RESEARCH ARTICLE
Comprehensive analysis of genes involved in the oxidative stress tolerance using yeast heterozygous deletion collection Natsumi Okada1, Jun Ogawa2 & Jun Shima1 1
Research Division of Microbial Sciences, Kyoto University, Kyoto, Japan; and 2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Correspondence: Jun Shima, Research Division of Microbial Sciences, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto, 606-8502, Japan. Tel.: +81 75 753 9545; fax: +81 75 753 9544; e-mail: [email protected] Received 25 September 2013; revised 19 December 2013; accepted 1 January 2014. Final version published online 03 February 2014. DOI: 10.1111/1567-1364.12136 Editor: Hyun Ah Kang
YEAST RESEARCH
Keywords Saccharomyces cerevisiae; oxidative stress tolerance; essential genes.
Abstract In Saccharomyces cerevisiae, oxidative stress plays an inhibitory role during industrial fermentation. Although previous reports have identified genes required for oxidative stress tolerance, employing the yeast genome-wide screening, these screenings used a homozygous mutant collection which did not include the essential genes whose deletions result in lethality. Here, we report a truly genome-wide screening for the genes required for oxidative stress tolerance, using a heterozygous mutant collection which includes both essential and nonessential genes. Approximately 6300 heterozygous deletion mutants were grown in the presence or absence of H2O2. The screening identified a total of 331 genes whose heterozygotes conferred hypersensitivity to H2O2, indicating that these genes are required for oxidative stress tolerance. Notably, among these genes, 71 were essential genes. We classified these 71 essential genes based on localization, indicating that the localization of gene products from these essential genes was enriched in the nucleus and nucleolus. Classification of these essential genes based on functional categorizations showed that rRNA synthesis and tRNA synthesis were over-represented, suggesting that nuclear function, such as RNA synthesis, is important in the response to oxidative stress. These results provide a helpful resource for the understanding of the molecular basis of oxidative stress-tolerant mechanisms.
Introduction Yeasts, such as Saccharomyces cerevisiae, are exposed to various severe environmental stresses during the industrial processes (Attfield, 1997; Shima & Takagi, 2009). These include freeze–thaw, high sugar concentrations, air drying, high osmotic pressure, and alcohol. Such stresses induce oxidative stress due to the decline in intracellular pH and excessive generation of reactive oxygen species (ROS), leading to cell damage and low fermentation ability (Attfield, 1997). Saccharomyces cerevisiae possesses several response systems to oxidative stress. Transcriptional factors such as Yap1, Msn2/4, and Skn7 are important in the response to oxidative stress (Attfield, 1997; Jamieson, 1998; Herrero et al., 2008; Morano et al., 2012). These factors are involved in the activation or expression of specific antiox-
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idants and thereby detoxification of the excess intracellular ROS and normalization of the internal pH. Alternatively, S. cerevisiae cells possess several groups of enzymes that act directly as ROS detoxifiers: Superoxide dismutases (SODs) play a role in the detoxification of superoxide anions; glutathione peroxidases (GPXs), catalases (Cta1 and Ctt1), and thioredoxin peroxidases (TSAs) reduce hydrogen peroxide (H2O2); and thioredoxins (TRXs) and thioredoxin reductases (TRRs) play a role in thiol reduction. In addition, loss of the proton-transporting activities of V-ATPases, which are responsible for maintaining cytosolic pH and ion homeostasis, is known to dampen the tolerance of yeast cells to various stresses including oxidative stress, suggesting that V-ATPases play a role in stress tolerance (Thorpe et al., 2004; Ando et al., 2007; Milgrom et al., 2007; Shima et al., 2008; Shima & Takagi, 2009). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Due to the fact that oxidative stress has a negative impact under various stress conditions, a comprehensive understanding of the mechanism of oxidative stress tolerance would provide useful insights for the further development of molecular breeding of industrial yeasts. Yeast deletion collections are a valuable resource for examining loss-of-function phenotypes of yeast genes (Giaever et al., 2002), which enables a genome-wide screening to clarify the relevance of the genes in oxidative stress tolerance (Higgins et al., 2002; Thorpe et al., 2004; Ando et al., 2007; Shima et al., 2008). However, previous genomewide screenings for stress response genes have used either haploid or homozygous deletion collections (Birrell et al., 2001; Warringer et al., 2003; Thorpe et al., 2004; Ando et al., 2006, 2007; Shima et al., 2008) and thereby have been limited to nonessential genes due to the lethality resulting from the deletion of essential genes. Therefore, although essential genes occupy about 20% of all genes, the relevance of essential genes to oxidative stress tolerance remains obscure. Alternatively, a heterozygous deletion collection of diploid yeast, developed by European Saccharomyces cerevisiae archive for functional analysis (EUROSCARF), is available (Giaever et al., 1999; Hillenmeyer et al., 2008). As the majority of heterozygotes show no obvious growth defects, the heterozygous deletion collection is a promising tool to enable the genome-wide screening which includes essential genes. In this study, with a view to a more comprehensive understanding of oxidative stress tolerance, we screened for the essential genes required for oxidative stress tolerance using a heterozygous deletion mutant collection. Our genome-wide screening verified the relevance of essential genes in oxidative stress tolerance. Furthermore, the classification of the identified essential genes by function and localization suggested that nuclear functions, such as RNA synthesis, were implied in a critical role for the oxidative stress response.
Materials and methods Yeast strains and growth conditions
Saccharomyces cerevisiae BY4743 (MATa/a his3D1/his3D1 leu2D0/leu2D0 lys2D0/LYS2 MET15/met15D0 ura3D0/ura3 D0) and a heterozygous collection of diploid deletion strains (MATa/a) constructed using the insertion of kanMX4 cassettes (geneticin resistance) as selective markers for the genome of BY4743 (Hillenmeyer et al., 2008) were obtained from EUROSCARF. Each strain was maintained in liquid YPD medium (yeast extract, 1%; peptone, 2%; glucose, 2%) supplemented with 200 mg mL 1 of geneticin (Sigma–Aldrich, St. Louis, MO).
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Identification of mutant strains hypersensitive to H2O2 stress
Sensitivity to oxidative stress was assessed using H2O2. The wild-type strain (BY4743) and deletion mutants were grown in 100 lL of YPD medium without geneticin in 96-well microtiter plates (VIOLAMO, Osaka, Japan) using a 48-pin replicator (Funakoshi, Tokyo, Japan) and then precultured at 30 °C for 2 days without shaking. Portions (c. 1 lL) of the preculture were inoculated into 100 lL of YPD and YPD containing 3 mM H2O2 media using a 48-pin replicator. After incubation at 30 °C for 17 h, the optical density of the culture at 600 nm (OD600 nm) was measured using a microtiter-plate reader (SpectraMax 250; Molecular Devices, San Diego, CA). Four parameters were used in the data analysis of the sensitivity of the deletion mutants to H2O2 stress: NM, defined as the OD600 nm of mutant strains in YPD medium; SM, defined as the OD600 nm of mutant strains in YPD medium with 3 mM H2O2; NW, defined as the OD600 nm of BY4743 (wild-type) strain in YPD medium; and SW, defined as the OD600 nm of BY4743 strain in YPD medium with 3 mM H2O2. Mutants that showed considerable growth inhibition in YPD medium (i.e. NM/NW < 0.1) were removed from further data analysis. The value of (SM/ SW)/(NM/NW) was used as the parameter for sensitivity to H2O2. In this calculation, NM/NW values were used for compensation of growth defects under nonstress conditions. Measurements for the identification of H2O2-sensitive mutant strains were performed in triplicate, and the average of the values was used for the evaluation of sensitivity. The statistical analysis was also performed using the two-tailed Student’s t-test. Mutants that exhibited a sensitivity value below 0.5 and a P-value < 0.05 were defined as sensitive to H2O2. Time-course growth measurements of the H2O2-sensitive mutants
The H2O2-sensitive mutants were cultured in YPD or YPD containing 3 mM H2O2 media, and the OD600nm was measured every hour after incubation at 30 °C for 0–20 h using a microtiter-plate reader. Data are presented as the means for triplicate experiments. Classification of genes based on gene function and subcellular localization of gene products
Genes involved in H2O2 tolerance were categorized using the Munich Information Center for Protein Sequences (MIPS; http://mips.helmholtz-muenchen.de/genre/proj/ yeast/) databases (Mewes et al., 2004) and Gene Ontology
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(GO) terms presented by the Saccharomyces Genome Database (SGD) via FunSpec (http://funspec.med.utoronto.ca/), with the P-value threshold set at 0.01. The P values calculated by FunSpec could be a good statistical indicator of the frequencies (Robinson et al., 2002).
Results Screening of genes required for tolerance to H2O2
To assess the sensitivity to oxidative stress, we used H2O2, a member of the ROS. First, to determine suitable measurement conditions for the evaluation of H2O2 sensitivity, we measured the growth of the wild-type BY4743 strain in YPD media containing various concentrations of H2O2 (0–5 mM). After 17-h incubation at 30 °C, the growth of the wild-type BY4743 strain was inhibited by c. 50% at an H2O2 concentration of 3 mM (Fig. 1). We also determined the growth of a trx2 heterozygous deletion mutant (TRX2/trx2D). TRX2 encodes thioredoxin II, and a trx2 deletion mutant (trx2D) has been reported to be hypersensitive to oxidative stress (Kuge & Jones, 1994). Growth of the TRX2/trx2D strain was severely impaired in the presence of 3 mM H2O2, indicating that the TRX2/ trx2D heterozygote was also hypersensitive to oxidative stress despite the heterozygous deletion of TRX2 (Fig. 1). Based on these results, we used YPD medium containing 3 mM H2O2 for further analysis, and the growth of the strains was measured after incubation for 17 h at 30 °C to screen for H2O2-sensitive mutant strains. Next, we used the heterozygous deletion mutant collection to identify H2O2-sensitive mutants. Approximately 6300 heterozygote strains derived from BY4743 were 1.0
: WT-YPD+0 mM : WT-YPD+3 mM : TRX2/trx2Δ-YPD+0 mM
Classification of 331 genes based on the MIPS database
Next, to understand the biological functions important for H2O2 tolerance, the 331 genes required for H2O2 tolerance were classified using the MIPS database via FunSpec. Classification of the genes based on the functional and
Frequency (number of mutants)
1000
: TRX2/trx2Δ-YPD+3 mM
Growth (OD600)
cultivated in the presence or absence of 3 mM H2O2, and their growth was observed. To evaluate the sensitivity to H2O2 quantitatively, we used the (SM/SW)/(NM/NW) value (see Materials and methods). Figure 2 shows the distribution of the (SM/SW)/(NM/NW) values of the deletion mutants in a frequency distribution plot, providing an overview of the effects of heterozygous gene deletions on H2O2 sensitivity. Among the c. 6300 heterozygous deletion mutant strains tested, 331 strains (c. 5.3% of all strains) showed H2O2 tolerance < 50% of that of the wild-type BY4743 strain. Therefore, we defined these 331 strains as hypersensitive to H2O2 stress, and the 331 genes whose heterozygous deletions conferred hypersensitivity to H2O2 were considered to be required for H2O2 tolerance. We focused on these 331 genes, required for H2O2 tolerance, for the subsequent analysis. Among the 331 genes, the essential and nonessential genes were 71 (c. 6.4% of all the essential genes) and 260 (c. 4.7% of all nonessential genes), respectively (Supporting Information, Table S1). In addition, we monitored and assessed the growth of approximately a third of the H2O2-sensitive mutants, 119 strains, at 0–20 h (Fig. S1). The growth of all mutants was inhibited in the presence of H2O2, supporting the validity of our screening analysis.
0.5
5
10
0.5 1.0 1.5 2.0 H2O2 sensitivity value (SM/SW)/(NM/NW)
15 (h)
Fig. 1. The effect of H2O2 on the growth of the wild-type and TRX2/ trx2D strains. Each strain was grown in YPD medium in the presence or absence of 3 mM H2O2, and the growth was monitored by measurement of the OD600 nm.
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Non-essential genes
500
0 0.0
0
Essential genes
Fig. 2. Frequency distribution of H2O2 sensitivity in the heterozygous mutant collection. H2O2 sensitivity is expressed as the value of (SM/ SW)/(NM/NW). The heterozygous mutants with values of (SM/SW)/ (NM/NW) which were < 0.5 were defined as H2O2-hypersensitive strains.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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(a)
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Ribosomal proteins All genes Genes required for H2O2 tolerance
Transport ATPases Oxidative stress response 0
(b)
5
(%)
Nucleus All genes Genes required for H2O2 tolerance
Vacuole Cytoplasm 0
10
20
30
40
50
(%)
Fig. 3. MIPS classification of the total genes required for H2O2 tolerance. The 331 essential and nonessential genes whose heterozygous deletions conferred H2O2-hypersensitivity are shown. All Saccharomyces cerevisiae genes were classified based on function (a) and subcellular localization (b), using the MIPS database.
subcellular localization categories is shown in Fig. 3, indicating that the genes required for H2O2 sensitivity were frequently included in the ‘ribosomal proteins’, ‘transport ATPases’ (Fig. 3a), ‘nucleus’, and ‘vacuole’ classes (Fig. 3b). Notably, the transport ATPase category included VMA3, VMA8, and VMA13, which encode the components of V-ATPase and have been previously reported to contribute to oxidative stress tolerance (Thorpe et al., 2004; Ando et al., 2007; Milgrom et al., 2007; Shima et al., 2008). Furthermore, using subcellular localization, the gene products were significantly assigned into the vacuole where V-ATPases are localized. Thus, these results were consistent with previous reports including genome-wide screening using a homozygous mutant collection (Thorpe et al., 2004; Ando et al., 2007; Shima et al., 2008), supporting the validity of our screening using heterozygous mutants. Classification of essential genes required for H2O2 stress tolerance by the MIPS database and GO terms
Next, we focused on the 71 essential genes required for H2O2 tolerance (listed in Table 1). To identify significantly (P < 0.01) enriched functions, cellular processes and subcellular localization, the 71 essential genes were also analyzed using the MIPS database via FunSpec (Fig. 4 and Table 2). Functional classification using the MIPS database revealed that the essential genes required for H2O2 tolerance were frequently included in the ‘rRNA processing’, ‘DNA binding’, ‘rRNA synthesis’, and ‘tRNA synthesis’ classes (Fig. 4a). Classification of the subcellular localization showed that a high number of gene products from essential genes required for H2O2 tolerance were located in the nucleus and nucleolus (Fig. 4b), which were related to the results of the functional classification that significantly included the nuclear functions (Fig. 4a). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Next, we also classified the 71 essential genes required for H2O2 tolerance based on the molecular functional, biological process and cellular component categories, using GO terms presented by the SGD (Fig. 5 and Table 2). Using the molecular functional categorization employing GO terms, the ‘RNA binding’, ‘DNA-directed RNA polymerase activity’, and ‘RNA-directed RNA polymerase activity’ classes were over-represented in the essential genes required for H2O2 tolerance (Fig. 5a). In addition, classification of the GO cellular component revealed that a high number of gene products from essential genes required for H2O2 tolerance were located in the nucleus and nucleolus (Fig. 5c). Based on biological processes, the 71 essential genes were predominantly categorized into rRNA processing, transcription, and translation processes (Fig. 5b), suggesting that RNA and protein synthesis, processed in the nucleus, might be related to H2O2 stress tolerance. Taken together, the classifications using GO terms were closely correlated with the classifications using the MIPS database, and both classifications suggested that the essential gene products which are localized in the nucleus or nucleolus and involved in RNA synthesis are critical in the response against oxidative stress.
Discussion So far, there have been several reports which have identified the genes required for oxidative stress tolerance by means of the genome-wide screening of yeast (Thorpe et al., 2004; Ando et al., 2007; Shima et al., 2008). However, these screenings relied on the null mutant collection and excluded the essential genes whose deletions result in lethality. In this study, we applied a heterozygous mutant collection to genome-wide screening for genes required for oxidative stress tolerance; this was the first use of a heterozygous mutant collection for this purpose. This approach identified a total of 331 genes required for the oxidative stress response, notably including 71 essential genes, under the same experimental conditions. Saccharomyces cerevisiae possesses antioxidant systems such as transcriptional activators and ROS-scavenging enzymes. The transcriptional activators, Yap1, Skn7, and Msn2/4, are well known to be involved in the response to oxidative stress in yeast. Indeed, our results also showed that the heterozygous deletions of YAP1 and SKN7 affected sensitivity to H2O2. MSN2/msn2D and MSN4/ msn4D strains did not exhibit hypersensitivity to H2O2, which agrees with previous reports that no obvious effect of the single null deletions of Msn2 and Msn4 was observed (Estruch & Carlson, 1993; Estruch, 2000). It is also known that the cytosolic thioredoxin system of S. cerevisiae is required as defense against externally added H2O2 (Attfield, 1997; Garrido & Grant, 2002; FEMS Yeast Res 14 (2014) 425–434
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Table 1. The essential genes required for H2O2 tolerance [the value of (SM/SW)/(NM/NW) < 0.5 and P-value < 0.05] Systematic name
Standard name
H2O2 sensitivity
P-value
YPL143W YPL235W YLR293C YDL193W YBL014C
RPL33A RVB2 GSP1 NUS1 RRN6
0.065 0.092 0.126 0.133 0.145
1.10E 5.79E 4.41E 1.01E 9.00E
13 09 07 05 07
YPR181C YDR353W YEL026W YDR404C YPR187W YDL043C YMR013C YEL035C YIL046W YDR145W YFL005W
SEC23 TRR1 SNU13 RPB7 RPO26 PRP11 SEC59 UTR5 MET30 TAF12 SEC4
0.160 0.178 0.227 0.235 0.242 0.256 0.261 0.302 0.320 0.325 0.327
1.51E 8.02E 4.72E 5.63E 8.11E 8.35E 1.08E 3.74E 4.50E 8.85E 1.56E
07 07 08 07 07 08 05 08 08 04 08
YGL030W YDR212W YDR362C
RPL30 TCP1 TFC6
0.330 0.344 0.345
5.59E 07 5.69E 06 6.23E 05
YMR309C YGL068W YGR216C
NIP1 MNP1 GPI1
0.346 0.351 0.352
1.03E 04 6.17E 05 8.50E 05
YPL131W YOL040C YDR373W
RPL5 RPS15 FRQ1
0.356 0.361 0.371
3.99E 04 4.28E 05 4.01E 03
YOR151C YPR086W YOR149C YPL124W YOR103C
RPB2 SUA7 SMP3 SPC29 OST2
0.378 0.381 0.381 0.385 0.385
1.42E 4.57E 6.34E 5.97E 1.23E
YKL189W YJL005W YKL165C
HYM1 CYR1 MCD4
0.387 0.389 0.391
7.29E 03 1.85E 04 9.82E 03
YDR365C YPR104C YHR128W YDR361C YLR163C YIL048W YOR335C YDR376W YDR064W YGR186W YGL097W YDR299W YDR390C
ESF1 FHL1 FUR1 BCP1 MAS1 NEO1 ALA1 ARH1 RPS13 TFG1 SRM1 BFR2 UBA2
0.398 0.405 0.414 0.417 0.418 0.423 0.424 0.428 0.429 0.431 0.437 0.440 0.449
1.12E 9.81E 1.60E 7.55E 4.15E 4.21E 2.46E 2.34E 7.93E 2.33E 3.70E 9.89E 1.65E
YGR113W
DAM1
0.449
7.55E 04
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Description
05 06 04 06 06
07 10 07 05 03 06 09 03 06 06 07 06 09
Ribosomal 60S subunit protein L33A ATP-dependent DNA helicase RNA processing and transport Putative prenyltransferase Component of the core factor (CF) rDNA transcription factor complex GTPase-activating protein, involved in ER to Golgi transport Cytoplasmic thioredoxin reductase RNA-binding protein RNA polymerase II subunit B16 RNA polymerase subunit ABC23 Subunit of the SF3a splicing factor complex Dolichol kinase Protein of unknown function F-box protein containing five copies of the WD40 motif Subunit (61/68 kDa) of TFIID and SAGA complexes Rab family GTPase essential for vesicle-mediated exocytic secretion and autophagy Ribosomal 60S subunit protein L30 Alpha subunit of chaperonin-containing T-complex One of six subunits of RNA polymerase III transcription initiation factor complex (TFIIIC) eIF3c subunit of the eukaryotic translation initiation factor 3 (eIF3) Protein associated with the mitochondrial nucleoid Membrane protein involved in the synthesis of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI) Ribosomal 60S subunit protein L5 Protein component of the small (40S) ribosomal subunit N-Myristoylated calcium-binding protein that may have a role in intracellular signaling through its regulation of the phosphatidylinositol 4-kinase Pik1p RNA polymerase II second largest subunit B150 Transcription factor TFIIB Alpha 1,2-mannosyltransferase Inner plaque spindle pole body (SPB) component Epsilon subunit of the oligosaccharyltransferase complex of the ER lumen Component of the RAM signaling network Adenylate cyclase Protein involved in glycosylphosphatidylinositol (GPI) anchor synthesis Nucleolar protein involved in pre-rRNA processing Regulator of ribosomal protein (RP) transcription Uracil phosphoribosyltransferase, synthesizes UMP from uracil Essential protein involved in nuclear export of Mss4p Smaller subunit of the mitochondrial processing protease (MPP) Putative aminophospholipid translocase (flippase) Cytoplasmic and mitochondrial alanyl-tRNA synthetase Oxidoreductase of the mitochondrial inner membrane Protein component of the small (40S) ribosomal subunit TFIIF (transcription factor II) largest subunit Nucleotide-exchange factor for Gsp1p Essential protein that is a component of 90S preribosomes Subunit of a heterodimeric nuclear SUMO-activating enzyme (E1) with Aos1p Essential subunit of the Dam1 complex (aka DASH complex)
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Table 1. Continued Systematic name
Standard name
H2O2 sensitivity
P-value
Description
YJL010C YNL103W YOR319W
NOP9 MET4 HSH49
0.449 0.450 0.452
6.37E 05 9.72E 11 3.29E 07
YOR116C YHR069C YNL313C YDL031W
RPO31 RRP4 EMW1 DBP10
0.461 0.462 0.465 0.466
9.43E 3.37E 8.66E 7.81E
04 06 07 04
YOL127W YKL154W YDR416W YDR288W YNL240C
RPL25 SRP102 SYF1 NSE3 NAR1
0.467 0.468 0.469 0.470 0.473
4.81E 1.90E 1.24E 1.07E 3.73E
04 02 06 04 03
YOR340C YMR296C YDR341C YNL312W YDL058W
RPA43 LCB1 RRS1 RFA2 USO1
0.474 0.476 0.478 0.481 0.483
4.65E 7.16E 4.91E 1.69E 1.79E
07 07 05 05 05
YDR182W YFR002W YLR009W YDR498C
CDC1 NIC96 RLP24 SEC20
0.484 0.488 0.491 0.494
8.10E 8.51E 2.35E 1.08E
06 08 11 05
YGL044C YDR339C
RNA15 FCF1
0.496 0.499
1.39E 02 2.60E 06
YPL128C
TBF1
0.499
2.32E 06
Essential subunit of U3-containing 90S preribosome Leucine-zipper transcriptional activator U2-snRNP-associated splicing factor with similarity to the mammalian splicing factor SAP49 RNA polymerase III largest subunit C160 Exosome noncatalytic core component Essential conserved protein with a role in cell wall integrity Putative ATP-dependent RNA helicase of the DEAD-box protein family Ribosomal 60S subunit protein L25 Signal recognition particle (SRP) receptor beta subunit Member of the NineTeen Complex (NTC) Component of the SMC5-SMC6 complex Component of the cytosolic iron-sulfur (FeS) protein assembly machinery RNA polymerase I subunit A43 Component of serine palmitoyltransferase Arginyl-tRNA synthetase Subunit of heterotrimeric replication protein A (RPA) Essential protein involved in the vesicle-mediated ER to Golgi transport step of secretion Putative lipid phosphatase of the endoplasmic reticulum Linker nucleoporin component of the nuclear pore complex (NPC) Essential protein with similarity to Rpl24Ap and Rpl24Bp Membrane glycoprotein v-SNARE involved in retrograde transport from the Golgi to the ER Component of the cleavage and polyadenylation factor I (CF I) Putative PINc domain nuclease required for early cleavages of 35S pre-rRNA and maturation of 18S rRNA gene Telobox containing general regulatory factor
Herrero et al., 2008). Consistent with this knowledge, the heterozygous deletions of TSA1, TRX2, and TRR1, which encode the cytosolic thioredoxin system, conferred hypersensitivity to H2O2. In contrast, although TSA2, TRX1, and TRR2 also encode thioredoxin systems, the heterozygous mutants of these genes were not hypersensitive to H2O2 as the value of (SM/SW)/(NM/NW) was more than 0.5. In addition, neither catalases (Ctt1 and Cta1) nor superoxide dismutases (Sod1 and Sod2) were required for H2O2 resistance under our experimental conditions. However, a previous study by Shima and colleagues using homozygous null mutants showed that cta1D, sod1D, sod2D, tsa2D, trx1D, trr2D, gpx1D, gpx2D, grx1D, and grx3D were not sensitive to oxidative stress (Shima et al., 2008), which is similar to our results. The H2O2 sensitivity values of heterozygous mutants for genes of the ROS-scavenging systems are presented in Table 3. Although it should be noted that our genome-wide screening relied on a heterozygous mutant collection, and therefore included the effects of the intact allele of a gene, our results clearly demonstrate that a heterozygous ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
(a)
rRNA processing DNA binding rRNA synthesis tRNA synthesis RNA binding Ribosomal proteins
General transcription activities Cellular signalling All genes Essential genes required for H2O2 tolerance
Modification with fatty acids Phospholipid metabolism 0
(b)
5
10
(%)
Nucleus All genes Essential genes required for H2O2 tolerance
Nucleolus 0
10
20
30
40
50 (%)
Fig. 4. MIPS classification of the essential genes required for H2O2 tolerance. The 71 essential genes whose heterozygous deletions conferred H2O2-hypersensitivity are shown. All Saccharomyces cerevisiae essential genes were classified based on function (a) and subcellular localization (b), using the MIPS database.
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Table 2. Functional and localization categories of the essential genes required for H2O2 tolerance at high frequencies Category MIPS functional classification rRNA processing DNA binding rRNA synthesis tRNA synthesis RNA binding Ribosomal proteins General transcription activities Cellular signaling Modification with fatty acids Phospholipid metabolism MIPS subcellular localization Nucleus
P-value 1.11E 2.66E 2.86E 6.88E 3.99E 4.63E 4.64E 5.16E 5.16E 5.96E
In category from cluster 05 04 04 04 03 03 03 03 03 03
4.44E 04
Nucleolus GO molecular function RNA binding
1.64E 03
DNA-directed RNA polymerase activity RNA-directed RNA polymerase activity Ribonucleoside binding Structural constituent of ribosome General RNA polymerase II transcription factor activity GO biological process rRNA processing Transcription, DNA-dependent
2.72E 2.45E 6.75E 2.20E 9.48E
Translation Ribosome biogenesis Transcription initiation from RNA polymerase II promoter GPI anchor biosynthetic process Transcription initiation, DNA-dependent Response to cadmium ion Response to arsenic-containing substance Cysteine biosynthetic process Ribosomal large subunit assembly Transcription from RNA polymerase II promoter GO cellular component Nucleus
1.05E 05 05 04 04 03 03
2.17E 04 4.98E 04 5.20E 04 2.20E 2.59E 3.56E 3.91E 3.91E 5.89E 7.02E 7.68E 8.02E
03 03 03 03 03 03 03 03 03
2.23E 05
Nucleolus
7.00E 05
DNA-directed RNA polymerase II, core complex U2-type prespliceosome Endoplasmic reticulum membrane ER to Golgi transport vesicle membrane Intracellular Golgi membrane U2 snRNP DNA-directed RNA polymerase I complex
2.45E 3.22E 6.64E 7.02E 7.13E 8.24E 9.54E 9.54E
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04 03 03 03 03 03 03 03
DBP10 ESF1 SNU13 RPL30 SRM1 RRP4 NOP9 GSP1 RVB2 FHL1 NSE3 RPB7 RFA2 RPO31 RPB2 RPA43 TBF1 RPO26 RRN6 TFC6 RPO31 RPA43 RPO26 TFC6 RPO31 FHL1 RPO26 PRP11 ESF1 RNA15 NOP9 RPL25 HSH49 RPL5 DBP10 RPS13 RPL30 MNP1 RPS15 RPL25 RPL5 RPL33A RRN6 TFC6 RPB7 MET4 RPB2 RPO26 FRQ1 SRM1 GPI1 GPI1 MCD4 SMP3 GPI1 MCD4 SEC59 SMP3 RRN6 PRP11 TAF12 NSE3 FCF1 TRR1 BCP1 TFC6 ESF1 UBA2 RPB7 SYF1 SNU13 NIC96 RNA15 SRM1 DAM1 TFG1 RRP4 MET30 NOP9 HYM1 GSP1 MET4 RFA2 EMW1 RPO31 RPB2 HSH49 RPA43 SPC29 TBF1 SUA7 FHL1 RPO26 RRN6 DBP10 BFR2 ESF1 SNU13 RRP4 NOP9 RPA43 DBP10 PRP11 ESF1 RPB7 SNU13 RPL30 RNA15 RRP4 NOP9 RPS15 RPL25 HSH49 ALA1 RPL5 RPB7 RPO31 RPB2 RPA43 RPO26 RPB7 RPB2 RPO26 RPO31 RPB2 RPS13 RPL30 MNP1 RLP24 RPS15 RPL25 RPL5 RPL33A TAF12 TFG1 SUA7 DBP10 BFR2 FCF1 ESF1 SNU13 RPL30 RRP4 NOP9 RVB2 RRN6 TAF12 TFC6 RPB7 TFG1 MET30 MET4 RPO31 RPB2 RPA43 TBF1 RVB2 SUA7 FHL1 RPO26 RPS13 YDR341C RPL30 MNP1 RLP24 NIP1 RPS15 RPL25 ALA1 RPL5 RPL33A DBP10 FCF1 ESF1 SNU13 NOP9 RLP24 RPS15 RPB7 TFG1 SUA7 GPI1 MCD4 SMP3 TAF12 SUA7 MET30 MET4 MET30 MET4 MET30 MET4 DBP10 RPL25 RPL5 TAF12 RPB7 RPB2 RPO26 RRN6 DBP10 PRP11 NUS1 TAF12 NSE3 BFR2 FCF1 BCP1 TFC6 ESF1 UBA2 RPB7 SYF1 SNU13 NIC96 RNA15 SRM1 DAM1 TFG1 RRP4 MET30 NOP9 RLP24 GSP1 MET4 NAR1 RFA2 EMW1 RPO31 RPB2 HSH49 RPA43 TBF1 RVB2 SUA7 FHL1 RPO26 RRN6 DBP10 BFR2 FCF1 ESF1 SNU13 RRP4 NOP9 RLP24 RPA43 FHL1 RPB7 RPB2 RPO26 PRP11 SYF1 HSH49 USO1 NUS1 SEC20 SRP102 MCD4 SEC59 LCB1 SMP3 SEC23 USO1 SEC23 PRP11 RPS13 SYF1 RPL30 MNP1 FUR1 HYM1 RPL25 RPL5 RPL33A USO1 FRQ1 NEO1 MCD4 SEC23 PRP11 HSH49 RPA43 RPO26
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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(a)
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RNA binding DNA-directed RNA polymerase activity All genes Essential genes required for H2O2 tolerance
RNA-directed RNA polymerase activity Ribonucleoside binding Structural constituent of ribosome General RNA polymerase II transcription factor activity
(b)
0
10
5
15
(%)
rRNA processing Transcription, DNA-dependent Translation Ribosome biogenesis
Transcription initiation from RNA polymerase II promoter GPI anchor biosynthetic process Transcription initiation, DNA-dependent Response to cadmium ion Response to arsenic-containing substance All genes Essential genes required for H2O2 tolerance
Cysteine biosynthetic process Ribosomal large subunit assembly Transcription from RNA polymerase II promoter
0
(c)
5
10
15
20 (%)
Nucleus Nucleolus DNA-directed RNA polymerase II, core complex U2-type prespliceosome Endoplasmic reticulum membrane ER to Golgi transport vesicle membrane Intracellular Golgi membrane
All genes Essential genes required for H2O2 tolerance
U2 snRNP DNA-directed RNA polymerase I complex
0
10
20
mutant collection is available for the genome-wide analysis for oxidative stress tolerance. Recently, an increasing number of studies have shown that V-ATPase activity is responsible for oxidative stress tolerance (Attfield, 1997; Thorpe et al., 2004; Ando et al., 2007; Milgrom et al., 2007; Shima et al., 2008; Shima & Takagi, 2009). Our results also show that heterozygous deletions of the genes encoding the components of V-ATPase, such as VMA3, VMA8, and VMA13, resulted in hypersensitivity to H2O2. Thus, our study supports the emerging role of V-ATPase in oxidative stress tolerance and the potency of V-ATPase activity for the breeding of yeasts with high fermentation ability (Hasegawa et al., 2012). The identified 71 essential genes correspond to 6.4% of the total essential genes. The ratio of essential genes required for oxidative stress tolerance to the total essential genes is higher than that of the nonessential genes required for oxidative stress tolerance (260 genes: 4.7% of total nonessential genes), implying a substantial role of essential genes in the response to oxidative stress. Thus, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
30
40
50 (%)
Fig. 5. GO classification of the essential genes required for H2O2 tolerance. The 71 essential genes required for H2O2 tolerance are shown. All Saccharomyces cerevisiae essential genes were classified based on molecular function (a), biological process (b), and cellular component (c), using GO terms.
Table 3. H2O2 sensitivity of heterozygous mutants with deletion of the genes involved in ROS-scavenging systems Mutant
H2O2 sensitivity value
Product of deleted gene
cta1 ctt1 sod1 sod2 trr1 trr2 trx1 trx2 tsa1 tsa2 gpx1 gpx2 gpx3 grx1 grx2 grx3 glr1 yap1 skn7 msn2 msn4
0.58 0.65 0.87 0.61 0.18 0.98 1.14 0.08 0.26 1.08 0.56 0.65 0.84 1.33 0.88 1.06 0.94 0.40 0.39 0.78 0.68
Catalase A Catalase T Superoxide dismutase Superoxide dismutase Thioredoxin reductase Thioredoxin reductase Thioredoxin Thioredoxin Thioredoxin peroxidase Thioredoxin peroxidase Glutathione peroxidase Glutathione peroxidase Glutathione peroxidase Glutaredoxin Glutaredoxin Glutaredoxin Glutathione oxidoreductase Transcriptional factor Transcriptional factor Transcriptional factor Transcriptional factor
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Impacts of oxidative stress on essential genes in yeast
our results provide the first evidence for the relevance of essential genes in oxidative stress tolerance. To deduce the cellular functions in which the essential genes required for oxidative stress tolerance are involved, we examined the distribution of the 71 genes in the functional categories based on MIPS and GO (Table 2, Figs 4 and 5). The functional categories such as rRNA synthesis and tRNA synthesis were over-represented in the essential genes required for oxidative stress tolerance. Using the classification based on localization, the localization of gene products from the essential genes required for oxidative stress tolerance was enriched in the nucleus and nucleolus. This is reasonable as tRNA and rRNA are synthesized in the nucleus and nucleolus, respectively. rRNA and tRNA are integral factors for protein synthesis. Therefore, our results suggest that tRNA and rRNA are important for oxidative stress tolerance, implying that protein synthesis is a key process in the response to oxidative stress. It is possible that a protein synthesis defect results in an insufficient synthesis of antioxidants, which precludes cells responding to stress efficiently. Thus, our results shed light on the role of essential genes in oxidative stress tolerance and represent a helpful resource for the comprehensive understanding of the molecular basis of oxidative stress-tolerant mechanisms.
Acknowledgements This work was supported financially by the Institute for Fermentation, Osaka (IFO). We thank Dr. Akira Ando (National Agriculture and Food Research Organization) for his helpful discussions. We also thank Dr. S. Matsuda for technical advice and enormously constructive comments.
References Ando A, Tanaka F, Murata Y, Takagi H & Shima J (2006) Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae. FEMS Yeast Res 6: 249–267. Ando A, Nakamura T, Murata Y, Takagi H & Shima J (2007) Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains. FEMS Yeast Res 7: 244–253. Attfield PV (1997) Stress tolerance: the key to effective strains of industrial baker’s yeast. Nat Biotechnol 15: 1351–1357. Birrell GW, Giaever G, Chu AM, Davis RW & Brown JM (2001) A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity. P Natl Acad Sci USA 98: 12608–12613.
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Estruch F (2000) Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol Rev 24: 469–486. Estruch F & Carlson M (1993) Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae. Mol Cell Biol 13: 3872–3881. Garrido EO & Grant CM (2002) Role of thioredoxins in the response of Saccharomyces cerevisiae to oxidative stress induced by hydroperoxides. Mol Microbiol 43: 993–1003. Giaever G, Shoemaker DD, Jones TW, Liang H, Winzeler EA, Astromoff A & Davis RW (1999) Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat Genet 21: 278–283. Giaever G, Chu AM, Ni L et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: 387–991. Hasegawa S, Ogata Tt K, Ando A, Takagi H & Shima J (2012) Overexpression of vacuolar H+-ATPase-related genes in bottom-fermenting yeast enhances ethanol tolerance and fermentation rates under high-gravity fermentation. J Inst Brew 118: 179–185. Herrero E, Ros J, Belli G & Cabiscol E (2008) Redox control and oxidative stress in yeast cells. Biochim Biophys Acta 1780: 1217–1235. Higgins VJ, Alic N, Thorpe GW, Breitenbach M, Larsson V & Dawes IW (2002) Phenotypic analysis of gene deletant strains for sensitivity to oxidative stress. Yeast 19: 203–214. Hillenmeyer ME, Fung E, Wildenhain J et al. (2008) The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 320: 362–365. Jamieson DJ (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14: 1511–1527. Kuge S & Jones N (1994) YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J 13: 655–664. Mewes HW, Amid C, Arnold R et al. (2004) MIPS: analysis and annotation of proteins from whole genomes. Nucleic Acids Res 32: D41–D44. Milgrom E, Diab H, Middleton F & Kane PM (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J Biol Chem 282: 7125–7136. Morano KA, Grant CM & Moye-Rowley WS (2012) The response to heat shock and oxidative stress in Saccharomyces cerevisiae. Genetics 190: 1157–1195. Robinson MD, Grigull J, Mohammad N & Hughes TR (2002) FunSpec: a web-based cluster interpreter for yeast. BMC Bioinformatics 3: 35. Shima J & Takagi H (2009) Stress-tolerance of baker’s-yeast (Saccharomyces cerevisiae) cells: stress-protective molecules and genes involved in stress tolerance. Biotechnol Appl Biochem 53: 155–164. Shima J, Ando A & Takagi H (2008) Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide
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screening of Saccharomyces cerevisiae deletion strains. Yeast 25: 179–190. Thorpe GW, Fong CS, Alic N, Higgins VJ & Dawes IW (2004) Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes. P Natl Acad Sci USA 101: 6564–6569. Warringer J, Ericson E, Fernandez L, Nerman O & Blomberg A (2003) High-resolution yeast phenomics resolves different physiological features in the saline response. P Natl Acad Sci USA 100: 15724–15729.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. The genes required for H2O2 tolerance (the value of (SM/SW)/(NM/NW) < 0.5 and P-value < 0.05). Fig. S1. Growth of the H2O2-sensitive mutants.
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Comprehensive analysis of genes involved in the oxidative stress tolerance using yeast heterozygous deletion collection Natsumi Okada1, Jun Ogawa2 & Jun Shima1 1
Research Division of Microbial Sciences, Kyoto University, Kyoto, Japan; and 2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
Correspondence: Jun Shima, Research Division of Microbial Sciences, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto, 606-8502, Japan. Tel.: +81 75 753 9545; fax: +81 75 753 9544; e-mail: [email protected] Received 25 September 2013; revised 19 December 2013; accepted 1 January 2014. Final version published online 03 February 2014. DOI: 10.1111/1567-1364.12136 Editor: Hyun Ah Kang
YEAST RESEARCH
Keywords Saccharomyces cerevisiae; oxidative stress tolerance; essential genes.
Abstract In Saccharomyces cerevisiae, oxidative stress plays an inhibitory role during industrial fermentation. Although previous reports have identified genes required for oxidative stress tolerance, employing the yeast genome-wide screening, these screenings used a homozygous mutant collection which did not include the essential genes whose deletions result in lethality. Here, we report a truly genome-wide screening for the genes required for oxidative stress tolerance, using a heterozygous mutant collection which includes both essential and nonessential genes. Approximately 6300 heterozygous deletion mutants were grown in the presence or absence of H2O2. The screening identified a total of 331 genes whose heterozygotes conferred hypersensitivity to H2O2, indicating that these genes are required for oxidative stress tolerance. Notably, among these genes, 71 were essential genes. We classified these 71 essential genes based on localization, indicating that the localization of gene products from these essential genes was enriched in the nucleus and nucleolus. Classification of these essential genes based on functional categorizations showed that rRNA synthesis and tRNA synthesis were over-represented, suggesting that nuclear function, such as RNA synthesis, is important in the response to oxidative stress. These results provide a helpful resource for the understanding of the molecular basis of oxidative stress-tolerant mechanisms.
Introduction Yeasts, such as Saccharomyces cerevisiae, are exposed to various severe environmental stresses during the industrial processes (Attfield, 1997; Shima & Takagi, 2009). These include freeze–thaw, high sugar concentrations, air drying, high osmotic pressure, and alcohol. Such stresses induce oxidative stress due to the decline in intracellular pH and excessive generation of reactive oxygen species (ROS), leading to cell damage and low fermentation ability (Attfield, 1997). Saccharomyces cerevisiae possesses several response systems to oxidative stress. Transcriptional factors such as Yap1, Msn2/4, and Skn7 are important in the response to oxidative stress (Attfield, 1997; Jamieson, 1998; Herrero et al., 2008; Morano et al., 2012). These factors are involved in the activation or expression of specific antiox-
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idants and thereby detoxification of the excess intracellular ROS and normalization of the internal pH. Alternatively, S. cerevisiae cells possess several groups of enzymes that act directly as ROS detoxifiers: Superoxide dismutases (SODs) play a role in the detoxification of superoxide anions; glutathione peroxidases (GPXs), catalases (Cta1 and Ctt1), and thioredoxin peroxidases (TSAs) reduce hydrogen peroxide (H2O2); and thioredoxins (TRXs) and thioredoxin reductases (TRRs) play a role in thiol reduction. In addition, loss of the proton-transporting activities of V-ATPases, which are responsible for maintaining cytosolic pH and ion homeostasis, is known to dampen the tolerance of yeast cells to various stresses including oxidative stress, suggesting that V-ATPases play a role in stress tolerance (Thorpe et al., 2004; Ando et al., 2007; Milgrom et al., 2007; Shima et al., 2008; Shima & Takagi, 2009). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Due to the fact that oxidative stress has a negative impact under various stress conditions, a comprehensive understanding of the mechanism of oxidative stress tolerance would provide useful insights for the further development of molecular breeding of industrial yeasts. Yeast deletion collections are a valuable resource for examining loss-of-function phenotypes of yeast genes (Giaever et al., 2002), which enables a genome-wide screening to clarify the relevance of the genes in oxidative stress tolerance (Higgins et al., 2002; Thorpe et al., 2004; Ando et al., 2007; Shima et al., 2008). However, previous genomewide screenings for stress response genes have used either haploid or homozygous deletion collections (Birrell et al., 2001; Warringer et al., 2003; Thorpe et al., 2004; Ando et al., 2006, 2007; Shima et al., 2008) and thereby have been limited to nonessential genes due to the lethality resulting from the deletion of essential genes. Therefore, although essential genes occupy about 20% of all genes, the relevance of essential genes to oxidative stress tolerance remains obscure. Alternatively, a heterozygous deletion collection of diploid yeast, developed by European Saccharomyces cerevisiae archive for functional analysis (EUROSCARF), is available (Giaever et al., 1999; Hillenmeyer et al., 2008). As the majority of heterozygotes show no obvious growth defects, the heterozygous deletion collection is a promising tool to enable the genome-wide screening which includes essential genes. In this study, with a view to a more comprehensive understanding of oxidative stress tolerance, we screened for the essential genes required for oxidative stress tolerance using a heterozygous deletion mutant collection. Our genome-wide screening verified the relevance of essential genes in oxidative stress tolerance. Furthermore, the classification of the identified essential genes by function and localization suggested that nuclear functions, such as RNA synthesis, were implied in a critical role for the oxidative stress response.
Materials and methods Yeast strains and growth conditions
Saccharomyces cerevisiae BY4743 (MATa/a his3D1/his3D1 leu2D0/leu2D0 lys2D0/LYS2 MET15/met15D0 ura3D0/ura3 D0) and a heterozygous collection of diploid deletion strains (MATa/a) constructed using the insertion of kanMX4 cassettes (geneticin resistance) as selective markers for the genome of BY4743 (Hillenmeyer et al., 2008) were obtained from EUROSCARF. Each strain was maintained in liquid YPD medium (yeast extract, 1%; peptone, 2%; glucose, 2%) supplemented with 200 mg mL 1 of geneticin (Sigma–Aldrich, St. Louis, MO).
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Identification of mutant strains hypersensitive to H2O2 stress
Sensitivity to oxidative stress was assessed using H2O2. The wild-type strain (BY4743) and deletion mutants were grown in 100 lL of YPD medium without geneticin in 96-well microtiter plates (VIOLAMO, Osaka, Japan) using a 48-pin replicator (Funakoshi, Tokyo, Japan) and then precultured at 30 °C for 2 days without shaking. Portions (c. 1 lL) of the preculture were inoculated into 100 lL of YPD and YPD containing 3 mM H2O2 media using a 48-pin replicator. After incubation at 30 °C for 17 h, the optical density of the culture at 600 nm (OD600 nm) was measured using a microtiter-plate reader (SpectraMax 250; Molecular Devices, San Diego, CA). Four parameters were used in the data analysis of the sensitivity of the deletion mutants to H2O2 stress: NM, defined as the OD600 nm of mutant strains in YPD medium; SM, defined as the OD600 nm of mutant strains in YPD medium with 3 mM H2O2; NW, defined as the OD600 nm of BY4743 (wild-type) strain in YPD medium; and SW, defined as the OD600 nm of BY4743 strain in YPD medium with 3 mM H2O2. Mutants that showed considerable growth inhibition in YPD medium (i.e. NM/NW < 0.1) were removed from further data analysis. The value of (SM/ SW)/(NM/NW) was used as the parameter for sensitivity to H2O2. In this calculation, NM/NW values were used for compensation of growth defects under nonstress conditions. Measurements for the identification of H2O2-sensitive mutant strains were performed in triplicate, and the average of the values was used for the evaluation of sensitivity. The statistical analysis was also performed using the two-tailed Student’s t-test. Mutants that exhibited a sensitivity value below 0.5 and a P-value < 0.05 were defined as sensitive to H2O2. Time-course growth measurements of the H2O2-sensitive mutants
The H2O2-sensitive mutants were cultured in YPD or YPD containing 3 mM H2O2 media, and the OD600nm was measured every hour after incubation at 30 °C for 0–20 h using a microtiter-plate reader. Data are presented as the means for triplicate experiments. Classification of genes based on gene function and subcellular localization of gene products
Genes involved in H2O2 tolerance were categorized using the Munich Information Center for Protein Sequences (MIPS; http://mips.helmholtz-muenchen.de/genre/proj/ yeast/) databases (Mewes et al., 2004) and Gene Ontology
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(GO) terms presented by the Saccharomyces Genome Database (SGD) via FunSpec (http://funspec.med.utoronto.ca/), with the P-value threshold set at 0.01. The P values calculated by FunSpec could be a good statistical indicator of the frequencies (Robinson et al., 2002).
Results Screening of genes required for tolerance to H2O2
To assess the sensitivity to oxidative stress, we used H2O2, a member of the ROS. First, to determine suitable measurement conditions for the evaluation of H2O2 sensitivity, we measured the growth of the wild-type BY4743 strain in YPD media containing various concentrations of H2O2 (0–5 mM). After 17-h incubation at 30 °C, the growth of the wild-type BY4743 strain was inhibited by c. 50% at an H2O2 concentration of 3 mM (Fig. 1). We also determined the growth of a trx2 heterozygous deletion mutant (TRX2/trx2D). TRX2 encodes thioredoxin II, and a trx2 deletion mutant (trx2D) has been reported to be hypersensitive to oxidative stress (Kuge & Jones, 1994). Growth of the TRX2/trx2D strain was severely impaired in the presence of 3 mM H2O2, indicating that the TRX2/ trx2D heterozygote was also hypersensitive to oxidative stress despite the heterozygous deletion of TRX2 (Fig. 1). Based on these results, we used YPD medium containing 3 mM H2O2 for further analysis, and the growth of the strains was measured after incubation for 17 h at 30 °C to screen for H2O2-sensitive mutant strains. Next, we used the heterozygous deletion mutant collection to identify H2O2-sensitive mutants. Approximately 6300 heterozygote strains derived from BY4743 were 1.0
: WT-YPD+0 mM : WT-YPD+3 mM : TRX2/trx2Δ-YPD+0 mM
Classification of 331 genes based on the MIPS database
Next, to understand the biological functions important for H2O2 tolerance, the 331 genes required for H2O2 tolerance were classified using the MIPS database via FunSpec. Classification of the genes based on the functional and
Frequency (number of mutants)
1000
: TRX2/trx2Δ-YPD+3 mM
Growth (OD600)
cultivated in the presence or absence of 3 mM H2O2, and their growth was observed. To evaluate the sensitivity to H2O2 quantitatively, we used the (SM/SW)/(NM/NW) value (see Materials and methods). Figure 2 shows the distribution of the (SM/SW)/(NM/NW) values of the deletion mutants in a frequency distribution plot, providing an overview of the effects of heterozygous gene deletions on H2O2 sensitivity. Among the c. 6300 heterozygous deletion mutant strains tested, 331 strains (c. 5.3% of all strains) showed H2O2 tolerance < 50% of that of the wild-type BY4743 strain. Therefore, we defined these 331 strains as hypersensitive to H2O2 stress, and the 331 genes whose heterozygous deletions conferred hypersensitivity to H2O2 were considered to be required for H2O2 tolerance. We focused on these 331 genes, required for H2O2 tolerance, for the subsequent analysis. Among the 331 genes, the essential and nonessential genes were 71 (c. 6.4% of all the essential genes) and 260 (c. 4.7% of all nonessential genes), respectively (Supporting Information, Table S1). In addition, we monitored and assessed the growth of approximately a third of the H2O2-sensitive mutants, 119 strains, at 0–20 h (Fig. S1). The growth of all mutants was inhibited in the presence of H2O2, supporting the validity of our screening analysis.
0.5
5
10
0.5 1.0 1.5 2.0 H2O2 sensitivity value (SM/SW)/(NM/NW)
15 (h)
Fig. 1. The effect of H2O2 on the growth of the wild-type and TRX2/ trx2D strains. Each strain was grown in YPD medium in the presence or absence of 3 mM H2O2, and the growth was monitored by measurement of the OD600 nm.
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Non-essential genes
500
0 0.0
0
Essential genes
Fig. 2. Frequency distribution of H2O2 sensitivity in the heterozygous mutant collection. H2O2 sensitivity is expressed as the value of (SM/ SW)/(NM/NW). The heterozygous mutants with values of (SM/SW)/ (NM/NW) which were < 0.5 were defined as H2O2-hypersensitive strains.
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Ribosomal proteins All genes Genes required for H2O2 tolerance
Transport ATPases Oxidative stress response 0
(b)
5
(%)
Nucleus All genes Genes required for H2O2 tolerance
Vacuole Cytoplasm 0
10
20
30
40
50
(%)
Fig. 3. MIPS classification of the total genes required for H2O2 tolerance. The 331 essential and nonessential genes whose heterozygous deletions conferred H2O2-hypersensitivity are shown. All Saccharomyces cerevisiae genes were classified based on function (a) and subcellular localization (b), using the MIPS database.
subcellular localization categories is shown in Fig. 3, indicating that the genes required for H2O2 sensitivity were frequently included in the ‘ribosomal proteins’, ‘transport ATPases’ (Fig. 3a), ‘nucleus’, and ‘vacuole’ classes (Fig. 3b). Notably, the transport ATPase category included VMA3, VMA8, and VMA13, which encode the components of V-ATPase and have been previously reported to contribute to oxidative stress tolerance (Thorpe et al., 2004; Ando et al., 2007; Milgrom et al., 2007; Shima et al., 2008). Furthermore, using subcellular localization, the gene products were significantly assigned into the vacuole where V-ATPases are localized. Thus, these results were consistent with previous reports including genome-wide screening using a homozygous mutant collection (Thorpe et al., 2004; Ando et al., 2007; Shima et al., 2008), supporting the validity of our screening using heterozygous mutants. Classification of essential genes required for H2O2 stress tolerance by the MIPS database and GO terms
Next, we focused on the 71 essential genes required for H2O2 tolerance (listed in Table 1). To identify significantly (P < 0.01) enriched functions, cellular processes and subcellular localization, the 71 essential genes were also analyzed using the MIPS database via FunSpec (Fig. 4 and Table 2). Functional classification using the MIPS database revealed that the essential genes required for H2O2 tolerance were frequently included in the ‘rRNA processing’, ‘DNA binding’, ‘rRNA synthesis’, and ‘tRNA synthesis’ classes (Fig. 4a). Classification of the subcellular localization showed that a high number of gene products from essential genes required for H2O2 tolerance were located in the nucleus and nucleolus (Fig. 4b), which were related to the results of the functional classification that significantly included the nuclear functions (Fig. 4a). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Next, we also classified the 71 essential genes required for H2O2 tolerance based on the molecular functional, biological process and cellular component categories, using GO terms presented by the SGD (Fig. 5 and Table 2). Using the molecular functional categorization employing GO terms, the ‘RNA binding’, ‘DNA-directed RNA polymerase activity’, and ‘RNA-directed RNA polymerase activity’ classes were over-represented in the essential genes required for H2O2 tolerance (Fig. 5a). In addition, classification of the GO cellular component revealed that a high number of gene products from essential genes required for H2O2 tolerance were located in the nucleus and nucleolus (Fig. 5c). Based on biological processes, the 71 essential genes were predominantly categorized into rRNA processing, transcription, and translation processes (Fig. 5b), suggesting that RNA and protein synthesis, processed in the nucleus, might be related to H2O2 stress tolerance. Taken together, the classifications using GO terms were closely correlated with the classifications using the MIPS database, and both classifications suggested that the essential gene products which are localized in the nucleus or nucleolus and involved in RNA synthesis are critical in the response against oxidative stress.
Discussion So far, there have been several reports which have identified the genes required for oxidative stress tolerance by means of the genome-wide screening of yeast (Thorpe et al., 2004; Ando et al., 2007; Shima et al., 2008). However, these screenings relied on the null mutant collection and excluded the essential genes whose deletions result in lethality. In this study, we applied a heterozygous mutant collection to genome-wide screening for genes required for oxidative stress tolerance; this was the first use of a heterozygous mutant collection for this purpose. This approach identified a total of 331 genes required for the oxidative stress response, notably including 71 essential genes, under the same experimental conditions. Saccharomyces cerevisiae possesses antioxidant systems such as transcriptional activators and ROS-scavenging enzymes. The transcriptional activators, Yap1, Skn7, and Msn2/4, are well known to be involved in the response to oxidative stress in yeast. Indeed, our results also showed that the heterozygous deletions of YAP1 and SKN7 affected sensitivity to H2O2. MSN2/msn2D and MSN4/ msn4D strains did not exhibit hypersensitivity to H2O2, which agrees with previous reports that no obvious effect of the single null deletions of Msn2 and Msn4 was observed (Estruch & Carlson, 1993; Estruch, 2000). It is also known that the cytosolic thioredoxin system of S. cerevisiae is required as defense against externally added H2O2 (Attfield, 1997; Garrido & Grant, 2002; FEMS Yeast Res 14 (2014) 425–434
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Table 1. The essential genes required for H2O2 tolerance [the value of (SM/SW)/(NM/NW) < 0.5 and P-value < 0.05] Systematic name
Standard name
H2O2 sensitivity
P-value
YPL143W YPL235W YLR293C YDL193W YBL014C
RPL33A RVB2 GSP1 NUS1 RRN6
0.065 0.092 0.126 0.133 0.145
1.10E 5.79E 4.41E 1.01E 9.00E
13 09 07 05 07
YPR181C YDR353W YEL026W YDR404C YPR187W YDL043C YMR013C YEL035C YIL046W YDR145W YFL005W
SEC23 TRR1 SNU13 RPB7 RPO26 PRP11 SEC59 UTR5 MET30 TAF12 SEC4
0.160 0.178 0.227 0.235 0.242 0.256 0.261 0.302 0.320 0.325 0.327
1.51E 8.02E 4.72E 5.63E 8.11E 8.35E 1.08E 3.74E 4.50E 8.85E 1.56E
07 07 08 07 07 08 05 08 08 04 08
YGL030W YDR212W YDR362C
RPL30 TCP1 TFC6
0.330 0.344 0.345
5.59E 07 5.69E 06 6.23E 05
YMR309C YGL068W YGR216C
NIP1 MNP1 GPI1
0.346 0.351 0.352
1.03E 04 6.17E 05 8.50E 05
YPL131W YOL040C YDR373W
RPL5 RPS15 FRQ1
0.356 0.361 0.371
3.99E 04 4.28E 05 4.01E 03
YOR151C YPR086W YOR149C YPL124W YOR103C
RPB2 SUA7 SMP3 SPC29 OST2
0.378 0.381 0.381 0.385 0.385
1.42E 4.57E 6.34E 5.97E 1.23E
YKL189W YJL005W YKL165C
HYM1 CYR1 MCD4
0.387 0.389 0.391
7.29E 03 1.85E 04 9.82E 03
YDR365C YPR104C YHR128W YDR361C YLR163C YIL048W YOR335C YDR376W YDR064W YGR186W YGL097W YDR299W YDR390C
ESF1 FHL1 FUR1 BCP1 MAS1 NEO1 ALA1 ARH1 RPS13 TFG1 SRM1 BFR2 UBA2
0.398 0.405 0.414 0.417 0.418 0.423 0.424 0.428 0.429 0.431 0.437 0.440 0.449
1.12E 9.81E 1.60E 7.55E 4.15E 4.21E 2.46E 2.34E 7.93E 2.33E 3.70E 9.89E 1.65E
YGR113W
DAM1
0.449
7.55E 04
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Description
05 06 04 06 06
07 10 07 05 03 06 09 03 06 06 07 06 09
Ribosomal 60S subunit protein L33A ATP-dependent DNA helicase RNA processing and transport Putative prenyltransferase Component of the core factor (CF) rDNA transcription factor complex GTPase-activating protein, involved in ER to Golgi transport Cytoplasmic thioredoxin reductase RNA-binding protein RNA polymerase II subunit B16 RNA polymerase subunit ABC23 Subunit of the SF3a splicing factor complex Dolichol kinase Protein of unknown function F-box protein containing five copies of the WD40 motif Subunit (61/68 kDa) of TFIID and SAGA complexes Rab family GTPase essential for vesicle-mediated exocytic secretion and autophagy Ribosomal 60S subunit protein L30 Alpha subunit of chaperonin-containing T-complex One of six subunits of RNA polymerase III transcription initiation factor complex (TFIIIC) eIF3c subunit of the eukaryotic translation initiation factor 3 (eIF3) Protein associated with the mitochondrial nucleoid Membrane protein involved in the synthesis of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI) Ribosomal 60S subunit protein L5 Protein component of the small (40S) ribosomal subunit N-Myristoylated calcium-binding protein that may have a role in intracellular signaling through its regulation of the phosphatidylinositol 4-kinase Pik1p RNA polymerase II second largest subunit B150 Transcription factor TFIIB Alpha 1,2-mannosyltransferase Inner plaque spindle pole body (SPB) component Epsilon subunit of the oligosaccharyltransferase complex of the ER lumen Component of the RAM signaling network Adenylate cyclase Protein involved in glycosylphosphatidylinositol (GPI) anchor synthesis Nucleolar protein involved in pre-rRNA processing Regulator of ribosomal protein (RP) transcription Uracil phosphoribosyltransferase, synthesizes UMP from uracil Essential protein involved in nuclear export of Mss4p Smaller subunit of the mitochondrial processing protease (MPP) Putative aminophospholipid translocase (flippase) Cytoplasmic and mitochondrial alanyl-tRNA synthetase Oxidoreductase of the mitochondrial inner membrane Protein component of the small (40S) ribosomal subunit TFIIF (transcription factor II) largest subunit Nucleotide-exchange factor for Gsp1p Essential protein that is a component of 90S preribosomes Subunit of a heterodimeric nuclear SUMO-activating enzyme (E1) with Aos1p Essential subunit of the Dam1 complex (aka DASH complex)
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Table 1. Continued Systematic name
Standard name
H2O2 sensitivity
P-value
Description
YJL010C YNL103W YOR319W
NOP9 MET4 HSH49
0.449 0.450 0.452
6.37E 05 9.72E 11 3.29E 07
YOR116C YHR069C YNL313C YDL031W
RPO31 RRP4 EMW1 DBP10
0.461 0.462 0.465 0.466
9.43E 3.37E 8.66E 7.81E
04 06 07 04
YOL127W YKL154W YDR416W YDR288W YNL240C
RPL25 SRP102 SYF1 NSE3 NAR1
0.467 0.468 0.469 0.470 0.473
4.81E 1.90E 1.24E 1.07E 3.73E
04 02 06 04 03
YOR340C YMR296C YDR341C YNL312W YDL058W
RPA43 LCB1 RRS1 RFA2 USO1
0.474 0.476 0.478 0.481 0.483
4.65E 7.16E 4.91E 1.69E 1.79E
07 07 05 05 05
YDR182W YFR002W YLR009W YDR498C
CDC1 NIC96 RLP24 SEC20
0.484 0.488 0.491 0.494
8.10E 8.51E 2.35E 1.08E
06 08 11 05
YGL044C YDR339C
RNA15 FCF1
0.496 0.499
1.39E 02 2.60E 06
YPL128C
TBF1
0.499
2.32E 06
Essential subunit of U3-containing 90S preribosome Leucine-zipper transcriptional activator U2-snRNP-associated splicing factor with similarity to the mammalian splicing factor SAP49 RNA polymerase III largest subunit C160 Exosome noncatalytic core component Essential conserved protein with a role in cell wall integrity Putative ATP-dependent RNA helicase of the DEAD-box protein family Ribosomal 60S subunit protein L25 Signal recognition particle (SRP) receptor beta subunit Member of the NineTeen Complex (NTC) Component of the SMC5-SMC6 complex Component of the cytosolic iron-sulfur (FeS) protein assembly machinery RNA polymerase I subunit A43 Component of serine palmitoyltransferase Arginyl-tRNA synthetase Subunit of heterotrimeric replication protein A (RPA) Essential protein involved in the vesicle-mediated ER to Golgi transport step of secretion Putative lipid phosphatase of the endoplasmic reticulum Linker nucleoporin component of the nuclear pore complex (NPC) Essential protein with similarity to Rpl24Ap and Rpl24Bp Membrane glycoprotein v-SNARE involved in retrograde transport from the Golgi to the ER Component of the cleavage and polyadenylation factor I (CF I) Putative PINc domain nuclease required for early cleavages of 35S pre-rRNA and maturation of 18S rRNA gene Telobox containing general regulatory factor
Herrero et al., 2008). Consistent with this knowledge, the heterozygous deletions of TSA1, TRX2, and TRR1, which encode the cytosolic thioredoxin system, conferred hypersensitivity to H2O2. In contrast, although TSA2, TRX1, and TRR2 also encode thioredoxin systems, the heterozygous mutants of these genes were not hypersensitive to H2O2 as the value of (SM/SW)/(NM/NW) was more than 0.5. In addition, neither catalases (Ctt1 and Cta1) nor superoxide dismutases (Sod1 and Sod2) were required for H2O2 resistance under our experimental conditions. However, a previous study by Shima and colleagues using homozygous null mutants showed that cta1D, sod1D, sod2D, tsa2D, trx1D, trr2D, gpx1D, gpx2D, grx1D, and grx3D were not sensitive to oxidative stress (Shima et al., 2008), which is similar to our results. The H2O2 sensitivity values of heterozygous mutants for genes of the ROS-scavenging systems are presented in Table 3. Although it should be noted that our genome-wide screening relied on a heterozygous mutant collection, and therefore included the effects of the intact allele of a gene, our results clearly demonstrate that a heterozygous ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
(a)
rRNA processing DNA binding rRNA synthesis tRNA synthesis RNA binding Ribosomal proteins
General transcription activities Cellular signalling All genes Essential genes required for H2O2 tolerance
Modification with fatty acids Phospholipid metabolism 0
(b)
5
10
(%)
Nucleus All genes Essential genes required for H2O2 tolerance
Nucleolus 0
10
20
30
40
50 (%)
Fig. 4. MIPS classification of the essential genes required for H2O2 tolerance. The 71 essential genes whose heterozygous deletions conferred H2O2-hypersensitivity are shown. All Saccharomyces cerevisiae essential genes were classified based on function (a) and subcellular localization (b), using the MIPS database.
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Table 2. Functional and localization categories of the essential genes required for H2O2 tolerance at high frequencies Category MIPS functional classification rRNA processing DNA binding rRNA synthesis tRNA synthesis RNA binding Ribosomal proteins General transcription activities Cellular signaling Modification with fatty acids Phospholipid metabolism MIPS subcellular localization Nucleus
P-value 1.11E 2.66E 2.86E 6.88E 3.99E 4.63E 4.64E 5.16E 5.16E 5.96E
In category from cluster 05 04 04 04 03 03 03 03 03 03
4.44E 04
Nucleolus GO molecular function RNA binding
1.64E 03
DNA-directed RNA polymerase activity RNA-directed RNA polymerase activity Ribonucleoside binding Structural constituent of ribosome General RNA polymerase II transcription factor activity GO biological process rRNA processing Transcription, DNA-dependent
2.72E 2.45E 6.75E 2.20E 9.48E
Translation Ribosome biogenesis Transcription initiation from RNA polymerase II promoter GPI anchor biosynthetic process Transcription initiation, DNA-dependent Response to cadmium ion Response to arsenic-containing substance Cysteine biosynthetic process Ribosomal large subunit assembly Transcription from RNA polymerase II promoter GO cellular component Nucleus
1.05E 05 05 04 04 03 03
2.17E 04 4.98E 04 5.20E 04 2.20E 2.59E 3.56E 3.91E 3.91E 5.89E 7.02E 7.68E 8.02E
03 03 03 03 03 03 03 03 03
2.23E 05
Nucleolus
7.00E 05
DNA-directed RNA polymerase II, core complex U2-type prespliceosome Endoplasmic reticulum membrane ER to Golgi transport vesicle membrane Intracellular Golgi membrane U2 snRNP DNA-directed RNA polymerase I complex
2.45E 3.22E 6.64E 7.02E 7.13E 8.24E 9.54E 9.54E
FEMS Yeast Res 14 (2014) 425–434
04 03 03 03 03 03 03 03
DBP10 ESF1 SNU13 RPL30 SRM1 RRP4 NOP9 GSP1 RVB2 FHL1 NSE3 RPB7 RFA2 RPO31 RPB2 RPA43 TBF1 RPO26 RRN6 TFC6 RPO31 RPA43 RPO26 TFC6 RPO31 FHL1 RPO26 PRP11 ESF1 RNA15 NOP9 RPL25 HSH49 RPL5 DBP10 RPS13 RPL30 MNP1 RPS15 RPL25 RPL5 RPL33A RRN6 TFC6 RPB7 MET4 RPB2 RPO26 FRQ1 SRM1 GPI1 GPI1 MCD4 SMP3 GPI1 MCD4 SEC59 SMP3 RRN6 PRP11 TAF12 NSE3 FCF1 TRR1 BCP1 TFC6 ESF1 UBA2 RPB7 SYF1 SNU13 NIC96 RNA15 SRM1 DAM1 TFG1 RRP4 MET30 NOP9 HYM1 GSP1 MET4 RFA2 EMW1 RPO31 RPB2 HSH49 RPA43 SPC29 TBF1 SUA7 FHL1 RPO26 RRN6 DBP10 BFR2 ESF1 SNU13 RRP4 NOP9 RPA43 DBP10 PRP11 ESF1 RPB7 SNU13 RPL30 RNA15 RRP4 NOP9 RPS15 RPL25 HSH49 ALA1 RPL5 RPB7 RPO31 RPB2 RPA43 RPO26 RPB7 RPB2 RPO26 RPO31 RPB2 RPS13 RPL30 MNP1 RLP24 RPS15 RPL25 RPL5 RPL33A TAF12 TFG1 SUA7 DBP10 BFR2 FCF1 ESF1 SNU13 RPL30 RRP4 NOP9 RVB2 RRN6 TAF12 TFC6 RPB7 TFG1 MET30 MET4 RPO31 RPB2 RPA43 TBF1 RVB2 SUA7 FHL1 RPO26 RPS13 YDR341C RPL30 MNP1 RLP24 NIP1 RPS15 RPL25 ALA1 RPL5 RPL33A DBP10 FCF1 ESF1 SNU13 NOP9 RLP24 RPS15 RPB7 TFG1 SUA7 GPI1 MCD4 SMP3 TAF12 SUA7 MET30 MET4 MET30 MET4 MET30 MET4 DBP10 RPL25 RPL5 TAF12 RPB7 RPB2 RPO26 RRN6 DBP10 PRP11 NUS1 TAF12 NSE3 BFR2 FCF1 BCP1 TFC6 ESF1 UBA2 RPB7 SYF1 SNU13 NIC96 RNA15 SRM1 DAM1 TFG1 RRP4 MET30 NOP9 RLP24 GSP1 MET4 NAR1 RFA2 EMW1 RPO31 RPB2 HSH49 RPA43 TBF1 RVB2 SUA7 FHL1 RPO26 RRN6 DBP10 BFR2 FCF1 ESF1 SNU13 RRP4 NOP9 RLP24 RPA43 FHL1 RPB7 RPB2 RPO26 PRP11 SYF1 HSH49 USO1 NUS1 SEC20 SRP102 MCD4 SEC59 LCB1 SMP3 SEC23 USO1 SEC23 PRP11 RPS13 SYF1 RPL30 MNP1 FUR1 HYM1 RPL25 RPL5 RPL33A USO1 FRQ1 NEO1 MCD4 SEC23 PRP11 HSH49 RPA43 RPO26
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(a)
N. Okada et al.
RNA binding DNA-directed RNA polymerase activity All genes Essential genes required for H2O2 tolerance
RNA-directed RNA polymerase activity Ribonucleoside binding Structural constituent of ribosome General RNA polymerase II transcription factor activity
(b)
0
10
5
15
(%)
rRNA processing Transcription, DNA-dependent Translation Ribosome biogenesis
Transcription initiation from RNA polymerase II promoter GPI anchor biosynthetic process Transcription initiation, DNA-dependent Response to cadmium ion Response to arsenic-containing substance All genes Essential genes required for H2O2 tolerance
Cysteine biosynthetic process Ribosomal large subunit assembly Transcription from RNA polymerase II promoter
0
(c)
5
10
15
20 (%)
Nucleus Nucleolus DNA-directed RNA polymerase II, core complex U2-type prespliceosome Endoplasmic reticulum membrane ER to Golgi transport vesicle membrane Intracellular Golgi membrane
All genes Essential genes required for H2O2 tolerance
U2 snRNP DNA-directed RNA polymerase I complex
0
10
20
mutant collection is available for the genome-wide analysis for oxidative stress tolerance. Recently, an increasing number of studies have shown that V-ATPase activity is responsible for oxidative stress tolerance (Attfield, 1997; Thorpe et al., 2004; Ando et al., 2007; Milgrom et al., 2007; Shima et al., 2008; Shima & Takagi, 2009). Our results also show that heterozygous deletions of the genes encoding the components of V-ATPase, such as VMA3, VMA8, and VMA13, resulted in hypersensitivity to H2O2. Thus, our study supports the emerging role of V-ATPase in oxidative stress tolerance and the potency of V-ATPase activity for the breeding of yeasts with high fermentation ability (Hasegawa et al., 2012). The identified 71 essential genes correspond to 6.4% of the total essential genes. The ratio of essential genes required for oxidative stress tolerance to the total essential genes is higher than that of the nonessential genes required for oxidative stress tolerance (260 genes: 4.7% of total nonessential genes), implying a substantial role of essential genes in the response to oxidative stress. Thus, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
30
40
50 (%)
Fig. 5. GO classification of the essential genes required for H2O2 tolerance. The 71 essential genes required for H2O2 tolerance are shown. All Saccharomyces cerevisiae essential genes were classified based on molecular function (a), biological process (b), and cellular component (c), using GO terms.
Table 3. H2O2 sensitivity of heterozygous mutants with deletion of the genes involved in ROS-scavenging systems Mutant
H2O2 sensitivity value
Product of deleted gene
cta1 ctt1 sod1 sod2 trr1 trr2 trx1 trx2 tsa1 tsa2 gpx1 gpx2 gpx3 grx1 grx2 grx3 glr1 yap1 skn7 msn2 msn4
0.58 0.65 0.87 0.61 0.18 0.98 1.14 0.08 0.26 1.08 0.56 0.65 0.84 1.33 0.88 1.06 0.94 0.40 0.39 0.78 0.68
Catalase A Catalase T Superoxide dismutase Superoxide dismutase Thioredoxin reductase Thioredoxin reductase Thioredoxin Thioredoxin Thioredoxin peroxidase Thioredoxin peroxidase Glutathione peroxidase Glutathione peroxidase Glutathione peroxidase Glutaredoxin Glutaredoxin Glutaredoxin Glutathione oxidoreductase Transcriptional factor Transcriptional factor Transcriptional factor Transcriptional factor
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Impacts of oxidative stress on essential genes in yeast
our results provide the first evidence for the relevance of essential genes in oxidative stress tolerance. To deduce the cellular functions in which the essential genes required for oxidative stress tolerance are involved, we examined the distribution of the 71 genes in the functional categories based on MIPS and GO (Table 2, Figs 4 and 5). The functional categories such as rRNA synthesis and tRNA synthesis were over-represented in the essential genes required for oxidative stress tolerance. Using the classification based on localization, the localization of gene products from the essential genes required for oxidative stress tolerance was enriched in the nucleus and nucleolus. This is reasonable as tRNA and rRNA are synthesized in the nucleus and nucleolus, respectively. rRNA and tRNA are integral factors for protein synthesis. Therefore, our results suggest that tRNA and rRNA are important for oxidative stress tolerance, implying that protein synthesis is a key process in the response to oxidative stress. It is possible that a protein synthesis defect results in an insufficient synthesis of antioxidants, which precludes cells responding to stress efficiently. Thus, our results shed light on the role of essential genes in oxidative stress tolerance and represent a helpful resource for the comprehensive understanding of the molecular basis of oxidative stress-tolerant mechanisms.
Acknowledgements This work was supported financially by the Institute for Fermentation, Osaka (IFO). We thank Dr. Akira Ando (National Agriculture and Food Research Organization) for his helpful discussions. We also thank Dr. S. Matsuda for technical advice and enormously constructive comments.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. The genes required for H2O2 tolerance (the value of (SM/SW)/(NM/NW) < 0.5 and P-value < 0.05). Fig. S1. Growth of the H2O2-sensitive mutants.
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