Nitric oxide-mediated antioxidative mechanism in yeast through the activation of the transcription factor Mac1

Abstract

The budding yeast Saccharomyces cerevisiae possesses various defense mechanisms against environmental stresses that generate reactive oxygen species, leading to growth inhibition or cell death. Our recent study showed a novel antioxidative mechanism mediated by nitric oxide (NO) in yeast cells, but the mechanism underlying the oxidative stress tolerance remained unclear. We report here one of the downstream pathways of NO involved in stress-tolerance mechanism in yeast. Our microarray and real-time quantitative PCR analyses revealed that exogenous NO treatment induced the expression of genes responsible for copper metabolism under the control of the transcription factor Mac1, including the CTR1 gene encoding high-affinity copper transporter. Our ChIP analysis also demonstrated that exogenous NO enhances the binding of Mac1 to the promoter region of target genes. Interestingly, we found that NO produced under high-temperature stress conditions increased the transcription level of the CTR1 gene. Furthermore, NO produced during exposure to high temperature also increased intracellular copper content, the activity of Cu,Zn-superoxide dismutase Sod1, and cell viability after exposure to high-temperature in a manner dependent on Mac1. NO did not affect the expression of the MAC1 gene, indicating that NO activates Mac1 through its post-translational modification. Based on the results shown here, we propose a novel NO-mediated antioxidative mechanism that Mac1 activated by NO induces the CTR1 gene, leading to an increase in cellular copper level, and then Cu(I) activates Sod1. This is the first report to unveil the mechanism of NO-dependent antioxidative system in yeast.

Figure 1. RT qPCR analysis of yeast cells treated with the exogenous NO.

S. cerevisiae BY4741 or BY4741Δmac1 cells treated or untreated with SNAP were subjected to RT qPCR analysis. The mRNA level of each gene was normalized to that of ACT1 and the relative induction fold compared to untreated cells was shown. The values are the means and standard deviations of three independent experiments. *p<0.05 by Student's t test.

Figure 2. ChIP analysis of yeast cells treated with the exogenous NO.

S. cerivisiae BY4741 and BY4741MAC1-TAP cells grown in SD medium with/without NO treatment or in LCM medium, which is the same as SD medium without supplementation of copper, were subjected to ChIP analysis. (A) Electrophoresis of amplified DNA corresponding to the promoter region of each gene. The CMD1 gene, which is not under the control of Mac1, was used as a negative control. (B) Quantification of DNA binding to Mac1 by densitometry. The signal from immunoprecipitated DNA was divided by the signal from the corresponding input DNA. The resultant values were normalized as the value in SD medium is 100%.

Figure 3. RT qPCR under high-temperature stress conditions.

S. cerevisiae L5685 or L5685Δmac1 cells were incubated at 39°C in the presence (100 mM) or absence of NAME followed by the process for RT qPCR analysis. The mRNA level of each gene was normalized to that of ALG9 and the relative induction fold compared to cells incubated at 25°C was shown. The values are the means and standard deviations of four independent experiments. ***p<0.001; **p<0.01; *p<0.05 by Student's t test.

Figure 4. Cell viability under high-temperature stress conditions.

Viability of S. cerevisiae L5685 or L5685Δmac1 cells was measured after exposure to high temperature in the presence (100 mM) or absence of NAME. Cell viability was expressed as percentages, calculated as follows: (no. of colonies after exposure to high temperature stress (39°C))/(no. of colonies after exposure to non-stress condition (25°C))×100. The values are the means and standard deviations of at least two independent experiments.

Figure 5. Intracellular copper content.

The intracellular copper content in S. cerevisiae L5685 or L5685Δmac1 cells was measured before or after exposure to 39°C in the absence (A) or presence (B) of 100 mM NAME. The values are the means and standard deviations of three independent experiments. *p<0.05 by Student's t test.

Figure 6. The activity of Cu,Zn-superoxide disumutase Sod1.

Sod1 activity in S. cerevisiae L5685 or L5685Δmac1 cells was measured before or after exposure to 39°C in the absence (A) or presence (B) of 100 mM NAME. The values are the means and standard deviations of three independent experiments. *p<0.05 by Student's t test.

Figure 7. A proposed model for the antioxidative mechanism through Mac1 activated by NO.

When yeast cells are exposed to high-temperature stress, NO is produced from arginine by NOS-like activity. NO-mediated Mac1 activation through its post-translational modification upregulates the expression of CTR1, which encodes a copper transporter. Protein name: Fre1, cupric reductase; Ctr1, copper ion transporter; Sod1, Cu,Zn-superoxide dismutase; Ccs1, copper chaperone; Mac1, transcription factor responsible for copper metabolism.