Genome-Wide Screening of Saccharomyces cerevisiae Genes Regulated by Vanillin
Genome-Wide Screening of Saccharomyces cerevisiae Genes Regulated by Vanillin
Journal of Microbiology and Biotechnology. 2015. Jan, 25(1): 50-56
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : September 22, 2014
  • Accepted : September 29, 2014
  • Published : January 28, 2015
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Eun-Hee, Park
Myoung-Dong, Kim

During pretreatment of lignocellulosic biomass, a variety of fermentation inhibitors, including acetic acid and vanillin, are released. Using DNA microarray analysis, this study explored genes of the budding yeast Saccharomyces cerevisiae that respond to vanillin-induced stress. The expression of 273 genes was upregulated and that of 205 genes was downregulated under vanillin stress. Significantly induced genes included MCH2 , SNG1 , GPH1 , and TMA10 , whereas NOP2 , UTP18 , FUR1 , and SPR1 were down regulated. Sequence analysis of the 5’-flanking region of upregulated genes suggested that vanillin might regulate gene expression in a stress response element (STRE)-dependent manner, in addition to a pathway that involved the transcription factor Yap1p. Retardation in the cell growth of mutant strains indicated that MCH2 , SNG1 , and GPH1 are intimately involved in vanillin stress response. Deletion of the genes whose expression levels were decreased under vanillin stress did not result in a notable change in S. cerevisiae growth under vanillin stress. This study will provide the basis for a better understanding of the stress response of the yeast S. cerevisiae to fermentation inhibitors.
Lignocellulosic biomass such as crop residues and wood chips is an abundant energy source that has the potential for utilization as a feedstock for the production of bioethanol [27] . The production of fuel ethanol from biomass has attracted attention as part of efforts to prevent global warming, protect the environment, and improve energy reserves [23] . The major components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Therefore, to obtain fermentable sugars, physical or chemical pretreatment processes such as steam explosion, diluteacid hydrolysis, ammonia freeze explosion, and enzymatic hydrolysis are necessary [20] . The degradation products formed by pretreatment of lignocellulose depend on both the biomass and pretreatment conditions such as temperature, time, pressure, pH, and addition of catalysts [6] . For economic reasons, dilute-acid hydrolysis is commonly used to prepare lignocellulose for enzymatic saccharification and fermentation [11] . As a result of the pretreatment, various inhibitory compounds are released from lignocellulose biomass, which reduce the ethanol yield and productivity during fermentation by microorganisms [1 , 20] . These inhibitory compounds belong to three groups: furans, weak acids, and phenolic compounds. The furan compounds 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde, which are formed by dehydration of hexoses and pentoses, respectively [7 , 33] , have been shown to reduce the enzymatic activity of alcohol dehydrogenase, aldehyde dehydrogenase, and pyruvate dehydrogenase [28] . Formic and levulinic acids are products of HMF breakdown, whereas acetic acid is formed by de-acetylation of hemicelluloses [1 , 7] . A wide range of phenolic compounds are generated due to lignin breakdown; vanillin is a major phenolic compound [1 , 8] .
Recent reports have shown that vanillin can effectively inhibit the growth of yeast and mold when tested in fruit purees and fruit-based agar systems [4 , 5] . Vanillin inhibited the growth of Saccharomyces cerevisiae , Zygosaccharomyces bailii , Debaryomyces hansenii , and Z. rouxii [24 , 32] .
Vanillin has been suggested to be a stronger inhibitor of growth and bioethanol fermentation than other inhibitors because it acts at low concentrations [9 , 20] . Improvement of S. cerevisiae tolerance to vanillin is important in the development of industrial strains for fuel ethanol production and vanillin synthesis [30] . Since vanillin is toxic to yeast, the development of vanillin-tolerant S. cerevisiae strains is a critical prerequisite for efficient bioethanol production. A mitochondrial superoxide dismutase mutant exhibits an enhanced resistance to vanillin [14] . Vanillin causes nuclear accumulation of Yap1, an oxidative stress–responsive transcription factor, and subsequent transcriptional activation of Yap1-regulated genes in S. cerevisiae [30] . These data indicate that a variety of factors affect S. cerevisiae resistance to vanillin, although the mechanism of vanillin tolerance remains obscure. Genome-wide, fitness-based screening methods have identified mutations that confer vanillin sensitivity and non-essential deletion mutants exhibiting vanillin sensitivity [9 , 14 , 19] . Despite the increasing interest in the inhibitory effects of vanillin, little is known about the genes regulated by vanillin and the mode of action of vanillin in S. cerevisiae .
In this study, S. cerevisiae genes whose expression levels are notably changed by vanillin were explored by DNA microarray analysis. In addition, the phenotypes of S. cerevisiae strains harboring deletions of the genes deregulated by vanillin stress were examined.
Materials and Methods
- Chemicals, Strains, and Cultivation Conditions
S. cerevisiae strains ( Table 1 ) were purchased from European Saccharomyces cerevisiae Archive for Functional Analysis and grown in YEPD (1% bacto-yeast extract, 2% bacto-proteose peptone, and 2% glucose) at 30℃ unless stated otherwise. Vanillin was purchased from Sigma-Aldrich (USA). To determine S. cerevisiae sensitivity to vanillin, various vanillin concentrations were added to the medium. Cell growth was quantified by measuring the optical density at 600 nm (OD 600 ). For microarray experiments, exponentially growing cells were harvested by filtration [31] and resuspended in YEPD containing 4 mM vanillin for 2 h. Cells were then cultured in 500 ml baffled flasks (Nalgene, USA) containing 100 ml of YEPD supplemented with various concentrations of vanillin at 30℃ for 30 h with shaking at 200 rpm. For plate growth assay, S. cerevisiae was grown in YEPD overnight and diluted to an OD 600 of 0.2. Then, 5-fold serial dilutions were prepared, and 10 µl of each dilution was spotted onto a YEPD plate containing 6 mM vanillin. The plates were incubated at 30℃ for 2 or 3 days [25] .
S. cerevisiaestrains used in this study.
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S. cerevisiae strains used in this study.
- Analytical Methods
Ethanol concentrations were determined by a QuantiChrom Ethanol Assay Kit (BioAssay Systems, USA). Glucose was measured by high-performance liquid chromatography (Shimadzu, Japan) on an HPX-87H column (Bio-Rad, USA); the mobile phase was 0.005 N H 2 SO 4 .
- RNA Extraction and DNA Microarray Analysis
Total RNA was extracted from cells exponentially growing in YEPD containing 4 mM vanillin. mRNA was purified using a PureLink RNA Kit (Ambion, USA) according to the manufacturer’s recommendations. Gene expression analysis was performed using a GeneChip Yeast Genome 2.0 Array (Affymetrix, USA) and GenPlex software (Istech, Korea). Statistical analysis for the normalization of microarray data was performed using methods described elsewhere [17 , 18] .
Results and Discussion
- Effects of Vanillin on Growth ofS. cerevisiae
To determine the appropriate concentration of vanillin for DNA microarray analysis, the sensitivity of S. cerevisiae BY4742 strain to vanillin was examined. S. cerevisiae growth was inhibited by vanillin at concentrations above 4 mM ( Fig. 1 ). At a vanillin concentration of 4 mM, the specific growth rate of the BY4742 strain was reduced by approximately 38%. Inhibition of cell growth by vanillin was also observed in shake-flask cultures ( Fig. 2 ). Inhibition of growth of the haploid BY4742 strain treated with vanillin was highly correlated with ethanol production, which is in line with the data reported for the diploid BY4743 strain [3] . The rates of both cell growth and ethanol production from glucose were significantly affected at 4 mM vanillin. Based on these results, RNA was prepared from wild-type S. cerevisiae BY4742 cells grown in the presence of 4 mM vanillin.
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Plate growth assay (A) and specific growth rate (B) of S. cerevisiae strain BY4742 in vanillin-containing media. For plate growth assay, cells were grown in YEPD overnight and diluted to an OD600 of 0.2. Then, 5-fold serial dilutions were prepared, and 10 µl of each dilution was spotted onto a YEPD plate. The plates were incubated at 30℃ for 2 days. Specific growth rates were determined at an exponential growth phase. Different letters in (B) indicate a significant difference between means.
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Cell growth profiles (OD600), glucose consumption, and ethanol production by S. cerevisiae BY4742. Shake-flask cultures were grown in YEPD supplemented with 0 mM (●), 2 mM (○), 4 mM (▼), 6 mM (△), 8 mM (■), or 10 mM (□) vanillin. Averages and standard errors from three independent experiments are shown.
- DNA Microarray Analysis
Vanillin-regulated genes were identified using DNA microarrays. Based on the comparison of statistically normalized test and control data from 5,744 probe sets for 5,845 genes present in S. cerevisiae , it was found that the expression of 273 genes was upregulated over 2-fold, whereas the expression of 205 genes was downregulated over 2-fold under vanillin stress ( Table 2 ). Expression of the genes involved in the citrate cycle was significantly affected by vanillin ( Fig. 3 ); 46% of the genes of the citrate cycle (such as IRC15 , CIT3 , PYC1 , SDH1 , SDH2 , LSC2 , and CIT1 ) were upregulated. However, the number of genes involved in glyoxylate and dicarboxylate metabolism whose expression was upregulated under vanillin stress was relatively small: out of 61 genes, only ICL2 , CIT3 , MLS1 , and CIT1 were upregulated. Microarray analysis also revealed that the expression of the glycogen phosphorylase 1 ( GPH1 ) gene, involved in glycogen metabolism, was increased 5.4-fold. The expression of GPH1 is known to be regulated by the stress-response element (STRE) [22] and Hog1p-mitogen-activated protein kinase-dependent pathway [34] .
S. cerevisiaegenes deregulated by vanillin.
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S. cerevisiae genes deregulated by vanillin.
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Functional categories of the genes whose expression levels are deregulated over 2-fold by vanillin (4 mM). The denominators and numerators in parentheses indicate the numbers of total genes and affected genes in each category, respectively.
The expression of 24 genes involved in ribosome biogenesis, including NOP2 , UTP18 , FUR1 , SPR1 , NOB1 , NMD3 , RNT1 , and IMP3 , was notably downregulated under vanillin stress. Nop2p is an RNA methyltransferase, which plays a role in rRNA processing and large ribosomal subunit biogenesis [16] . Vanillin was reported to affect ribosome assembly [19] , a process that also involves NOP2 , UTP18 , and PWP1 [8] . Four genes involved in autophagy [21] , ATG3 , ATG7 , ATG8 and ATG13 , were slightly upregulated.
It is interesting to note that MCH2 , SNG1 , and TMA10 were strongly upregulated under vanillin stress. Mch2p, a monocarboxylate transporter-homologous (MCH) family protein, is involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate, and acetate across the plasma membrane [26] . However, the function of Mch2p in stress response has not been studied intensively. It might be involved in the transport of vanillin, a phenolic aldehyde that has aldehyde, hydroxyl, and ether functional groups [26] . SNG1 has been previously described as a gene involved in nitrosoguanidine resistance [13] ; its overexpression causes resistance to 6-azauracil in S. cerevisiae [12] . The expression of TMA10 , which is increased under DNA-replication stress [10] , was also upregulated by vanillin. Transcription of the HSP42 gene, which encodes a cytosolic small heat shock protein with chaperone activity [15] , was also increased. The HSP42 transcript is detectable at the basal level under all culture conditions, but its expression is induced by stresses such as heat shock, salt shock, and starvation [15 , 36] . This upregulation of HSP42 transcription is mediated by the transcription factors Hsf1p and Msn2p/Msn4p that respectively bind to heat shock elements [2] and stress response elements in the HSP42 promoter [35] . Semiquantitative polymerase chain reaction confirmed that the expression of the genes listed in Table 2 was significantly changed by vanillin (data not shown).
Vanillin regulates gene expression in a Yap1p-dependent manner [30] . Sequence analysis indicated that the 5’-flanking region of MCH2 contains one putative Yap1pbinding sequence (5’-TT/GACTAA-3’) [29] , 508 nucleotides upstream of the initiation codon ( Fig. 4 ). A putative Yap1p-binding sequence was also found 98 nucleotides upstream of the initiation codon of GPH1 ( Fig. 4 ). These observations suggest that the expression of MCH2 and GPH1 may be regulated in a Yap1p-dependent manner. Three putative STRE sequences were found in the 5’-flanking region of GPH1 , but not in that of MCH2 , suggesting that regulation of GPH1 expression is more complex. An interesting finding of this study is that the expression of genes that have no Yap1p-binding sequences in their 5’-flanking regions was strongly induced by vanillin. It cannot be excluded that other transcription factors such as Msn2p/Msn4p related to the STRE-mediated pathway play a crucial role in vanillin stress. Identification of the transcription factors and their roles would also be important for understanding the response of S. cerevisiae to other stresses.
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Alignment of stress response elements (STRE) and Yap1p-binding sequence in the 5’-flanking regions of the genes regulated by vanillin in S. cerevisiae. Conserved nucleotides are shaded in black. Numbers indicate nucleotide positions upstream of the translation initiation codon (ATG) of each gene.
- Phenotypes of MutantS. cerevisiaeStrains
The role of the genes whose expression was affected by vanillin stress was examined in a plate growth assay using mutant strains of S. cerevisiae BY4742 ( Fig. 5 ). Deletion of MCH2 , SNG1 , or GPH1 , but not of any other genes listed in Table 1 , resulted in notable growth defects at an elevated vanillin concentration (8 mM). These data suggest that the elevated expression of MCH2 , SNG1 , and GPH1 under vanillin stress may be required for the response to vanillin. Deletion of the genes whose expression was downregulated under vanillin stress did not result in a notable change in growth of S. cerevisiae strains either in a haploid or a diploid genetic background (data not shown). These results warrant further studies to unveil the genetic regulation underlying the vanillin stress response in S. cerevisiae .
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Growth phenotypes of S. cerevisiae strains on vanillin-containing medium. S. cerevisiae strains with the indicated mutation in a haploid genetic background were used for the growth assay. Cells were grown to mid-log phase in YEPD and diluted to an OD600 of 0.2 with the same medium. Then, 5-fold serial dilutions were spotted onto a YEPD plate and cells were incubated at 30℃ for 3 days.
In conclusion, the expression of genes involved in a wide range of cellular processes was affected by vanillin. The results of this study will be useful for developing more stress-tolerant S. cerevisiae strains for bioethanol fermentation.
This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009477)” Rural Development Administration, Republic of Korea, and the Ministry of Trade, Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the Promoting Regional specialized Industry (Project No. A0059 00747).
Almeida JRM , Modig T , Petersson A , Hägerdal BH , Lidén G , Grauslund MFG 2007 Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates bySaccharomyces cerevisiae. J. Chem. Technol. Biotechnol. 82 340 - 349    DOI : 10.1002/jctb.1676
Amoros M , Estruch F 2001 Hsf1p and Msn2/4p cooperate in the expression ofSaccharomyces cerevisiaegenesHSP26andHSP104in a gene- and stress type-dependent manner. Mol. Microbiol. 39 1523 - 1532    DOI : 10.1046/j.1365-2958.2001.02339.x
Brachmann CB , Davies A , Cost GJ , Caputo E , Li J , Hieter P , Boeke JD 1998 Designer deletion strains derived fromSaccharomyces cerevisiaeS288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14 115 - 132
Cerrutti P , Alzamora SM 1996 Inhibitory effects of vanillin on some food spoilage yeasts in laboratory media and fruit purees. Int. J. Food Microbiol. 29 379 - 386    DOI : 10.1016/0168-1605(95)00026-7
Cerrutti P , Alzamora SM , Vidales SL 1997 Vanillin as an antimicrobial for producing shelf-stable strawberry puree. J. Food Sci. 62 608 - 610    DOI : 10.1111/j.1365-2621.1997.tb04442.x
Cortez DV , Roberto IC 2010 Individual and interaction effects of vanillin and syringaldehyde on the xylitol formation byCandida guilliermondii. Bioresour. Technol. 101 1858 - 1865    DOI : 10.1016/j.biortech.2009.09.072
Dunlop AP 1948 Furfural formation and behavior. Ind. Eng. Chem. 40 204 - 209    DOI : 10.1021/ie50458a006
Endo A , Nakamura T , Ando A , Tokuyasu K , Shima J 2008 Genome-wide screening of the genes required for tolerance to vanillin, which is a potential inhibitor of bioethanol fermentation, inSaccharomyces cerevisiae. Biotechnol. Biofuels 1 3 -    DOI : 10.1186/1754-6834-1-3
Endo A , Nakamura T , Shima J 2009 Involvement of ergosterol in tolerance to vanillin, a potential inhibitor of bioethanol fermentation, inSaccharomyces cerevisiae. FEMS Microbiol. Lett. 299 95 - 99    DOI : 10.1111/j.1574-6968.2009.01733.x
Fleischer TC , Weaver CM , McAfee KJ , Jennings JL , Link AJ 2006 Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes. Genes Dev. 20 1294 - 1307    DOI : 10.1101/gad.1422006
Galbe M , Zacchi G 2002 A review of the production of ethanol from softwood. Appl. Microbiol. Biotechnol. 59 618 - 628    DOI : 10.1007/s00253-002-1058-9
García-López MC , Mirón-García MC , Garrido-Godino AI , Mingorance C , Navarro F 2010 Overexpression of SNG1 causes 6-azauracil resistance inSaccharomyces cerevisiae. Curr. Genet. 56 251 - 263    DOI : 10.1007/s00294-010-0297-z
Grey M , Pich CT , Haase E , Brendel M 1995 SNG1- a new gene involved in nitrosoguanidine resistance inSaccharomyces cerevisiae. Mutat. Res. 346 207 - 214    DOI : 10.1016/0165-7992(95)90037-3
Hansen EH , Moller BL , Kock GR , Bunner CM , Kristensen C , Jensen OR 2009 De novobiosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker’s yeast (Saccharomyces cerevisiae). Appl. Environ. Microbiol. 75 2765 - 2774    DOI : 10.1128/AEM.02681-08
Haslbeck M , Braun N , Stromer T , Richter B , Model N , Weinkauf S , Buchner J 2004 Hsp42 is the general small heat shock protein in the cytosol ofSaccharomyces cerevisiae. EMBO J. 23 638 - 649    DOI : 10.1038/sj.emboj.7600080
Hong B , Wu K , Brockenbrough JS , Wu P , Aris JP 2001 Temperature sensitivenop2alleles defective in synthesis of 25S rRNA and large ribosomal subunits inSaccharomyces cerevisiae. Nucleic Acids Res. 29 2927 - 2937    DOI : 10.1093/nar/29.14.2927
Hubbell E , Liu WM , Mei R 2002 Robust estimators for expression analysis. Bioinformatics 18 1585 - 1592    DOI : 10.1093/bioinformatics/18.12.1585
Irizarry RA , Bolstad BM , Collin F , Cope LM , Hobbs B , Speed TP 2003 Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31 15 -    DOI : 10.1093/nar/gng015
Iwaki A , Ohnuki S , Suga Y , Izawa S , Ohya Y 2013 Vanillin inhibits translation and induces messenger ribonucleoprotein (mRNP) granule formation inSaccharomyces cerevisiae: application and validation of high-content, image-based profiling. PLoS One 8 e61748 -    DOI : 10.1371/journal.pone.0061748
Klinke HB , Thomsen AB , Ahring BK 2004 Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl. Microbiol. Biotechnol. 66 10 - 26    DOI : 10.1007/s00253-004-1642-2
Klionsky DJ , Cregg JM , Dunn WA , Emr SD , Sakai Y , Sandoval IV 2003 A unified nomenclature for yeast autophagy-related genes. Dev. Cell 5 539 - 545    DOI : 10.1016/S1534-5807(03)00296-X
Kobayashi N , McEntee K 1993 Identification ofcisand trans components of a novel heat shock stress regulatory pathway inSaccharomyces cerevisiae. Mol. Cell Biol. 13 248 - 256
Lin T , Tanaka S 2006 Ethanol fermentation from biomass resources: current state and prospects. Appl. Microbiol. Biotechnol. 69 627 - 642    DOI : 10.1007/s00253-005-0229-x
López-Malo A , Alzamora SM , Argaiz A 1995 Effect of natural vanillin on germination time and radial growth of moulds in fruit-based agar systems. Food Microbiol. 12 213 - 219    DOI : 10.1016/S0740-0020(95)80100-6
Mahmud SA , Hirasawa T , Furusawa C , Yoshikawa K , Shimizu H 2012 Understanding the mechanism of heat stress tolerance caused by high trehalose accumulation inSaccharomyces cerevisiaeusing DNA microarray. J. Biosci. Bioeng. 113 526 - 528    DOI : 10.1016/j.jbiosc.2011.11.028
Makuc J , Paiva S , Schauen M , Krämer R , André B , Casal M 2001 The putative monocarboxylate permeases of the yeastSaccharomyces cerevisiaedo not transport monocarboxylic acids across the plasma membrane. Yeast 18 1131 - 1143    DOI : 10.1002/yea.763
Minique H , Faaij A , vanden Broek R , Berndes G , Gielen D , Turkenburg W 2003 Exploration of the ranges of the global potential of biomass for energy. Biomass Bioenergy 25 119 - 133    DOI : 10.1016/S0961-9534(02)00191-5
Modig T , Liden G , Taherzadeh MJ 2002 Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem. J. 363 769 - 776    DOI : 10.1042/0264-6021:3630769
Mulford KE , Fassler JS 2011 Association of the Skn7 and Yap1 transcription factors in theSaccharomyces cerevisiaeoxidative stress response. Eukaryot. Cell 10 761 - 769    DOI : 10.1128/EC.00328-10
Nguyen TTM , Iwaki A , Ohya Y , Izawa S 2014 Vanillin cause the activation of Yap1 and mitocondrial fragmentation inSaccharomyces cerevisiae. J. Biosci. Bioeng. 117 33 - 38    DOI : 10.1016/j.jbiosc.2013.06.008
Park EH , Lee HY , Ryu YW , Seo JH , Kim MD 2011 Role of osmotic and salt stress in the expression of erythrose reductase inCandida magnoliae. J. Microbiol. Biotechnol. 21 1064 - 1068    DOI : 10.4014/jmb.1105.05029
Rivera-Carriles K , Argaiz A , Palou E , Lopez-Malo A 2005 Synergistic inhibitory effect of citral with selected phenolics againstZygosaccharomyces bailii. J. Food Prot. 68 602 - 606
Srokol Z , Bouche AG , van Estrik A , Strik RC , Maschmeyer T , Peters JA 2004 Hydrothermal upgrading of biomass to biofuel; studies on some monosaccharide model compounds. Carbohydr. Res. 339 1717 - 1726    DOI : 10.1016/j.carres.2004.04.018
Sunnarborg SW , Miller SP , Unnikrishnan I , LaPorte DC 2001 Expression of the yeast glycogen phosphorylase gene is regulated by stress-response elements and by the HOG MAP kinase pathway. Yeast 18 1505 - 1514    DOI : 10.1002/yea.752
Treger JM , Schmitt AP , Simon JR , McEntee K 1998 Transcriptional factor mutations reveal regulatory complexities of heat shock and newly identified stress genes inSaccharomyces cerevisiae. J. Biol. Chem. 273 26875 - 26879    DOI : 10.1074/jbc.273.41.26875
Trotter EW , Kao CM , Berenfeld L , Botstein D , Petsko GA , Gray JV 2002 Misfolded proteins are competent to mediate a subset of the responses to heat shock inSaccharomyces cerevisiae. J. Biol. Chem. 277 44817 - 44825    DOI : 10.1074/jbc.M204686200