。该程序可以确定参与广泛线粒体过程的 101 种 Trx 目标候选蛋白。此外，生化检测表明，潜在的 Trx 靶蛋白之一，替代氧化酶，实际上是由 Trx 调节氧化还原。这项研究提供了对线粒体生物学中通过 Trx 系统介导的多种功能的调节机制的见解。
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.