Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae
Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae
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DOI：
10.1128/mcb.18.5.2514
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发表时间：
1998-05-01
影响因子：
5.3
通讯作者：
Thiele, DJ
中科院分区：
文献类型：
--
作者：
Peña, MMO;Koch, KA;Thiele, DJ
铜的本质是有毒的，需要对铜稳态机制进行严格的调节，以确保细胞中有足够的铜来驱动基本的生化过程，同时防止积累到有毒水平。在酿酒酵母中，营养铜传感器Mac1p调节高亲和力Cu(I)摄取基因CTR1、CTR3和FRE1的铜依赖性表达，而有毒铜传感器Ace1p调节解毒基因CUP1、CRS5和SOD1对铜的转录激活。在这项研究中，我们描述了铜摄取和解毒途径的串联调节，以响应生长培养基中铜离子浓度的长期升高。添加CuSO4后，CTR3的mRNA水平迅速降低到原来基础水平的8倍，而ace1p介导的CUP1的转录激活是快速有效的，但是短暂的。由Ace1p DNA结合域-单纯疱疹病毒VP16转激活域融合驱动的CUP1表达也是短暂的，这表明这种调节模式是通过调节Ace1p铜激活的DNA结合域发生的。CUP1启动子的体内硫酸二甲酯足迹分析表明，Ace1p短暂占据金属响应元件，与CUP1 mRNA表达平行。Mac1p突变体对Cu(I)转运基因铜依赖性抑制的折射分析这些研究(i)证明了营养和有毒铜金属调节转录因子Mac1p和Ace1p必须以动态方式感知和响应铜离子，以适当调节铜离子的稳态；（ii）建立了野生型Mac1p在有毒铜水平存在下生存的需求。
The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.