# MET18 Deficiency Increases the Sensitivity of Yeast to Oxidative Stress and Shortens Replicative Lifespan by Inhibiting Catalase Activity

Authors: Ya-qin Chen; Xin-guang Liu; Wei Zhao; Hongjing Cui; Jie Ruan; Yuan Yuan; Zhiguang Tu
Journal: BioMed Research International
 (2017)

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## Abstract

YeastMET18, a subunit of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery which is responsible for the maturation of Fe/S proteins, has been reported to participate in the oxidative stress response. However, the underlying molecular mechanisms remain unclear. In this study, we constructed aMET18/met18Δ heterozygous mutant yeast strain and found thatMET18deficiency in yeast cells impaired oxidative stress resistance as evidenced by increased sensitivity to hydrogen peroxide (H2O2) and cumene hydroperoxide (CHP). Mechanistically, the mRNA levels of catalase A(CTA1) and catalase T(CTT1) as well as the total catalase activity were significantly reduced inMET18-deficient cells. In contrast, overexpression ofCTT1 orCTA1inMET18-deficient cells significantly increased the intracellular catalase activity and enhanced the resistance ability against H2O2 and CHP. In addition,MET18 deficiency diminished the replicative capacity of yeast cells as evidenced by the shortened replicative lifespan, which can be restored byCTT1 overexpression, but not byCTA1, in theMET18-deficient cells. These results suggest thatMET18, in a catalase-dependent manner, plays an essential role in enhancing the resistance of yeast cells to oxidative stress and increasing the replicative capacity of yeast cells.


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## Body

## 1. Introduction

Oxygen (O2) is crucial for the livelihood of organisms grown aerobically. However, exposure to higher O2 concentration can increase the production of reactive oxygen species (ROS), including hydroxyl radical (OH−), hydrogen peroxide (H2O2), and superoxide anions (O2−), which may exert detrimental effects on cell growth. ROS can also be generated by redox-cycling chemicals and heavy metals in the environment, as well as by the endogenous metabolic process. In addition, the effects of environmental oxygen concentrations on the intracellular ROS production are highly dependent upon the culture medium, the cells themselves, the metabolic state of the cells, and the presence of any antioxidative compound or enzyme and other known and unknown factors [1–3]. It has been shown that culturing cells in air (21% O2) exposes the cells to much higher levels of oxygen than what they are usually submitted to under physiological conditions (2~5% O2) [2]. Cells normally have oxidant defense systems to protect themselves against ROS or oxidative stress. However, the underlying mechanisms remain largely unknown [3, 4].YeastMET18, the homologue of methyl-methanesulfonate sensitivity protein 19(MMS19) in human, is a conserved component of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery in eukaryotes [5–8]. From yeasts to plant cells to human cells, the CIA machinery is generally located in the cytoplasm and responsible for the maturation of Fe/S proteins through catalyzing the synthesis of Fe/S clusters [9, 10]. In the CIA pathway,MET18 and other subunits, such asCIA1,CIA2, and nuclear architecture-related protein 1(NAR1), form a CIA complex in whichMET18 functions as a delivering agent by directly interacting with the target protein and transferring the Fe/S cluster to the protein, leading to the maturation of Fe/S proteins [11, 12].MET18could form complexes with Dos2, Rki1, and Cdc20 in order to participate in various cell processes such as DNA repair, RNA polymerase transcription, chromosome segregation, and maintenance of telomere length, among others [13]. Therefore,MET18 has a number of roles in cell metabolism.Previous studies have suggested that some components of CIA machinery,CIA2 andNAR1, are associated with yeast response to oxidative stress and thus regulate the replicative lifespan (RLS) of yeast. InCIA2/cia2Δ mutants, the deficiency ofCIA2 enhances the sensitivity of yeast to the oxidant cumene hydroperoxide (CHP), resulting in an impaired oxidative stress tolerance and thus a decreased RLS [14, 15]. Similarly,NAR1-deficient yeast cells have been found to be more sensitive to O2−-generating compound paraquat (PQ) and have shorter RLS than wild-type (WT) yeast strain [16]. Any deficiency in the CIA pathway will impact the activities of enzymes involved in ROS and oxidative stress management [13].SinceMET18 is also a component of CIA, we hypothesized thatMET18 may play an important role in yeast response to oxidative stress. To confirm this hypothesis, we performed the functional assay forMET18 mutation by constructing aMET18/met18Δheterozygous mutant yeast strain. Our results demonstrate for the first time thatMET18 deficiency may increase the response sensitivity of yeast to oxidative stress and shorten the RLS through suppressing the expression and activity of catalases.


## 2. Materials and Methods

### 2.1. Yeast Strains and Culture

The yeast strains used in this study are listed in Table1. All strains were isogenic to BY4743 and were stored in liquid yeast peptone dextrose (YPD; 1% yeast extract, 2% peptone, and 2% glucose; Oxoid, Basingstoke, UK) medium mixed with equal volume of 50% (v/v) glycerol at −80°C. For all experiments, cells were removed from frozen stock and streaked onto solid YPD plate followed by an incubation for 2~3 days at 30°C. Then the single colonies were picked and grown in liquid YPD until the exponential phase using an orbital shaker where yeast cells were shaken at 30°C and 180 rpm.Table 1
Yeast strains in this study.
Strain name
Genotype
Comments
Source
BY4743
MATa/MATαhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2/lys2Δ0 met15Δ0/met15 ura3Δ0/ura3Δ0
Wild-type strain
Gift from Matt Kaeberlein
GDMUB1001
BY4743MET18::URA3
Deletion of one copy ofMET18 in BY4743
This experiment
GDMUB1002
BY4743 + pAUR123
BY4743 harboring pAUR123
This experiment
GDMUB1003
BY4743MET18::URA3 +pAUR123
GDMUB1001 harboring pAUR123
This experiment
GDMUB1004
BY4743MET18::URA3 +pAUR123-CTA1
GDMUB1001 harboring pAUR123-CTA1
This experiment
GDMUB1005
BY4743MET18::URA3 +pAUR123-CTT1
GDMUB1001 harboring pAUR123-CTT1
This experiment


### 2.2.MET18/met18Δ Mutants and Plasmids Construction

TheMET18/met18Δ mutants were constructed as previously described [14]. Briefly,URA3 cassette was amplified by polymerase chain reaction (PCR) from pRS306 vector using the following primers: 5′-TGT TTT AAC TGG GAA AAA GCG GAA CAA TTG GGC CTT ACA AGA TTG TAC TGA GAG TGC AC-3′ (forward) and 5′-CGT GCT CAT CAA TGT GAA CAA ATT ATT AAA TAC AAG CGT CTG TGC GGT ATT TCA CAC CG-3′ (reverse) (Invitrogen, Shanghai, China). Then the PCR products were transformed into BY4743 to replace one copy ofMET18 by homologous recombination, and the transformants were selected on SD-URA medium (Clontech, Mountain View, CA, USA). The heterozygousMET18/met18Δ cells were verified by PCR using the following primers: 5′-TGT GGC TGT CGT TTC GTG G-3′ (forward) and 5′-TAC AGT TTC CAC TGC GAA CAC A-3′ (reverse) (Invitrogen, Shanghai, China).The entire coding regions of catalase A(CTA1) and catalase T(CTT1) were amplified from genomic DNA of BY4743 using primers flanked bySacI andXbaI restriction sites (CTT1 primers: 5′-TAT GTC GAC ATG AAC GTG TTC GGT AAA AAA G-3′ (forward) and 5′-GCG TCT AGA TTA ATT GGC ACT TGC AAT GG-3′ (reverse);CTA1 primers: 5′-ATA GTC GAC ATG TCG AAA TTG GGA CAA GA-3′ (forward) and 5′-CGC TCT AGA TCA AAA TTT GGA GTT ACT CG-3′ (reverse)) followed by digestion withSacI andXbaI (Takara, Otsu, Shiga, Japan) and ligation into pAUR123 vector (Takara, Otsu, Shiga, Japan) to generate pAUR123-CTT1 and pAUR123-CTA1expressingCTT1 andCTA1, respectively. Subsequently, pAUR123-CTT1 or pAUR123-CTA1 was transformed intoMET18/met18Δmutants. The empty pAUR123 vector was used as a negative control. The transformants were selected on YPD or SD-URA media containing aureobasidin A (Sigma-Aldrich, St. Louis, MO, USA) and then verified by PCR using the following primers: 5′-TCT GCA CAA TAT TTC AAG C-3′ (forward) and 5′-TTC GTT TTA AAA CCT AAG AGT CAC-3′ (reverse).


### 2.3. Real-Time PCR

Total RNA was isolated from yeast cells using a Yeast RNAiso Kit (Takara, Otsu, Shiga, Japan) followed by cDNA synthesis using a Transcriptor First-Strand cDNA Synthesis Kit with gDNA Eraser (Takara, Otsu, Shiga, Japan). Real-time PCR was performed using SYBR® Premix Ex Taq™ (Takara, Otsu, Shiga, Japan) and the primers are shown in Table2. Data were normalized to the internal controlPRP8[14].Table 2
The real-time PCR primers used in this study.
Gene
Primers
Sequence
PRP8
Forward
TCATGGCTGCGTCTGAAGTA
Reverse
GGCTCAAACCCTTCCGATAG
MET18
Forward
TGCTGGAAGTTGTCGTTGC
Reverse
TCGTTTTTGGAGAGGTGGTC
CTT1
Forward
GATTCCGTTCTACAAGCCAGAC
Reverse
GGAGTATGGACATCCCAAGTTTC
CTA1
Forward
CCAACAGGACAGACCCATTC
Reverse
TTACCCAAAACGCGGTAGAG


### 2.4. Spot Assay

The sensitivity of yeast to oxidant stressors, including H2O2, CHP, and PQ (Sigma-Aldrich, St. Louis, MO, USA), was determined by spot assay. Yeast cells were cultured to exponential phase (OD600 = 2.0) in liquid YPD followed by a 10-fold dilution with sterile water. Four additional 10-fold serial dilutions were performed, and 5 μL of each dilution was inoculated onto solid YPD plates supplemented with indicated stressors. The plates were incubated at 30°C for 2~5 days for colony formation.


### 2.5. Enzyme Activity Assay and Determination of Intracellular H2O2 Level

Yeast cells in exponential phase were harvested and washed twice with cold sterile water followed by resuspension in lysis buffer containing acid-washed glass beads and 20 cycles of 10 s vortexing plus 20 s cooling [14]. After centrifugation at 12000 rpm for 15 min, the supernatants were collected and used for an enzyme activity assay and determination of intracellular H2O2 levels. The protein concentration was determined using the BCA protein Assay Kit (Sangon Biotech, Shanghai, China) following the manufacturer’s instruction. The total superoxide dismutase (SOD) activity and the catalase activity were determined using a Total Cellular SOD Activity Kit (Dojindo Molecular Technologies, Rockville, MD, USA) and a Catalase Assay Kit (Beyotime Biotechnology, Shanghai, China), respectively. The intracellular H2O2 level was determined using an H2O2 Assay Kit (Beyotime Biotechnology, Shanghai, China).


### 2.6. Replicative Lifespan (RLS) Assay

RLS assay was performed as previously described [15]. Briefly, cells were thawed from frozen stocks and grown on freshly made YPD plate for 2 days and then patched onto the second YPD plate and incubated for an additional 24 h. Cells were then restruck on the third YPD plate and incubated for about 12 h, and then the virgin buds were isolated and used for RLS analysis.


### 2.7. Statistical Analysis

All experiments were repeated at least three times. Data were expressed as the mean ± standard deviation (SD). Statistical significance was assessed by calculatingp values using a two-tailed Student’s t-test or Wilcoxon rank-sum test. A value of p<0.05 was considered statistically significant.


## 2.1. Yeast Strains and Culture

The yeast strains used in this study are listed in Table1. All strains were isogenic to BY4743 and were stored in liquid yeast peptone dextrose (YPD; 1% yeast extract, 2% peptone, and 2% glucose; Oxoid, Basingstoke, UK) medium mixed with equal volume of 50% (v/v) glycerol at −80°C. For all experiments, cells were removed from frozen stock and streaked onto solid YPD plate followed by an incubation for 2~3 days at 30°C. Then the single colonies were picked and grown in liquid YPD until the exponential phase using an orbital shaker where yeast cells were shaken at 30°C and 180 rpm.Table 1
Yeast strains in this study.
Strain name
Genotype
Comments
Source
BY4743
MATa/MATαhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2/lys2Δ0 met15Δ0/met15 ura3Δ0/ura3Δ0
Wild-type strain
Gift from Matt Kaeberlein
GDMUB1001
BY4743MET18::URA3
Deletion of one copy ofMET18 in BY4743
This experiment
GDMUB1002
BY4743 + pAUR123
BY4743 harboring pAUR123
This experiment
GDMUB1003
BY4743MET18::URA3 +pAUR123
GDMUB1001 harboring pAUR123
This experiment
GDMUB1004
BY4743MET18::URA3 +pAUR123-CTA1
GDMUB1001 harboring pAUR123-CTA1
This experiment
GDMUB1005
BY4743MET18::URA3 +pAUR123-CTT1
GDMUB1001 harboring pAUR123-CTT1
This experiment


## 2.2.MET18/met18Δ Mutants and Plasmids Construction

TheMET18/met18Δ mutants were constructed as previously described [14]. Briefly,URA3 cassette was amplified by polymerase chain reaction (PCR) from pRS306 vector using the following primers: 5′-TGT TTT AAC TGG GAA AAA GCG GAA CAA TTG GGC CTT ACA AGA TTG TAC TGA GAG TGC AC-3′ (forward) and 5′-CGT GCT CAT CAA TGT GAA CAA ATT ATT AAA TAC AAG CGT CTG TGC GGT ATT TCA CAC CG-3′ (reverse) (Invitrogen, Shanghai, China). Then the PCR products were transformed into BY4743 to replace one copy ofMET18 by homologous recombination, and the transformants were selected on SD-URA medium (Clontech, Mountain View, CA, USA). The heterozygousMET18/met18Δ cells were verified by PCR using the following primers: 5′-TGT GGC TGT CGT TTC GTG G-3′ (forward) and 5′-TAC AGT TTC CAC TGC GAA CAC A-3′ (reverse) (Invitrogen, Shanghai, China).The entire coding regions of catalase A(CTA1) and catalase T(CTT1) were amplified from genomic DNA of BY4743 using primers flanked bySacI andXbaI restriction sites (CTT1 primers: 5′-TAT GTC GAC ATG AAC GTG TTC GGT AAA AAA G-3′ (forward) and 5′-GCG TCT AGA TTA ATT GGC ACT TGC AAT GG-3′ (reverse);CTA1 primers: 5′-ATA GTC GAC ATG TCG AAA TTG GGA CAA GA-3′ (forward) and 5′-CGC TCT AGA TCA AAA TTT GGA GTT ACT CG-3′ (reverse)) followed by digestion withSacI andXbaI (Takara, Otsu, Shiga, Japan) and ligation into pAUR123 vector (Takara, Otsu, Shiga, Japan) to generate pAUR123-CTT1 and pAUR123-CTA1expressingCTT1 andCTA1, respectively. Subsequently, pAUR123-CTT1 or pAUR123-CTA1 was transformed intoMET18/met18Δmutants. The empty pAUR123 vector was used as a negative control. The transformants were selected on YPD or SD-URA media containing aureobasidin A (Sigma-Aldrich, St. Louis, MO, USA) and then verified by PCR using the following primers: 5′-TCT GCA CAA TAT TTC AAG C-3′ (forward) and 5′-TTC GTT TTA AAA CCT AAG AGT CAC-3′ (reverse).


## 2.3. Real-Time PCR

Total RNA was isolated from yeast cells using a Yeast RNAiso Kit (Takara, Otsu, Shiga, Japan) followed by cDNA synthesis using a Transcriptor First-Strand cDNA Synthesis Kit with gDNA Eraser (Takara, Otsu, Shiga, Japan). Real-time PCR was performed using SYBR® Premix Ex Taq™ (Takara, Otsu, Shiga, Japan) and the primers are shown in Table2. Data were normalized to the internal controlPRP8[14].Table 2
The real-time PCR primers used in this study.
Gene
Primers
Sequence
PRP8
Forward
TCATGGCTGCGTCTGAAGTA
Reverse
GGCTCAAACCCTTCCGATAG
MET18
Forward
TGCTGGAAGTTGTCGTTGC
Reverse
TCGTTTTTGGAGAGGTGGTC
CTT1
Forward
GATTCCGTTCTACAAGCCAGAC
Reverse
GGAGTATGGACATCCCAAGTTTC
CTA1
Forward
CCAACAGGACAGACCCATTC
Reverse
TTACCCAAAACGCGGTAGAG


## 2.4. Spot Assay

The sensitivity of yeast to oxidant stressors, including H2O2, CHP, and PQ (Sigma-Aldrich, St. Louis, MO, USA), was determined by spot assay. Yeast cells were cultured to exponential phase (OD600 = 2.0) in liquid YPD followed by a 10-fold dilution with sterile water. Four additional 10-fold serial dilutions were performed, and 5 μL of each dilution was inoculated onto solid YPD plates supplemented with indicated stressors. The plates were incubated at 30°C for 2~5 days for colony formation.


## 2.5. Enzyme Activity Assay and Determination of Intracellular H2O2 Level

Yeast cells in exponential phase were harvested and washed twice with cold sterile water followed by resuspension in lysis buffer containing acid-washed glass beads and 20 cycles of 10 s vortexing plus 20 s cooling [14]. After centrifugation at 12000 rpm for 15 min, the supernatants were collected and used for an enzyme activity assay and determination of intracellular H2O2 levels. The protein concentration was determined using the BCA protein Assay Kit (Sangon Biotech, Shanghai, China) following the manufacturer’s instruction. The total superoxide dismutase (SOD) activity and the catalase activity were determined using a Total Cellular SOD Activity Kit (Dojindo Molecular Technologies, Rockville, MD, USA) and a Catalase Assay Kit (Beyotime Biotechnology, Shanghai, China), respectively. The intracellular H2O2 level was determined using an H2O2 Assay Kit (Beyotime Biotechnology, Shanghai, China).


## 2.6. Replicative Lifespan (RLS) Assay

RLS assay was performed as previously described [15]. Briefly, cells were thawed from frozen stocks and grown on freshly made YPD plate for 2 days and then patched onto the second YPD plate and incubated for an additional 24 h. Cells were then restruck on the third YPD plate and incubated for about 12 h, and then the virgin buds were isolated and used for RLS analysis.


## 2.7. Statistical Analysis

All experiments were repeated at least three times. Data were expressed as the mean ± standard deviation (SD). Statistical significance was assessed by calculatingp values using a two-tailed Student’s t-test or Wilcoxon rank-sum test. A value of p<0.05 was considered statistically significant.


## 3. Results

### 3.1.MET18 Deficiency Enhances the Cellular Response of Yeast to Oxidative Stress

To investigate the role ofMET18 in yeast response to oxidative stress, we constructed aMET18/met18Δ mutant strain which was confirmed by a real-time PCR detection ofMET18- andURA3-specific PCR products. As shown in Figure 1(a), the mRNA level ofMET18 was decreased by more than 50% inMET18-deficient cells compared with WT cells, indicating that the expression ofMET18 mRNA inMET18/met18Δmutants was successfully suppressed by the replacement of one copy ofMET18 withURA3 gene.Figure 1
Effects ofMET18 deficiency on yeast response to oxidative stress. (a) mRNA expression ofMET18 under unstressed condition was determined by real-time PCR. (b) Tenfold dilution series of WT andMET18/met18Δcells were spotted on YPD plates with or without 0.03% MMS and then incubated at 30°C for 2~3 days to detect the cellular response to MMS. (c) Tenfold dilution series of WT andMET18/met18Δcells were spotted on YPD plates with 0.225 mM CHP or 7 mM H2O2 and incubated at 30°C for 2~5 days to detect the cellular response to CHP and H2O2. Results shown are representative of three independent experiments, p∗∗<0.01 versus WT cells (n=3).
(a)
(b)
(c)We then sought to examine the cellular response ofMET18/met18Δmutants to oxidative stressors, including H2O2 and CHP. A DNA-damaging agent methyl-methanesulfonate (MMS) was used as a positive control [17]. As shown in Figures 1(b) and 1(c), the significant decreases in yeast growth in response to MMS, H2O2, and CHP were observed inMET18/met18Δmutants, compared with those in WT cells, suggesting thatMET18 deficiency may increase the oxidative stress response in yeast cells.


### 3.2.MET18 Deficiency Decreases Catalase Activity While Increasing Intracellular H2O2 Levels

To further examine whetherMET18 is involved in enzymatic antioxidant defense against oxidative stress, we determined the activities of SOD and catalases which are responsible for ROS scavenging [18, 19]. As shown in Figures 2(a) and 2(b), the activity of total SOD inMET18/met18Δmutants was comparable to that in WT strain, whereas the total catalase activity was significantly decreased by more than 50% inMET18-deficient cells compared with WT strain. Meantime, the mRNA expression of bothCTT1 andCTA1was significantly downregulated inMET18-deficient cells (Figure 2(c)). Conversely, H2O2 levels inMET18/met18Δmutants were higher than those in WT strain (Figure 2(d)), suggesting an increased oxidative stress inMET18/met18Δmutants. These results indicate thatMET18 deficiency may downregulate antioxidant defense enzymes, leading to the increased oxidative stress.Figure 2
Effects ofMET18 deficiency on enzymatic defense system against oxidative stress. Total SOD activity (a), catalase activity (b),CTA1 andCTT1 mRNA levels (c), and intracellular H2O2 levels (d) under unstressed condition were determined and compared between WT andMET18/met18Δcells. Results shown are representative of three independent experiments. p∗<0.05, p∗∗<0.01 versus WT cells (n=3).
(a)
(b)
(c)
(d)


### 3.3. EitherCTT1 orCTA1 Restores theMET18 Deficiency-Suppressed Oxidative Stress Resistance in Yeast

To investigate if the increased oxidative stress response inMET18/met18Δ mutants is attributable to inhibition of catalases, pAUR123-CTT1 or pAUR123-CTA1 was transfected intoMET18/met18Δ mutants to generateCTT1- orCTA1-overexpressing mutants. As shown in Figure 3(a), the catalase activity inMET18/met18Δ mutants overexpressingCTT1(Δ+CTT1) orCTA1(Δ+CTA1) was significantly increased as compared with the empty vector-transfected WT strain (WT + p) or the empty vector-transfected mutant strain (Δ+ p). Furthermore,CTT1- orCTA1-overexpression also reduced the intracellular levels of H2O2 (Figure 3(b)), consistent with our findings (Figure 2). Importantly,CTT1- orCTA1-overexpression greatly enhanced the resistance ofMET18/met18Δ mutants against H2O2 and CHP (Figure 3(c)), leading to an increased cell growth. In contrast,CTT1- orCTA1-overexpression did not appear to promote cell growth under unstressed conditions (Figure 3(c)), suggesting the important role of catalases in oxidative stress response ofMET18/met18Δ mutants. These results indicate that inhibition ofCTT1orCTA1activity may be a major mechanism underlying the impaired oxidative response inMET18-deficient yeasts.Figure 3
Effects ofCTT1 orCTA1 on oxidative stress resistance inMET18/met18Δ cells. Catalase activity (a) and the intracellular H2O2 levels (b) under unstressed conditions were determined and compared among pAUR123-transfected WT strain (WT + p), pAUR123-, andCTT1- andCTA1-transfectedMET18/met18Δ mutants ((Δ + p), (Δ +CTT1), and (Δ +CTA1), resp.). p∗<0.05 versus WT cells (n=3). (c) Tenfold dilution series of WT + p, Δ + p, Δ +CTT1, or Δ +CTA1 were spotted on YPD plates with or without the stressors, as indicated, and incubated at 30°C for 2~3 days to detect the cellular response. Results shown are representative of three independent experiments.
(a)
(b)
(c)


### 3.4.CTT1, but NotCTA1, Restores the Lifespan ofMET18-Deficient Yeast

To further examine ifMET18 has effects on yeast aging, we performed a RLS assay in WT strains andMET18/met18Δ mutants. The results showed thatMET18/met18Δ mutants had a shortened RLS compared with the WT strain BY4743 (mean lifespan: 23.1 versus 36.2) (Figure 4(a)). Of note, overexpression ofCTT1 rescuedMET18 deficiency-induced RLS defects, whereas overexpression ofCTA1 did not appear to affect RLS inMET18/met18Δ mutants (Figure 4(b)), suggesting thatMET18 deficiency leads to shortened RLS or aging in yeast, at least in part, through inhibition of catalases.Figure 4
Effect ofCTT1 orCTA1 on RLS inMET18/met18Δmutants. (a) RLS of WT andMET18/met18Δ cells; (b) RLS of WT andMET18/met18Δcells transfected with pAUR123,CTT1,orCTA1. Results shown are representative of three independent experiments.
(a)
(b)


## 3.1.MET18 Deficiency Enhances the Cellular Response of Yeast to Oxidative Stress

To investigate the role ofMET18 in yeast response to oxidative stress, we constructed aMET18/met18Δ mutant strain which was confirmed by a real-time PCR detection ofMET18- andURA3-specific PCR products. As shown in Figure 1(a), the mRNA level ofMET18 was decreased by more than 50% inMET18-deficient cells compared with WT cells, indicating that the expression ofMET18 mRNA inMET18/met18Δmutants was successfully suppressed by the replacement of one copy ofMET18 withURA3 gene.Figure 1
Effects ofMET18 deficiency on yeast response to oxidative stress. (a) mRNA expression ofMET18 under unstressed condition was determined by real-time PCR. (b) Tenfold dilution series of WT andMET18/met18Δcells were spotted on YPD plates with or without 0.03% MMS and then incubated at 30°C for 2~3 days to detect the cellular response to MMS. (c) Tenfold dilution series of WT andMET18/met18Δcells were spotted on YPD plates with 0.225 mM CHP or 7 mM H2O2 and incubated at 30°C for 2~5 days to detect the cellular response to CHP and H2O2. Results shown are representative of three independent experiments, p∗∗<0.01 versus WT cells (n=3).
(a)
(b)
(c)We then sought to examine the cellular response ofMET18/met18Δmutants to oxidative stressors, including H2O2 and CHP. A DNA-damaging agent methyl-methanesulfonate (MMS) was used as a positive control [17]. As shown in Figures 1(b) and 1(c), the significant decreases in yeast growth in response to MMS, H2O2, and CHP were observed inMET18/met18Δmutants, compared with those in WT cells, suggesting thatMET18 deficiency may increase the oxidative stress response in yeast cells.


## 3.2.MET18 Deficiency Decreases Catalase Activity While Increasing Intracellular H2O2 Levels

To further examine whetherMET18 is involved in enzymatic antioxidant defense against oxidative stress, we determined the activities of SOD and catalases which are responsible for ROS scavenging [18, 19]. As shown in Figures 2(a) and 2(b), the activity of total SOD inMET18/met18Δmutants was comparable to that in WT strain, whereas the total catalase activity was significantly decreased by more than 50% inMET18-deficient cells compared with WT strain. Meantime, the mRNA expression of bothCTT1 andCTA1was significantly downregulated inMET18-deficient cells (Figure 2(c)). Conversely, H2O2 levels inMET18/met18Δmutants were higher than those in WT strain (Figure 2(d)), suggesting an increased oxidative stress inMET18/met18Δmutants. These results indicate thatMET18 deficiency may downregulate antioxidant defense enzymes, leading to the increased oxidative stress.Figure 2
Effects ofMET18 deficiency on enzymatic defense system against oxidative stress. Total SOD activity (a), catalase activity (b),CTA1 andCTT1 mRNA levels (c), and intracellular H2O2 levels (d) under unstressed condition were determined and compared between WT andMET18/met18Δcells. Results shown are representative of three independent experiments. p∗<0.05, p∗∗<0.01 versus WT cells (n=3).
(a)
(b)
(c)
(d)


## 3.3. EitherCTT1 orCTA1 Restores theMET18 Deficiency-Suppressed Oxidative Stress Resistance in Yeast

To investigate if the increased oxidative stress response inMET18/met18Δ mutants is attributable to inhibition of catalases, pAUR123-CTT1 or pAUR123-CTA1 was transfected intoMET18/met18Δ mutants to generateCTT1- orCTA1-overexpressing mutants. As shown in Figure 3(a), the catalase activity inMET18/met18Δ mutants overexpressingCTT1(Δ+CTT1) orCTA1(Δ+CTA1) was significantly increased as compared with the empty vector-transfected WT strain (WT + p) or the empty vector-transfected mutant strain (Δ+ p). Furthermore,CTT1- orCTA1-overexpression also reduced the intracellular levels of H2O2 (Figure 3(b)), consistent with our findings (Figure 2). Importantly,CTT1- orCTA1-overexpression greatly enhanced the resistance ofMET18/met18Δ mutants against H2O2 and CHP (Figure 3(c)), leading to an increased cell growth. In contrast,CTT1- orCTA1-overexpression did not appear to promote cell growth under unstressed conditions (Figure 3(c)), suggesting the important role of catalases in oxidative stress response ofMET18/met18Δ mutants. These results indicate that inhibition ofCTT1orCTA1activity may be a major mechanism underlying the impaired oxidative response inMET18-deficient yeasts.Figure 3
Effects ofCTT1 orCTA1 on oxidative stress resistance inMET18/met18Δ cells. Catalase activity (a) and the intracellular H2O2 levels (b) under unstressed conditions were determined and compared among pAUR123-transfected WT strain (WT + p), pAUR123-, andCTT1- andCTA1-transfectedMET18/met18Δ mutants ((Δ + p), (Δ +CTT1), and (Δ +CTA1), resp.). p∗<0.05 versus WT cells (n=3). (c) Tenfold dilution series of WT + p, Δ + p, Δ +CTT1, or Δ +CTA1 were spotted on YPD plates with or without the stressors, as indicated, and incubated at 30°C for 2~3 days to detect the cellular response. Results shown are representative of three independent experiments.
(a)
(b)
(c)


## 3.4.CTT1, but NotCTA1, Restores the Lifespan ofMET18-Deficient Yeast

To further examine ifMET18 has effects on yeast aging, we performed a RLS assay in WT strains andMET18/met18Δ mutants. The results showed thatMET18/met18Δ mutants had a shortened RLS compared with the WT strain BY4743 (mean lifespan: 23.1 versus 36.2) (Figure 4(a)). Of note, overexpression ofCTT1 rescuedMET18 deficiency-induced RLS defects, whereas overexpression ofCTA1 did not appear to affect RLS inMET18/met18Δ mutants (Figure 4(b)), suggesting thatMET18 deficiency leads to shortened RLS or aging in yeast, at least in part, through inhibition of catalases.Figure 4
Effect ofCTT1 orCTA1 on RLS inMET18/met18Δmutants. (a) RLS of WT andMET18/met18Δ cells; (b) RLS of WT andMET18/met18Δcells transfected with pAUR123,CTT1,orCTA1. Results shown are representative of three independent experiments.
(a)
(b)


## 4. Discussion

In the present study, we constructed aMET18/met18Δ mutant using the WT BY4743 yeast strain and examined the response ofMET18-deficient yeast to oxidative stress caused by H2O2 and CHP. Our results suggest thatMET18 deficiency increases the cellular response of yeast to H2O2 and CHP and shortens RLS through downregulation of catalasesCTT1 andCTA1.It has been reported thatMET18 participates in oxidative stress responses [20, 21]; however, the underlying mechanism remains unknown. Oxidative stress is termed as a disruption of cellular redox balance due to the failure of oxidant defense system to scavenge excess ROS which can be generated from normal cellular metabolism in living organisms and is a potential damage to cells [22]. In our study, the total catalase activity as well as the mRNA levels of bothCTT1 andCTA1was significantly decreased inMET18/met18Δ mutant compared with the WT strain (Figures 2(b) and 2(c)), resulting in an increased level of intracellular H2O2 in mutant cells (Figure 2(d)). These results indicate that the cellular redox balance is abolished inMET18-deficient cells due to the decreased antioxidants (catalase) and increased ROS (H2O2) levels.SinceMET18 is a component of the CIA machinery and plays an important role in the maturation of Fe/S proteins [5, 6], one possible explanation for the downregulation of catalase is that the Fe/S cluster transfer required for catalase maturation is inhibited inMET18-deficient yeast, resulting in H2O2 accumulation and an increase in the oxidant burden of the cells. In addition,MET18 could be responsible for or has a role to play in Fe/S cluster transfer or epigenetic modifications in yeasts, affecting their metabolism [7, 13, 23]. Indeed, Wang et al. [13] showed thatMET18 can regulate active DNA methylation and methylation pathways inArabidopsis. Buzas et al. [23] showed thatAtDRE2, a member of the CIA, had epigenetic functions that are independent of the CIA. Duan et al. [7] showed thatMET18-dependent transfer of the iron cluster was required forROS1-mediated active DNA methylation. Nevertheless, additional studies will be necessary to specify the role ofMET18 in oxidative stress of the yeasts.H2O2, at high concentrations, primarily contributes to oxidative damage [20, 21]. Dismutation of O2− by SOD is a major source of H2O2 [24]. However, in our study, the activity of SOD inMET18/met18Δmutants did not appear to be different from that in WT strain (Figure 2(a)), suggesting that other enzymes or factors than SOD are responsible for the increased H2O2 level inMET18-deficient cells. Catalases may downregulate the intracellular level of H2O2 by catalyzing the decomposition of H2O2 into water and oxygen [25]. Accordingly, the increased H2O2 level and enhanced response to oxidative stress have been observed in yeast cells lackingCTA1 andCTT1 [24, 25], which is consistent with our findings. It seems that, compared with WT strains,MET18/met18Δ mutants are more sensitive to the oxidative stress caused by H2O2 and CHP due to the decreased catalase activity and the subsequent H2O2 accumulation.There are conflicting findings in regard to the role of catalases in yeast response to H2O2. Some studies show that the activity ofCTT1is essential to protect yeast cells against H2O2 challenge, whereas the activity ofCTA1 is dispensable [25]. On the other hand, some studies indicate that bothCTT1 andCTA1mutants are sensitive to H2O2, suggesting thatCTT1 andCTA1are equally important for antioxidative response of yeast to H2O2 stress [26]. Our study reveals that the overexpression of eitherCTT1 orCTA1 inMET18/met18Δ mutants can effectively scavenge the accumulated intracellular H2O2 and thus enhances the cellular resistance to CHP and H2O2 (Figure 3).Catalase activity is also closely associated with lifespan regulation in various species. It has been reported that overexpression of human catalase targeted to mitochondria can extend mouse lifespan by eliminating mitochondrial H2O2 [2]. Consistently, reduced mitochondrial ROS as well as increased antioxidant enzyme activities of SOD, catalase, and glutathione peroxidase 1 has been observed in long-lived white-footed mice compared with common laboratory mice [27]. The role of catalase in the yeast replicative aging may be complicated by genetic background (haploid or diploid) and nutrient availability (glucose or ethanol) [28]. Several lines of evidence show that catalases and excess H2O2 exert positive and negative effects on RLS in yeast, respectively [29]. Our study found that loss of one copy ofMET18 leads to the reduction in yeast RLS which can be rescued byCTT1, but not byCTA1 (Figure 4), indicating that inactivity ofCTT1 is a driving force of senescence inMET18/met18Δ mutants. These findings are in agreement with the previous study thatCTT1, but notCTA1, is required for the maintenance of RLS in yeast cultured in glucose-containing media [30].MET18 deficiency leads to shortened RLS or aging in yeast, at least in part, through inhibition of catalases. It is known thatCTT1 andCTA1 are not submitted to the same regulatory mechanisms in response to nutrient availability and oxidative stress [30]. This could explain, at least in part, whyCTT1 andCTA1 did not have the same effect on RLS in the present study, but additional studies are necessary to address this issue. Nevertheless, Rona et al. [2] showed thatCTT1 overexpression increased the lifespan of calorie-restrictedS. cerevisiae deficient inSod1 through increased peroxide degradation catalyzed by catalase. Similarly, Zhao et al. [31] showed thatCTT1 overexpression increased the replicative lifespan ofS. cerevisiae sensitive to MMS and deficient inKSP1. On the other hand, Ohtsuka et al. [32] showed thatCTT1 was required for the H2O2-resistant phenotype, but thatCTT1 overexpression did not increase the chronological lifespan of the yeast. These previous studies provide some clues about the role of catalases as modulators of replicative lifespan inS. cerevisiae.


## 5. Conclusions

In conclusion, our study demonstrates for the first time thatMET18deficiency in yeast impairs oxidative stress tolerance and shortens lifespan, at least partly, through the inhibition of catalase activity and accumulation of intracellular H2O2. Further investigation is needed to examine the role ofMET18 in other Fe/S transfer-associated processes such as epigenetic modifications in eukaryotes.



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*Source: 7587395-2017-07-30.xml*