Drakulic Tamara, Temple Mark D, Guido Ron, Jarolim Stefanie, Breitenbach Michael, Attfield Paul V, Dawes Ian W
Ramaciotti Centre for Gene Function Analysis and School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, Sydney, Australia.
FEMS Yeast Res. 2005 Dec;5(12):1215-28. doi: 10.1016/j.femsyr.2005.06.001. Epub 2005 Jul 28.
Saccharomyces cerevisiae mutants lacking oxidative stress response genes were used to investigate which genes are required under normal aerobic conditions to maintain cellular redox homeostasis, using intracellular glutathione redox potential (glutathione E(h)) to indicate the redox environment of the cells. Levels of reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) were also assessed by FACS using dihydroethidium and rhodamine 123 as fluorescent probes. Cells became more oxidised as strains shifted from exponential growth to stationary phase. During both phases the presence of reduced thioredoxin and the activity of glutathione reductase were important for redox homeostasis. Thioredoxin reductase contributed less during exponential phase when there was a strong requirement for active Yap1p transcription factor, but was critical during stationary phase. The absence of ROS detoxification systems, such as catalases or superoxide dismutases, had a lesser effect on glutathione E(h), but a more pronounced effect on ROS levels and MMP. These results reflect the major shift in ROS generation as cells switch from fermentative to respiratory metabolism and also showed that there was not a strong correlation between ROS production, MMP and cellular redox environment. Heterogeneity was detected in populations of strains with compromised anti-oxidant defences, and as cells aged they shifted from one cell type with low ROS content to another with much higher intracellular ROS.
利用缺乏氧化应激反应基因的酿酒酵母突变体，以细胞内谷胱甘肽氧化还原电位（谷胱甘肽E（h））来指示细胞的氧化还原环境，研究在正常有氧条件下维持细胞氧化还原稳态需要哪些基因。还使用二氢乙锭和罗丹明123作为荧光探针，通过流式细胞术评估活性氧（ROS）水平和线粒体膜电位（MMP）。随着菌株从指数生长期转变为稳定期，细胞变得更加氧化。在这两个阶段中，还原型硫氧还蛋白的存在和谷胱甘肽还原酶的活性对氧化还原稳态都很重要。在对活性Yap1p转录因子有强烈需求的指数生长期，硫氧还蛋白还原酶的作用较小，但在稳定期至关重要。过氧化氢酶或超氧化物歧化酶等ROS解毒系统的缺失对谷胱甘肽E（h）的影响较小，但对ROS水平和MMP的影响更为明显。这些结果反映了细胞从发酵代谢转变为呼吸代谢时ROS产生的主要变化，也表明ROS产生、MMP与细胞氧化还原环境之间没有很强的相关性。在抗氧化防御受损的菌株群体中检测到异质性，并且随着细胞老化，它们从一种ROS含量低的细胞类型转变为另一种细胞内ROS含量高得多的细胞类型。
