Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan.
FEMS Yeast Res. 2019 Aug 1;19(5). doi: 10.1093/femsyr/foz052.
The transcriptional factor Msn2 plays a pivotal role in response to environmental stresses by activating the transcription of stress-responsive genes in Saccharomyces cerevisiae. Our previous studies demonstrate that intracellular proline acts as a key protectant against various stresses. It is unknown, however, whether Msn2 is involved in proline homeostasis in S. cerevisiae cells. We here found that MSN2-overexpressing (MSN2-OE) cells showed higher sensitivity to a toxic analogue of proline, l-azetidine-2-carboxylic acid (AZC), as well as to the other amino acid toxic analogues, than wild-type cells. Overexpression of MSN2 increased the intracellular content of AZC, suggesting that Msn2 positively regulates the uptake of proline. Among the known proline permease genes, GNP1 was shown to play a predominant role in the AZC toxicity. Based on quantitative real-time PCR and western blot analyses, the overexpression of MSN2 did not induce any increases in the transcript levels of GNP1 or the other proline permease genes, while the amount of the Gnp1 protein was markedly increased in MSN2-OE cells. Microscopic observation suggested that the endocytic degradation of Gnp1 was impaired in MSN2-OE cells. Thus, this study sheds light on a novel link between the Msn2-mediated global stress response and the amino acid homeostasis in S. cerevisiae.
转录因子 Msn2 通过激活酿酒酵母中应激反应基因的转录，在应对环境应激中发挥关键作用。我们之前的研究表明，细胞内脯氨酸作为一种关键的保护剂，对抗各种应激。然而，目前尚不清楚 Msn2 是否参与酿酒酵母细胞中脯氨酸的动态平衡。我们在这里发现，过表达 Msn2（MSN2-OE）的细胞对脯氨酸的有毒类似物 l-azetidine-2-羧酸（AZC）以及其他氨基酸有毒类似物的敏感性高于野生型细胞。MSN2 的过表达增加了 AZC 的细胞内含量，表明 Msn2 正向调节脯氨酸的摄取。在已知的脯氨酸通透酶基因中，GNP1 被证明在 AZC 毒性中起主要作用。基于定量实时 PCR 和 Western blot 分析，MSN2 的过表达并没有诱导 GNP1 或其他脯氨酸通透酶基因的转录水平增加，而 Gnp1 蛋白的量在 MSN2-OE 细胞中明显增加。显微镜观察表明，MSN2-OE 细胞中 Gnp1 的内吞降解受损。因此，这项研究揭示了 Msn2 介导的全局应激反应与酿酒酵母中氨基酸动态平衡之间的新联系。
