Aroca Angeles, Schneider Markus, Scheibe Renate, Gotor Cecilia, Romero Luis C
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, Sevilla, Spain.
Department of Plant Physiology, Osnabrück University, Osnabrück, Germany.
Plant Cell Physiol. 2017 Jun 1;58(6):983-992. doi: 10.1093/pcp/pcx056.
Hydrogen sulfide is an important signaling molecule comparable with nitric oxide and hydrogen peroxide in plants. The underlying mechanism of its action is unknown, although it has been proposed to be S-sulfhydration. This post-translational modification converts the thiol groups of cysteines within proteins to persulfides, resulting in functional changes of the proteins. In Arabidopsis thaliana, S-sulfhydrated proteins have been identified, including the cytosolic isoforms of glyceraldehyde-3-phosphate dehydrogenase GapC1 and GapC2. In this work, we studied the regulation of sulfide on the subcellular localization of these proteins using two different approaches. We generated GapC1-green fluorescent protein (GFP) and GapC2-GFP transgenic plants in both the wild type and the des1 mutant defective in the l-cysteine desulfhydrase DES1, responsible for the generation of sulfide in the cytosol. The GFP signal was detected in the cytoplasm and the nucleus of epidermal cells, although with reduced nuclear localization in des1 compared with the wild type, and exogenous sulfide treatment resulted in similar signals in nuclei in both backgrounds. The second approach consisted of the immunoblot analysis of the GapC endogenous proteins in enriched nuclear and cytosolic protein extracts, and similar results were obtained. A significant reduction in the total amount of GapC in des1 in comparison with the wild type was determined and exogenous sulfide significantly increased the protein levels in the nuclei in both plants, with a stronger response in the wild type. Moreover, the presence of an S-sulfhydrated cysteine residue on GapC1 was demonstrated by mass spectrometry. We conclude that sulfide enhances the nuclear localization of glyceraldehyde-3-phosphate dehydrogenase.
硫化氢是植物中一种重要的信号分子，可与一氧化氮和过氧化氢相媲美。尽管有人提出其作用机制是S-硫巯基化，但具体机制尚不清楚。这种翻译后修饰将蛋白质中半胱氨酸的巯基转化为过硫化物，导致蛋白质功能发生变化。在拟南芥中，已鉴定出S-硫巯基化蛋白，包括甘油醛-3-磷酸脱氢酶GapC1和GapC2的胞质同工型。在这项工作中，我们使用两种不同的方法研究了硫化物对这些蛋白质亚细胞定位的调节。我们在野生型和l-半胱氨酸脱硫酶DES1缺陷的des1突变体中都构建了GapC1-绿色荧光蛋白（GFP）和GapC2-GFP转基因植物，DES1负责在细胞质中产生硫化物。在表皮细胞的细胞质和细胞核中检测到了GFP信号，尽管与野生型相比，des1中的核定位有所减少，并且外源性硫化物处理在两种背景下的细胞核中都产生了类似的信号。第二种方法包括对富集的细胞核和细胞质蛋白提取物中GapC内源蛋白的免疫印迹分析，并获得了类似的结果。与野生型相比，des1中GapC的总量显著降低，外源性硫化物显著增加了两种植物细胞核中的蛋白水平，野生型的反应更强。此外，通过质谱法证实了GapC1上存在一个S-硫巯基化的半胱氨酸残基。我们得出结论，硫化物增强了甘油醛-3-磷酸脱氢酶的核定位。
