Intracellular dynamism of transcription factors and signal cross-talk in the stress response in yeast

A)硫氧还蛋白在酿酒酵母YAP1转录因子调控中的作用YAP1是酿酒酵母中一种类似AP-1、b-Zip的转录因子，调节多种抗氧化酶基因的表达。当酵母细胞暴露在氧化应激下时，YAP1集中在细胞核中。我们发现，如果硫氧还蛋白(一种硫醇-二硫化物氧化还原酶)被破坏，YAP1就会结构性地集中在细胞核中。如果硫氧还蛋白被破坏(trx1/trx2)，细胞内谷胱甘肽含量会增加，然而，即使细胞内谷胱甘肽水平很高，这种突变体也表现出对氧化应激的敏感性增加。这可能是由于缺乏硫氧还蛋白。b)谷胱甘肽在酵母热休克应激反应中的作用。我们发现，如果酵母细胞被转移到更高的温度，氧气呼吸速率就会增加，从而提高细胞内的氧化水平。此外，我们发现Expr…热休克增加了谷胱甘肽合成酶GSH1和GSH2的表达，细胞内谷胱甘肽含量也增加。在gsh1突变体中，热休克处理导致呼吸缺陷率很高。C)酿酒酵母谷胱甘肽过氧化物酶的遗传分析。在酿酒酵母基因组序列数据库中发现了三种谷胱甘肽过氧化物酶(GPx)的同源物。我们破坏了每个同源基因，并分析了每个基因产物的功能。发现gpx3基因是结构性表达的，并且gpx3突变体对氧化应激敏感。相反，氧化应激以依赖YAP1的方式诱导GPX2基因的表达。根据对硫氧还蛋白过氧化物酶突变体(TSAL)的表型分析，GPX2基因产物可能作为硫氧还蛋白过氧化物酶的备份系统。较少

a)Role of thioredoxin in regulation of Yap1 transcription factor in Saccharomyces cerevisiae.Yap1 is an AP-1-like, b-Zip transcription factor in S. cerevisiae, and it regulates the expression of many genes encoding antioxidant enzymes. Yap1 is concentrated in the nucleus when the yeast cells are exposed to oxidative stress. We found that Yap1 is constitutively concentrated in the nucleus if thioredoxin, a thiol-disulfide oxidoreductase, was disrupted. Intracellular glutathione contents are increased if thioredoxin was destroyed (trx1△/trx2△), however, such a mutant showed increased sensitivity to oxidative stress even though intracellular glutathione levels are high. This was presumably due to the lack of thioredoxin.b)Role of glutathione in heat shock stress response in yeast.We found that oxygen respiration rate is increased if the yeast S. cerevisiae cells are shifted to higher temperature, and consequently intracellular oxidation level was increased. Additionally we found that expr … More ession of GSH1 and GSH2, both encoding glutathione-synthesizing enzymes, is increased by heat shock, and intracellular glutathione contents are also increased. In a gsh1 mutant, heat shock treatment induced the respiration deficiency at high rate. These results suggest that the increased production of glutathione under heat shock protects mitochondrial DNA from oxidative damage.c)Genetic analysis of glutathione peroxidase in S. cerevisiae.Three glutathione peroxidase (GPx) homologs were found in the yeast S.cerevisiae genome in the genome sequence database of this microorganism. We disrupted each of these homolog genes, and analyzed the function of each gene product. The GPX3 gene was found to be constitutively expressed, and the gpx3△ mutant was hypersensitive to oxidative stress. On the contrary, expression of the GPX2 gene was induced by oxidative stress in a Yap1-dependent fashion. Based on the analysis of the phenotype of a thioredoxin peroxidase mutant (tsal△), the GPX2 gene product was likely to function as a backup system for the thioredoxin peroxidase. Less