PDF(Pubmed)
Abstract：
Translational elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803. However, the sensitivity to ROS of chloroplast-localized EF-Tu (cpEF-Tu) of plants remains to be elucidated. In the present study, we generated a recombinant cpEF-Tu protein of Arabidopsis thaliana and examined its sensitivity to ROS in vitro. In cpEF-Tu that lacked a bound nucleotide, one of the two cysteine residues, Cys149 and Cys451, in the mature protein was sensitive to oxidation by H2O2, with the resultant formation of sulfenic acid. The translational activity of cpEF-Tu, as determined with an in vitro translation system, derived from Escherichia coli, that had been reconstituted without EF-Tu, decreased with the oxidation of a cysteine residue. Replacement of Cys149 with an alanine residue rendered cpEF-Tu insensitive to inactivation by H2O2, indicating that Cys149 might be the target of oxidation. In contrast, cpEF-Tu that had bound either GDP or GTP was less sensitive to oxidation by H2O2 than nucleotide-free cpEF-Tu. The addition of thioredoxin f1, a major thioredoxin in the Arabidopsis chloroplast, to oxidized cpEF-Tu allowed the reduction of Cys149 and the reactivation of cpEF-Tu, suggesting that the oxidation of cpEF-Tu might be a reversible regulatory mechanism that suppresses the chloroplast translation system in a redox-dependent manner.
摘要：
平移延伸率EF-Tu,将氨基酰基tRNA传递到核糖体，容易被蓝细菌中的活性氧（ROS）灭活。PCC6803。然而,植物叶绿体定位的EF-Tu（cpEF-Tu）对ROS的敏感性尚待阐明。在本研究中,我们产生了拟南芥的重组cpEF-Tu蛋白，并在体外检查了其对ROS的敏感性。在缺乏结合核苷酸的cpEF-Tu中，两个半胱氨酸残基之一，Cys149和Cys451，在成熟卵白中对H2O2氧化敏感，并由此生成次磺酸。cpEF-Tu的翻译活性,根据体外翻译系统的确定，来自大肠杆菌，没有EF-Tu的重组，随着半胱氨酸残基的氧化而降低。用丙氨酸残基替换Cys149使cpEF-Tu对H2O2的失活不敏感，表明Cys149可能是氧化的目标。相比之下,与不含核苷酸的cpEF-Tu相比，已结合GDP或GTP的cpEF-Tu对H2O2的氧化敏感性较低。添加拟南芥叶绿体中主要的硫氧还蛋白f1，氧化的cpEF-Tu允许Cys149的还原和cpEF-Tu的重新激活，表明cpEF-Tu的氧化可能是一种可逆的调节机制，以氧化还原依赖的方式抑制叶绿体翻译系统。
