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

Yeast MET18, 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 a MET18/met18Δ heterozygous mutant yeast strain and found that MET18 deficiency 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 in MET18-deficient cells. In contrast, overexpression of CTT1 or CTA1 in MET18-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 by CTT1 overexpression, but not by CTA1, in the MET18-deficient cells. These results suggest that MET18, 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.


Introduction
Oxygen (O 2 ) is crucial for the livelihood of organisms grown aerobically. However, exposure to higher O 2 concentration can increase the production of reactive oxygen species (ROS), including hydroxyl radical (OH − ), hydrogen peroxide (H 2 O 2 ), and superoxide anions (O 2− ), 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][2][3]. It has been shown that culturing cells in air (21% O 2 ) exposes the cells to much higher levels of oxygen than what they are usually submitted to under physiological conditions (2∼5% O 2 ) [2]. Cells normally have oxidant defense systems to protect themselves against ROS or oxidative stress. However, the underlying mechanisms remain largely unknown [3,4].
Yeast MET18, 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][6][7][8]. From yeasts to plant cells to human cells, the CIA machinery is generally located in the cytoplasm and responsible for the maturation 2 BioMed Research International of Fe/S proteins through catalyzing the synthesis of Fe/S clusters [9,10]. In the CIA pathway, MET18 and other subunits, such as CIA1, CIA2, and nuclear architecture-related protein 1 (NAR1), form a CIA complex in which MET18 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]. MET18 could 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 and NAR1, are associated with yeast response to oxidative stress and thus regulate the replicative lifespan (RLS) of yeast. In CIA2/cia2Δ mutants, the deficiency of CIA2 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 O 2− -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].
Since MET18 is also a component of CIA, we hypothesized that MET18 may play an important role in yeast response to oxidative stress. To confirm this hypothesis, we performed the functional assay for MET18 mutation by constructing a MET18/met18Δ heterozygous mutant yeast strain. Our results demonstrate for the first time that MET18 deficiency may increase the response sensitivity of yeast to oxidative stress and shorten the RLS through suppressing the expression and activity of catalases.

Yeast Strains and
Culture. The yeast strains used in this study are listed in Table 1. 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.

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 SYBR5 Premix Ex Taq6 (Takara, Otsu, Shiga, Japan) and the primers are shown in Table 2. Data were normalized to the internal control PRP8 [14].

Spot
Assay. The sensitivity of yeast to oxidant stressors, including H 2 O 2 , CHP, and PQ (Sigma-Aldrich, St. Louis, MO, USA), was determined by spot assay. Yeast cells were cultured to exponential phase (OD 600 = 2.0) in liquid YPD followed by a 10-fold dilution with sterile water. Four additional 10fold serial dilutions were performed, and 5 L 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.

Enzyme Activity Assay and Determination of Intracellular H 2 O 2 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 H 2 O 2 levels.

MET18 Deficiency Enhances the Cellular Response of Yeast to Oxidative Stress.
To investigate the role of MET18 in yeast response to oxidative stress, we constructed a MET18/met18Δ mutant strain which was confirmed by a real-time PCR detection of MET18-and URA3-specific PCR products. As shown in Figure 1(a), the mRNA level of MET18 was decreased by more than 50% in MET18-deficient cells compared with WT cells, indicating that the expression of MET18 mRNA in MET18/met18Δ mutants was successfully suppressed by the replacement of one copy of MET18 with URA3 gene.
We then sought to examine the cellular response of MET18/met18Δ mutants to oxidative stressors, including H 2 O 2 and CHP. A DNA-damaging agent methylmethanesulfonate (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, H 2 O 2 , and CHP were observed in MET18/met18Δ mutants, compared with those in WT cells, suggesting that MET18 deficiency may increase the oxidative stress response in yeast cells.

MET18 Deficiency Decreases Catalase Activity While
Increasing Intracellular H 2 O 2 Levels. To further examine whether MET18 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 in MET18/met18Δ mutants was comparable to that in WT strain, whereas the total catalase activity was significantly decreased by more than 50% in MET18-deficient cells compared with WT strain. Meantime, the mRNA expression of both CTT1 and CTA1 was significantly downregulated in MET18-deficient cells (Figure 2(c)). Conversely, H 2 O 2 levels in MET18/met18Δ mutants were higher than those in WT strain (Figure 2(d)), suggesting an increased oxidative stress in MET18/met18Δ mutants. These results indicate that MET18 deficiency may downregulate antioxidant defense enzymes, leading to the increased oxidative stress.

Either CTT1 or CTA1 Restores the MET18 Deficiency-Suppressed Oxidative Stress Resistance in Yeast.
To investigate if the increased oxidative stress response in MET18/met18Δ mutants is attributable to inhibition of catalases, pAUR123-CTT1 or pAUR123-CTA1 was transfected into MET18/met18Δ mutants to generate CTT1-or CTA1-overexpressing mutants. As shown in Figure 3(a), the catalase activity in MET18/ met18Δ mutants overexpressing CTT1 (Δ + CTT1) or CTA1 (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-or CTA1-overexpression also reduced the intracellular levels of H 2 O 2 (Figure 3(b)), consistent with our findings ( Figure 2). Importantly, CTT1-or CTA1-overexpression greatly enhanced the resistance of MET18/met18Δ mutants against H 2 O 2 and CHP (Figure 3(c)), leading to an increased cell growth. In contrast, CTT1-or CTA1-overexpression did not appear to promote cell growth under unstressed conditions (Figure 3(c)), suggesting the important role of catalases in oxidative stress response of MET18/met18Δ mutants. These results indicate that inhibition of CTT1 or CTA1 activity may be a major mechanism underlying the impaired oxidative response in MET18-deficient yeasts.  (Figure 4(a)). Of note, overexpression of CTT1 rescued MET18 deficiencyinduced RLS defects, whereas overexpression of CTA1 did not appear to affect RLS in MET18/met18Δ mutants (Figure 4(b)), suggesting that MET18 deficiency leads to shortened RLS or aging in yeast, at least in part, through inhibition of catalases.

Discussion
In the present study, we constructed a MET18/met18Δ mutant using the WT BY4743 yeast strain and examined the response of MET18-deficient yeast to oxidative stress caused by H 2 O 2 and CHP. Our results suggest that MET18 deficiency increases the cellular response of yeast to H 2 O 2 and CHP and shortens RLS through downregulation of catalases CTT1 and CTA1. It has been reported that MET18 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 both CTT1 and CTA1 was significantly decreased in MET18/met18Δ mutant compared with the WT strain (Figures 2(b) and 2(c)), resulting in an increased level of intracellular H 2 O 2 in mutant cells (Figure 2(d)). These results indicate that the cellular redox balance is abolished in MET18-deficient cells due to the decreased antioxidants (catalase) and increased ROS (H 2 O 2 ) levels.
Since MET18 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 in MET18-deficient yeast, resulting in H 2 O 2 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 that MET18 can regulate active DNA methylation and methylation pathways in Arabidopsis. Buzas et al. [23] showed that AtDRE2, a member of the CIA, had epigenetic functions that are independent of the CIA. Duan et al. [7] showed that MET18-dependent transfer of the iron cluster was required for ROS1-mediated active DNA methylation. Nevertheless, additional studies will be necessary to specify the role of MET18 in oxidative stress of the yeasts.
H 2 O 2 , at high concentrations, primarily contributes to oxidative damage [20,21]. Dismutation of O 2− by SOD is a major source of H 2 O 2 [24]. However, in our study, the activity of SOD in MET18/met18Δ mutants 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 H 2 O 2 level in MET18-deficient cells. Catalases may downregulate the intracellular level of H 2 O 2 by catalyzing the decomposition of H 2 O 2 into water and oxygen [25]. Accordingly, the increased H 2 O 2 level and enhanced response to oxidative stress have been observed in yeast cells lacking CTA1 and CTT1 [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 H 2 O 2 and CHP due to the decreased catalase activity and the subsequent H 2 O 2 accumulation.
There are conflicting findings in regard to the role of catalases in yeast response to H 2 O 2 . Some studies show that the activity of CTT1 is essential to protect yeast cells against H 2 O 2 challenge, whereas the activity of CTA1 is dispensable [25]. On the other hand, some studies indicate that both CTT1 and CTA1 mutants are sensitive to H 2 O 2 , suggesting that CTT1 and CTA1 are equally important for antioxidative response of yeast to H 2 O 2 stress [26]. Our study reveals that the overexpression of either CTT1 or CTA1 in MET18/met18Δ mutants can effectively scavenge the accumulated intracellular H 2 O 2 and thus enhances the cellular resistance to CHP and H 2 O 2 ( 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 H 2 O 2 [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 H 2 O 2 exert positive and negative effects on RLS in yeast, respectively [29]. Our study found that loss of one copy of MET18 leads to the reduction in yeast RLS which can be rescued by CTT1, but not by CTA1 (Figure 4), indicating that inactivity of CTT1 is a driving force of senescence in MET18/met18Δ mutants. These findings are in agreement with the previous study that CTT1, but not CTA1, 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 that CTT1 and CTA1 are not submitted to the same regulatory mechanisms in response to nutrient availability and oxidative stress [30]. This could explain, at least in part, why CTT1 and CTA1 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 that CTT1 overexpression increased the lifespan of calorie-restricted S. cerevisiae deficient in Sod1 through increased peroxide degradation catalyzed by catalase. Similarly, Zhao et al. [31] showed that CTT1 overexpression increased the replicative lifespan of S. cerevisiae sensitive to MMS and deficient in KSP1. On the other hand, Ohtsuka et al. [32] showed that CTT1 was required for the H 2 O 2 -resistant phenotype, but that CTT1 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 in S. cerevisiae.

Conclusions
In conclusion, our study demonstrates for the first time that MET18 deficiency in yeast impairs oxidative stress tolerance and shortens lifespan, at least partly, through the inhibition of catalase activity and accumulation of intracellular H 2 O 2 . Further investigation is needed to examine the role of MET18 in other Fe/S transfer-associated processes such as epigenetic modifications in eukaryotes.