Yoshida Keisuke, Hara Satoshi, Hisabori Toru
From the Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075, Japan.
From the Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, and.
J Biol Chem. 2015 Jun 5;290(23):14278-88. doi: 10.1074/jbc.M115.647545. Epub 2015 Apr 15.
Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network.
基于硫氧还蛋白（Trx）系统的氧化还原调节被认为可确保叶绿体中各种功能的光响应控制。据报道，有五种Trx亚型存在于叶绿体中，但它们在Trx靶蛋白氧化还原调节中的功能多样性仍不清楚。为了直接解决这个问题，我们在体外和体内研究了几种叶绿体硫醇调节酶的Trx依赖性氧化还原变化。使用一系列拟南芥重组蛋白进行的体外测定为Trx对氧化还原调节的选择性以及先前建议的基础提供了新的见解。最值得注意的是，通过将硫醇状态的区分与质谱和活性测量相结合，我们确定了NADP-苹果酸脱氢酶还原激活的一个未被表征的方面；该酶中含有的两对氧化还原活性半胱氨酸即使在单个多肽内也通过不同的Trx利用而被还原。在我们的体外测定中，Trx-f有效地还原了此处分析的所有硫醇调节酶。然后，我们使用拟南芥野生型和Trx-f缺陷型植物研究了这些体外发现的体内生理相关性。在Trx-f缺陷型植物中，果糖-1,6-二磷酸酶的光还原部分受损，但Trx-f缺陷对硫醇调节酶氧化还原行为的总体影响并不像体外数据预期的那样显著。我们的结果为Trx系统的体内功能提供了支持，也突出了叶绿体氧化还原网络的复杂性和可塑性。
