An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: oxidative stress-induced arginine synthesis and its physiological role

Saccharomyces cerevisiae S 1278b has the MPR1 gene encoding the N -acetyltrans-ferase Mpr1 that acetylates the proline metabolism intermediate D 1 -pyrroline-5-carboxylate (P5C)/glutamate- g -semialdehyde (GSA) in vitro . In addition, Mpr1 protects cells from various oxidative stresses by regulating the levels of intracellular reactive oxygen species (ROS). However, the relationship between P5C/GSA acetylation and antioxidative mechanism involving Mpr1 remains unclear. Here, we report the synthesis of oxidative stress-induced arginine via P5C/GSA acetylation catalyzed by Mpr1. Gene disruption analysis revealed that Mpr1 converts P5C/GSA into N -acetyl-GSA for arginine synthesis in the mitochondria, indicating that Mpr1 mediates the proline and arginine metabolic pathways. More impor-tantly, Mpr1 regulate ROS generation by acetylating toxic P5C/GSA. Under oxidative stress conditions, the transcription of PUT1 encoding the proline oxidase Put1 and MPR1 was strongly induced, and consequently, the arginine content was signiﬁcantly increased. We also found that two deletion mutants ( D mpr1 / 2 and D put1 ) were more sensitive to high-temperature stress than the wild-type strain, but that direct treatment with arginine restored the cell viability of these mutants. These results suggest that Mpr1-dependent arginine synthesis confers stress tolerance. We propose an antioxidative mechanism that is involved in stress-induced arginine synthesis requiring Mpr1 and Put1. yet unknown mechanism. Additionally, Mpr1 would regulate P5C-mediated ROS generation by acetylating P5C/GSA, although the pathway that P5C/GSA induces ROS production is unknown.


Introduction
In the yeast Saccharomyces cerevisiae S1278b, we discovered genes involved in the detoxification of the proline analogue azetidine-2-carboxylate (AZC) (Takagi et al., 2000;Shichiri et al., 2001). These genes, MPR1 and MPR2 (S1278b gene for proline-analogue resistance), encode the N-acetyltransferase Mpr1 that converts AZC into N-acetyl-AZC. Only one base change occurs between MPR1 and MPR2, and both genes have similar functions. Although MPR1 is missing in the genome project strain S. cerevisiae S288C, its homologous genes were found in the genomes of other yeasts and fungi, and AZC acetyltransferase activity has already been detected in a number of yeast strains (Wada et al., 2008). These results suggest that MPR1 homologues are widely present in yeast and fungal strains, and thus these homo-logues are most likely derived from a common ancestral gene.
However, it is unlikely that AZC is a natural substrate for Mpr1 because AZC occurs only in certain plants (Fowden, 1956;Fowden & Bryant, 1959;Troxler & Brown, 1974). Recently, we found that Mpr1 protects yeast cells by reducing the intracellular reactive oxygen species (ROS) levels under oxidative stress conditions, such as H 2 O 2 , heat shock, freeze-thaw or ethanol treatment (Nomura & Takagi, 2004;Du & Takagi, 2005. Exposure to a high temperature also produces ROS in the mitochondria, which cause oxidative stress in yeast cells (Davidson et al., 1996;Moraitis & Curran, 2004. We also isolated two Mpr1 variants with improved enzymatic functions (K63R and F65L) (Iinoya et al., 2009). The K63R and F65L variant exhibited higher catalytic efficiency and thermal stability than that of the wild-type Mpr1, respectively. Overexpression of the Mpr1 variants decreased intracellular ROS levels and increased cell viability under oxidative stress conditions compared with the wild-type Mpr1. In terms of biotechnological applications, it is worth noting that the expression of the Mpr1 variants rendered baker's yeast cells more tolerant to air-drying stress, leading to increased fermentation ability in dough (Sasano et al., 2010).
In addition to AZC, Mpr1 acetylates either D 1 -pyrroline-5-carboxylate (P5C) or, more likely, its equilibrium compound glutamate-g-semialdehyde (GSA) in vitro (Nomura & Takagi, 2004). The proline metabolism intermediate P5C/ GSA is generated in the cytoplasm and mitochondria when proline is enzymatically synthesized and degraded, respectively ( Fig. 1) (Adams & Frank, 1980). In terms of structural formulae, GSA could be acetylated by Mpr1 and converted into N-acetyl-GSA, an arginine biosynthesis intermediate ( Fig. 1). Therefore, Mpr1 would mediate the proline and arginine metabolic pathways via the acetylation of P5C/ GSA. A nutritionally important amino acid arginine is involved in stress resistance in various organisms (Morita et al., 2002;Shima et al., 2003;Ma et al., 2008;Petrovic et al., 2008). In baker's yeast, disruption of CAR1 encoding arginase, which participates in the first committed step of arginine degradation, increased intracellular arginine levels and enhanced freeze tolerance (Shima et al., 2003). Under oxidative stress conditions, such as H 2 O 2 and freeze-thaw stress, yeast cells accumulated arginine Momose et al., 2010), but the synthetic pathway and function of arginine are poorly understood.
In this study, gene disruption analysis revealed that Mpr1 acetylates P5C/GSA to yield N-acetyl-GSA for arginine synthesis in the mitochondria. Under oxidative stress conditions, we found that PUT1, which encodes the proline oxidase Put1, and MPR1 are induced to convert proline into arginine via N-acetyl-GSA. We also obtained evidence indicating that increased arginine from proline contributes to oxidative stress tolerance in yeast cells. Thus, we propose an antioxidative mechanism involving Put1 and Mpr1 via proline/arginine metabolism.
The centromere-based low-copy-number plasmids pRS414 and pRS416 (Stratagene, La Jolla, CA) harboring TRP1 and URA3, respectively, were used for complementing the auxotrophic markers or for expressing enhanced green fluorescent protein (EGFP)-fused Mpr1. The media used for the growth of S. cerevisiae were a synthetic minimal medium SD (2% glucose and 0.67% Bacto yeast nitrogen base without amino acids) (Difco Laboratories, Detroit, MI) and a nutrient medium YPD (2% glucose, 1% yeast extract, and 2% peptone). When required, appropriate amino acids were added to SD medium for auxotrophic strains. When necessary, 2% agar was added to solidify the medium.

Western blot analysis
The mitochondrial fractions were isolated from strain as described previously (Glick & Pon, 1995) using glycerol as a carbon source instead of lactate. Western blot analysis was performed with the cytosolic and mitochondrial fractions using anti-3-phosphoglycerate kinase (PGK) (Invitrogen, Carlsbad, CA), anti-pyruvate dehydrogenase (PDH) complex E1-a subunit (Invitrogen), and anti-Mpr1 antibodies as described previously (Shichiri et al., 2001). PGK and PDH complex E1-a subunit were used as marker proteins for cytosolic and mitochondrial fractions, respectively.

Plasmid construction
The primers used are listed in Supporting Information, Table S1. Plasmid pRS-MPR1SS was first constructed by subcloning of the 3.7-kb SacI-SacI fragment containing MPR1 from pMH1 (Takagi et al., 2000) into the SacI site of pRS416 to express GFP-fused Mpr1. For inserting EGFP into the C-terminus of Mpr1, the linearized pRS-MPR1SS truncating the termination codon of MPR1 was prepared by inverse-PCR with primers SmaI-MPR1-Fw containing the SmaI site and SacII-MPR1-Rv containing the SacII site, whereas the EGFP gene lacking the initiation codon was amplified by PCR using a pEGFP Vector (Clontech, Mountain View, CA) as a template with primers SacII-EGFP-Fw containing the SacII site and SmaI-EGFP-TAA-Rv containing the SmaI site. Both PCR products were digested with SacII and SmaI, mixed, and ligated. The plasmid in which the EGFP gene was fused to the carboxyl terminus of Mpr1 was designated as pRS-MPR1-EGFP. For inserting EGFP into the N-terminus of Mpr1, the linearized pRS-MPR1SS truncating the initiation codon of MPR1 and the EGFP gene fragment lacking the termination codon were prepared by PCR with primers SmaI-5 0 MPR1-Fw containing the SmaI site and SacII-5 0 MPR1-Rv containing the SacII site for pRS-MPR1SS and SacII-EGFP-ATG-Fw containing the SacII site and SmaI-EGFP-Rv containing the SmaI site for the EGFP gene, and pRS-EGFP-MPR1 was constructed in a similar manner as above. To express an intact EGFP gene under control of the MPR1 promoter, the ORF of MPR1 was removed from pRS-MPR1SS by inverse-PCR using pRS-MPR1SS as a template with the primers SmaI-3 0 MPR1-Fw and SacII-5 0 MPR1-Rv, whereas the EGFP gene was amplified by PCR using pEGFP as a template with primers SacII-EGFP-ATG-Fw and SmaI-EGFP-TAA-Rv, and then pRS-EGFP was constructed in a similar manner as above.
The DNA fragments of ARG2 and ARG8 were amplified by PCR performed with chromosomal DNA of L5685 and the primers ARG2-Fw and ARG2-Rv for ARG2 or ARG8-Fw and ARG8-Rv for ARG8. The unique amplified DNA fragments containing ARG2 and ARG8 were blunt-ended and ligated into the EcoRV site and the SmaI and HincII sites of pBluescript II SK1 (Toyobo, Osaka, Japan) to construct pARG2 and pARG8, respectively. Plasmid pARG2U or pARG8U was constructed by deleting the EcoRV fragment in ARG2 from pARG2 and inserting the 1.7-kb HpaI-SmaI fragment containing URA3 of YEp24 or deleting the StuI-ClaI fragment in ARG8 from pARG8 and inserting the 1.1-kb ClaI-SmaI fragment containing URA3 of YEp24, respectively, by blunt-end ligation.

Localization test
LD1014ura3 cells harboring pRS-MPR1-EGFP, pRS-EGFP-MPR1, or pRS-EGFP were grown to the early stationary phase (OD 600 nm of 3.0-4.0) in SD medium at 25 1C and subjected to 40 nM MitoTracker s Orange (Invitrogen) for 30 min to visualize the mitochondria. The cells were then harvested, washed with 0.9% NaCl, and viewed using an Axiovert 200 M microscope (Carl Zeiss, Oberkochen, Germany) with a Â 100 oil immersion objective and an AxioCam MRm CCD camera (Carl Zeiss).
The yeast Mpr1-mediated antioxidative mechanism Disruption of ARG2 and ARG8 The primers used are listed in Table S1. For the ARG2 or ARG8 disruption, the DNA fragment harboring arg2<URA3 or arg8<URA3 was amplified by PCR with the primers ARG2-Fw and ARG-Rv or ARG8-Fw and ARG8-Rv using pARG2U or pARG8U as a template, respectively, and integrated into the ARG2 or the ARG8 locus in L5685, and LD1014ura3 by transformation. The Ura 1 and Arg À phenotype was screened and the correct disruption event was verified by PCR using chromosomal DNA of each mutant as a template.

Oxidative stress tolerance test
Yeast cells were cultured to the exponential growth phase (OD 600 nm of 1.0) in SD medium at 25 1C and exposed to 39 1C in SD medium. Stress-treated cells were diluted in distilled water, and aliquots were plated on YPD plates. After incubation at 30 1C for 2 days, the survival rates were expressed as percentages, calculated as follows: (no. of colonies after exposure to oxidative stress)/(no. of colonies before exposure to oxidative stress) Â 100.

Real-time quantitative PCR analysis
The primers used are listed in Table S1. Yeast cells were disrupted with glass beads in a Multi-Beads Shocker (MB601U, Yasui Kikai, Osaka, Japan). Total RNA was prepared using an RNeasy Mini Kit (Qiagen, Valencia, CA) and was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). For real-time quantitative PCR, the synthesized cDNA was amplified with oligonucleotide primers for each gene using a 7300 Real-Time PCR System (Applied Biosystems). Reactions contained Power SYBR PCR Master Mix (Applied Biosystems), forward and reverse primers (0.1 mM each), and a cDNA template (20 ng). The cycle thresholds for each gene were normalized to ACT1 and the relative induction fold compared with unstressed cells was shown, for which one indicates no change in abundance.

Intracellular contents of amino acids and P5C
Yeast cells were harvested, washed twice with 50 mM potassium phosphate buffer (pH 7.4), and resuspended in 500 mL of distilled water. The suspension was transferred to boiling water and intracellular amino acids were extracted by boiling for 15 min. After centrifugation (10 min at 15 000 g), amino acids in each supernatant were subsequently quantified using an amino acid analyzer (JLC-500/ V, JEOL, Tokyo, Japan). P5C in each supernatant was measured by monitoring the amount of the P5C-o-aminobenzaldehyde complex using its extinction coefficient (2710 M À1 cm À1 ) as described previously (Nomura & Taka-gi, 2004). The contents of amino acids and P5C were expressed as mmol g À1 dry cell weight (DW).

Measurement of the intracellular oxidation level
The level of intracellular ROS induced in a cell during oxidative stress was measured using the oxidant-sensitive probe 2 0 ,7 0 -dichlorofluorescin diacetate (DCF-DA) (Molecular Probes, Eugene, OR). Exponential yeast cells were incubated at 25 1C for 30 min in SD medium containing 10 mM DCF-DA in the dark and then subjected to oxidative stress. The harvested cells were washed twice with 50 mM potassium phosphate buffer (pH 7.4), resuspended in 500 mL of distilled water, and disrupted with glass beads in a Multi-Beads Shocker. Cell extracts (50 mL) were mixed in 450 mL of distilled water, and the fluorescence was measured with l EX = 504 nm and l EM = 524 nm using a fluorescence spectrophotometer (F-7000; Hitachi, Tokyo, Japan). The value of l EM = 524 nm was normalized by protein in the mixture. The protein concentration was determined using the Bradford assay. The intensity of fluorescence of strain L5685 before exposure to oxidative stress (0 min) was relatively considered to be 100%.

Effect of arginine treatment on yeast cells under high-temperature stress
Exponential yeast cells were treated with 100 mM arginine and subjected to high-temperature stress (at 39 1C for 14 h). The harvested cells were washed twice with 50 mM potassium phosphate buffer (pH 7.4) and the survival rate was determined.
Preparation and treatment of P5C/GSA DL-P5C/GSA was synthesized by periodate oxidation of DL-hydroxylysine and purified using Dowex 50 (Dow Chemical, Midland, MI), as described previously (Williams & Frank, 1975). For the P5C/GSA cytotoxicity assay, yeast cells were grown to the exponential phase (OD 600 nm of 1.0) in SD medium at 25 1C and exposed to different concentrations of P5C/GSA for 2 h in SD medium. The P5C/GSA cytotoxicity was determined by cell viability and intracellular ROS visualization. For the visualization, cells were treated with DCF-DA and viewed using an Axiovert 200 M microscope (Carl Zeiss) with a Â 100 oil immersion objective and an AxioCam MRm CCD camera (Carl Zeiss).

Mpr1 converts P5C/GSA into N-acetyl-GSA in the mitochondria for arginine synthesis
In vitro studies of bacterially expressed Mpr1 have demonstrated that Mpr1 can acetylate the proline metabolic intermediate P5C/GSA (Nomura & Takagi, 2004). However, it remains unclear whether Mpr1 acetylates P5C/GSA in vivo. The subcellular localization of Mpr1 is also unknown. To examine the subcellular localization of Mpr1, we performed a Western blot analysis using the cytosolic and mitochondrial fractions prepared from the wild-type strain L5685 (MPR1/2) and the mpr1/2-disrupted strain LD1014 (Dmpr1/2). As shown in Fig. 2a, Mpr1 was detected not only in the cytosolic but also in the mitochondrial fractions from L5685. However, no bands corresponding to the Mpr1 protein were detected in either fraction from LD1014. Furthermore, the genes of Mpr1 fused with EGFP at the C-terminus (Mpr1-EGFP) or the N-terminus (EGFP-Mpr1) were constructed and expressed in the mpr1/2-disrupted strain LD1014ura3. Interestingly, we clearly observed Mpr1-EGFP in the cytosol and mitochondria in cells harboring pRS-MPR1-EGFP (Fig. 2b). In contrast, EGFP-Mpr1 was localized in both the cytosol and the vacuole, as in the case of EGFP expression only in LD1014ura3 (Fig. S1). As shown in Fig. 2c, the transformant cells expressing Mpr1-EGFP were able to grow on an AZC-containing medium as well as the transformant harboring pRS-MPR1SS. However, cells Total cytosolic (5 mg) and mitochondrial (5 mg) proteins were subjected to a 15% polyacrylamide gel, and Mpr1, PGK, and the PDH complex E1a subunit were detected using each antibody. PGK and the PDH complex E1-a subunit were used as marker proteins for the cytosolic and mitochondrial fractions, respectively. The arrow on the left indicates the position of Mpr1. (b) Localization of the EGFP-fused Mpr1 proteins. LD1014ura3 harboring pRS-MPR1-EGFP was grown in SD medium and observed in the fluorescence microscope for the localization of EGFP-fused Mpr1 (EGFP). Cell morphology was observed through differential interference contrast (DIC) and was also stained with MitoTracker to visualize the mitochondria.
(c) Growth phenotypes on SD agar medium of strains expressing Mpr1. After cultivation in liquid SD medium at 30 1C for 24 h, approximately 10 6 cells of each strain, and serial dilutions of 10 À1 -10 À4 (from left to right) were spotted onto SD agar medium in the absence and presence of 300 mg mL À1 AZC (1AZC). The plates were incubated at 30 1C for 3 days.
The yeast Mpr1-mediated antioxidative mechanism expressing EGFP-Mpr1 exhibited a growth defect in AZCcontaining medium. These results suggest that the N-terminal region in Mpr1 plays important roles in the translocation into the mitochondria and in the catalytic function.
In S. cerevisiae, arginine is synthesized in eight steps beginning with the formation of N-acetyl-glutamate from glutamate catalyzed by N-acetyl-glutamate synthase (the ARG2 gene product) (Fig. 1). In the fourth step of arginine biosynthesis, N-acetyl-GSA is converted into N-acetylornithine catalyzed by N-acetyl-ornithine aminotransferase (the ARG8 gene product) in the mitochondria. Therefore, the disruption of ARG2 or ARG8 in S. cerevisiae S288C and S1278b causes an arginine requirement phenotype ( Vandenbol & Portetelle, 1999;Abadjieva et al., 2001). To investigate whether P5C/GSA is the intracellular substrate for Mpr1, we examined the growth phenotypes of arg2-and arg8-disrupted S. cerevisiae S1278b. We found that Mpr1 clearly restored growth in arg2-deficient cells, even in the absence of arginine, but could not compensate the arg8 disruption blocking the downstream pathway of N-acetyl-GSA (Fig. 3). These results indicate that Mpr1 mediates both the proline and the arginine metabolic pathways by acetylating P5C/GSA in the mitochondria (Fig. 1).

MPR1 and PUT1 induce arginine synthesis under oxidative stress conditions
In addition to P5C/GSA acetylation, Mpr1 has been shown to confer tolerance to oxidative stress, such as H 2 O 2 , heat shock, freezing, or ethanol treatment, on yeast cells (Nomura & Takagi, 2004;Du & Takagi, 2005. However, the relationship between P5C/GSA acetylation and the antioxidative mechanism involving Mpr1 remains unclear. We then conducted a real-time quantitative PCR analysis to determine the transcription of MPR1 and the genes involved in proline and arginine metabolism (PUT1, PUT2, PRO1, PRO2, and PRO3, ARG1, ARG2, ARG3, ARG4, ARG5.6, ARG7, ARG8, and ARG11) in L5685 cells. As shown in Fig. 4, MPR1 and PUT1 were strongly induced with exposure to a high temperature (39 1C), whereas the transcription of PUT2 encoding P5C dehydrogenase remained unaffected by this treatment. Both PUT1 and PUT2 are known to be upregulated by excess proline under the control of the transcription activator Put3 (Morita et al., 2002). It would be of interest to examine whether MPR1 and PUT1 induction might occur by a novel mechanism. Interestingly, we found that the ARG1, ARG3, ARG4, ARG8, and ARG11 genes in the downstream of N-acetyl-GSA were strongly induced by the exposure to a high temperature (39 1C), whereas the ARG2, ARG5.6, and ARG7 genes were significantly unaffected or slightly changed by this treatment. There was no significant induction of PRO1, PRO2, or PRO3 required for proline synthesis (data not shown). Similar expression profiles were obtained with LD1014 and the put1-disrupted strain (Dput1) L5685Dput1 (data not shown), although the expression of MPR1 or PUT1 was not detected in LD1014 or L5685Dput1, respectively.
To examine whether proline or arginine metabolism is influenced by changes in gene expression in cells, we determined the levels of several amino acids and P5C in L5685, LD1014, and L5685Dput1 cells after exposure to a high temperature (39 1C) (Table 1). Interestingly, the arginine content in L5685 cells was significantly increased with decreasing concentrations of proline, in response to elevated temperature, as compared with cells under the nonstress condition (25 1C). In contrast, LD1014 cells maintained their arginine content, but accumulated higher levels of P5C/GSA and glutamate after exposure to 39 1C than did the nonstressed cells. On the other hand, the arginine content in LD1014ura3 cells harboring pMH1 (LD10141 MPR1) was significantly increased with decreasing concentrations of proline, in response to elevated temperature as in the case of L5685 cells. In L5685Dput1 cells, no change was observed in the arginine content, similar to the findings with LD1014 cells, but the intracellular proline level increased in response to high-temperature stress. These results suggest that S. cerevisiae S1278b cells induce arginine synthesis from proline via the upregulation of MPR1 and PUT1 under oxidative stress conditions.
Increased arginine confers oxidative stress tolerance on S. cerevisiae R1278b cells Mpr1 was found to protect yeast cells by reducing the intracellular ROS levels under oxidative stress conditions (Nomura & Takagi, 2004;Du & Takagi, 2005. To investigate whether increased arginine levels contribute to oxidative stress tolerance, we measured the viability and the ROS levels of L5685, LD1014, and L5685Dput1 cells after exposure to a high temperature (39 1C) (Fig. 5). The viability of L5685 cells was significantly higher than that in LD1014 and L5685Dput1 cells after exposure to 39 1C for 8 h, and L5685 cells showed a 2-to 10-fold increase in the survival rate after 20 h of treatment, as compared with that of LD1014 or L5685Dput1 cells under the same condition (Fig. 5a). As shown in Fig. 5b, crude extracts from yeast cells exhibited a significant increase in fluorescence in response to incubation at 39 1C, suggesting that exposure to a high temperature induces toxicity via the generation of intracellular ROS. Corresponding to the survival rate findings, the oxidation level of L5685 cells after exposure to 39 1C for 14 h declined to 30-40% of that in LD1014 and L5685Dput1 cells under the same condition (Fig. 5b). In addition, the viability and the ROS level of LD1014ura3 cells carrying pMH1 (LD10141MPR1) were higher and lower than that of LD1014 cells, respectively. We then examined the effect of the direct treatment of arginine on stress tolerance in yeast cells (Fig. 6). There was no significant difference in the viability of L5685 and LD10141MPR1 cells in the presence or absence of arginine. It is worth noting that the addition of arginine restored the survival rate of LD1014 and L5685Dput1 cells, the level being close to that in L5685 cells (Fig. 6a). The addition of arginine also restored the ROS level of LD1014 and L5685Dput1 cells (Fig. 6b), whereas there was no significant difference in the ROS level of L5685 and LD10141MPR1 cells in the presence or absence of arginine. These results suggest that Mpr1-dependent arginine synthesis is important for oxidative stress tolerance in S. cerevisiae S1278b cells.

Mpr1 regulates ROS generation by acetylating toxic P5C
We also found that the sensitivity of LD1014 cells to heat stress was much higher than that of L5685Dput1 cells ( Fig.  5a and b). In LD1014 cells, P5C/GSA was accumulated after  Fig. 4. Relative transcription levels of MPR1 and the genes involved in proline and arginine metabolism under a high-temperature stress condition. L5685 (pRS416, pRS414) cells were grown in SD medium at 25 1C to OD 600 nm of 1.0, exposed to 39 1C for 4 h, and then processed for RNA isolation and real-time quantitative PCR analysis. The mRNA level of each gene was normalized to that of ACT1 in the same sample and expressed as an average induction fold in stress-treated cells of 39 1C (shaded bar) vs. untreated cells (white bar). The values are the means and SDs of nine independent experiments. The intracellular contents of amino acids and P5C were determined after exposure to 25 and 39 1C.
The values are the means and SDs of nine independent experiments.
The yeast Mpr1-mediated antioxidative mechanism exposure to a high temperature, as compared with that in L5685 or L5685Dput1 cells (Table 1). The accumulation of P5C/GSA is suggested to induce the formation of ROS, which, by unknown mechanisms, could directly or indirectly trigger oxidative stress (Nomura & Takagi, 2004). To investigate whether Mpr1 detoxifies P5C/GSA, we examined the effect of the direct treatment with P5C/GSA on the cell viability and intracellular ROS level (Fig. 7). Interestingly, the cell viability in LD1014 was significantly decreased after incubation with P5C/GSA, whereas little toxicity of P5C/ GSA was observed in L5685 (Fig. 7a). Corresponding to the data of the survival rates, after incubation with P5C/GSA, a large amount of ROS was detected in LD1014 (Fig. 7b). In addition, the overexpression of Mpr1 complemented an mpr1/2 mutant in terms of the cell viability and the ROS level. These results suggest that Mpr1 detoxify P5C/ GSA by N-acetylation under oxidative stress conditions, in addition to the involvement of a novel arginine synthetic pathway.

Discussion
In this study, we showed that S. cerevisiae S1278b cells induce arginine synthesis via proline oxidation by Put1 and P5C/GSA acetylation by Mpr1 in the mitochondria. In addition, here, we propose that an increased arginine is important for conferring oxidative stress tolerance through an unknown mechanism (Fig. 8). This is the first study to demonstrate Mpr1-mediated arginine synthesis and its physiological role in yeast. How does Arg work for the oxidative stress tolerance? As shown in Fig 6, stress tolerance in the mpr1/2-disrupted strain (Dmpr1/2) LD1014 and the put1-disrupted strain (Dput1) L5685Dput1 increased by arginine treatment, while stress tolerance in the wild-type strain L5685 (MPR1 MPR2) did not change by arginine treatment. This result indicates that arginine accumulation in L5685 is enough for tolerance and that arginine is not directly the antioxidant, because if arginine itself is directly the antioxidant, an increased arginine probably causes more tolerance in L5685. In mammals and plants, it is known that arginine is converted to nitric oxide (NO) by NO synthase (NOSs) and that NO is  considered to be a widespread signaling molecule that confers oxidative stress tolerance by enhancing cellular antioxidative activity (Jobgen et al., 2006;Petrovic et al., 2008;Martin et al., 2009;Tossi et al., 2009). In S. cerevisiae, NO may be involved in stress response pathways, such as under high hydrostatic pressure and copper stress conditions (Shinyashiki et al., 2000;Domitrovic et al., 2003). In addition, it has been suggested that S.cerevisiae cells produce NO by classical NOS-like activity (Kanadia et al., 1998), but little is known about the role of NO due to the lack of DNA sequences that are homologous to human or plant NOSs (Osorio et al., 2007). In fact, we obtained evidence suggesting that NO is produced by an increased arginine via NOSlike activity and contributes to oxidative stress tolerance in yeast cells (unpublished data). Arginine-derived NO might be a key molecule in the antioxidative mechanism. We think that stress-induced arginine synthesis is firstly used for NO production. The arginine remaining is then used for newly protein synthesis, leading to cell growth. Consequently, it might take a long duration of time of over 40 h to start the growth (Fig. 3). Under oxidative stress conditions, our results revealed that proline is oxidized to P5C/GSA catalyzed by Put1, after which P5C/GSA is converted to Mpr1-catalyzed N-acetyl-GSA (Fig. 1). In brief, arginine is synthesized by the following route: proline ! P5C/GSA ! N-acetyl-GSA ! ! ornithine ! ! ! arginine, under an oxidative stress condition. This process clearly involves the upregulation of PUT1 and MPR1 ( Fig. 4 and Table 1). Mpr1 and Put1 are key enzymes for stress-induced arginine synthesis from proline, but the P5C dehydrogenase Put2, which converts P5C/GSA into glutamate, is not such a pivotal enzyme. Under nonstress conditions, arginine is synthesized from glutamate (Herzfeld & Raper, 1976) (Fig. 1). In contrast, stress-induced arginine synthesis may depend on Mpr1 instead of on Put2 in S. cerevisiae S1278b. As shown in Table 1, the intracellular proline level of L5685Dput1 (Dput1) increased in response to high-temperature treatment. However, the genes involved in proline synthesis (PRO1, PRO2, and PRO3) were not induced by exposure to a high temperature. Interestingly, this result suggests that yeast cells could increase the proline level without de novo synthesis. The arginine content in LD1014 cells did not increase in response to incubation at 39 1C, whereas LD1014 cells showed a twofold increase in the glutamate content, probably because P5C/GSA could have been oxidized to glutamate by Put2. However, in L5685 cells, no change in the glutamate content was observed. These results suggest that Mpr1, rather than Put2, preferentially converts P5C/GSA to N-acetyl-GSA under an oxidative stress condition. The increase of the glutamate content in LD1014 cells also suggests that Put2 can oxidize P5C/GSA to glutamate even under an oxidative stress condition. Therefore, we propose that the activity of Mpr1 and Put2 for the substrate P5C/ GSA would be regulated under an oxidative stress condition.
We also have a question as to why LD1014 showed higher sensitivity than that of L5685Dput1 at 39 1C. There is a significant difference in the P5C/GSA content between two strains after exposure to a high temperature (Table 1). LD1014 cells accumulated four times higher P5C/GSA levels (6.06 mmol g À1 DW) than did L5685Dput1 (1.59 mmol g À1 DW). You can see that P5C/GSA is toxic to yeast cells in terms of the viability and the ROS level (Fig. 7). We propose that Mpr1 protects yeast cells from P5C/GSA through N-acetylation. Therefore, two strains (LD1014 and L5685Dput1) are deficient in the stress-induced arginine synthesis, but LD1014 cells are exposed to more severe stress than did L5685Dput1 cells at 39 1C. The next question would be whether or not glutamate is converted into arginine under stress conditions. The first step of arginine synthesis from glutamate is catalyzed by the N-acetyl-glutamate synthase Arg2, but its activity is strongly subject to feedback inhibition by arginine (Alonso & Rubio, 1989;Funck et al., 2008). Therefore, it is possible that yeast cells do not induce arginine synthesis during exposure to stress, even if the glutamate content increases. In S. sereviciae S1278b, Mpr1, not Put2, contributed to the increase in the arginine content via a bypass of the first step of arginine synthesis. Based on the fact that the MPR1 homologue genes are widely present in yeasts and fungi, these lower eukaryotes are expected to possess a unique pathway for arginine biosynthesis under oxidative stress conditions. We found that the ARG1, ARG3, ARG4, ARG8, and ARG11 genes in the downstream of N-acetyl-GSA were strongly induced by the exposure to a high temperature (39 1C) (Fig. 4). Four of the arginine biosynthetic genes (ARG1, ARG3, ARG5.6, and ARG8) are clearly regulated by the ArgR complex repressor (Yoon et al., 2004). The ArgR complex mediates the repression of the specific ARG genes in response to arginine. The upregulation of ArgR-dependent genes suggests that the repression from ArgR would be partly canceled under oxidative stress conditions. Further work is needed to analyze the ArgR regulation under oxidative stress conditions. Interestingly, the mpr1/2-disrupted strain LD1014 was hypersensitive to P5C/GSA (Fig. 7). This suggests that Mpr1 detoxifies P5C/GSA under oxidative stress conditions. Genetic approaches, such as overexpression of proline oxidase and deficiency of P5C dehydrogenase activity, have suggested Arginine Stress tolerance ? Fig. 8. Proposed model for Mpr1-mediated oxidative stress tolerance in Saccharomyces cerevisiae S1278b. Under oxidative stress conditions, the transcription of PUT1 and MPR1 is upregulated to convert proline into arginine via P5C/GSA and N-acetyl-GSA. An increased arginine confers oxidative stress tolerance on yeast cells by a yet unknown mechanism. Additionally, Mpr1 would regulate P5C-mediated ROS generation by acetylating P5C/GSA, although the pathway that P5C/GSA induces ROS production is unknown.
the toxicity of P5C/GSA. Overexpression of proline oxidase induced apoptosis through the generation of ROS in mammalian cells (Donald et al., 2001;Maxwell & Rivera, 2003). The growth of a mutant deficient in P5C dehydrogenase was inhibited by a high concentration of proline through the generation of ROS (Deuschle et al., 2001;Nomura & Takagi, 2004). These effects were probably due to an increase in toxic amounts of P5C/GSA. In fact, the direct treatment with P5C/ GSA induces cell death (Hellmann et al., 2000;Maxwell & Davis, 2000). The mechanism of P5C/GSA's toxicity is poorly understood. The question arises as to how P5C accumulation induces ROS generation. In the proline metabolic pathway, there seems to be a reaction(s), which could produce ROS. For instance, it is probable that ROS is produced through the reaction catalyzed by the mitochondrial inner membrane enzyme proline oxidase, which may require oxygen molecules and cytochrome c, whereas P5C dehydrogenase is believed to use NAD or NADP as a coenzyme. The other possibility is that ROS is spontaneously generated from P5C/ GSA, which is a very unstable compound. For the above purposes, the P5C/GSA-hypersensitive strain LD1014 would be a good model organism.
We showed that Mpr1 is primarily present in the cytosol; moreover, it appears that some Mpr1 proteins are imported into the mitochondria from the cytosol (Fig. 2). The abnormal subcellular localization and inability for AZC detoxification of EGFP-Mpr1 suggested that the N-terminal region of Mpr1 plays important roles in the translocation into the mitochondria and in the catalytic function. In fact, the recombinant EGFP-Mpr1 showed little AZC acetyltransferase activity (data not shown). Because Mpr1 has no typical mitochondrial targeting signal and is not fully localized into the mitochondria, Mpr1 may be imported into the mitochondria by an unknown translocation system. It is considered that Mpr1 and Mpr2 play similar roles in both AZC acetylation and antioxidative activities, because there is only one amino acid change at position 85 between two enzymes (Takagi et al., 2000;Sasano et al., 2010).
In yeast strains lacking MPR1/2 such as S. cerevisiae S288C, the ornithine aminotransferase Car2 is considered to be an enzyme involved in arginine synthesis under stress conditions. Real-time quantitative PCR analysis suggested that CAR2 from the S288C background strain is induced in response to oxidative stresses, but the CAR2 of S1278b is not fully expressed (unpublished data). It will still be necessary to analyze yeast strains with and without MPR1/2 in order to investigate the novel antioxidative mechanism reported here.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Fig. S1. Localization of the EGFP-fused Mpr1 variants. Table S1. Primers used in this study.
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