Abstract

During the production of wine and beer, the yeast Saccharomyces cerevisiae can encounter an environment that is deficient in zinc, resulting in a ‘sluggish’ or a ‘stuck’ ferment. It has been shown that the Zap1p-transcription factor induces the expression of a regulon in response to zinc deficiency; however, it was evident that a separate regulon was also activated during zinc deficiency in a Zap1p-independent manner. This study discovered the Msn2p and Msn4p (Msn2/4p) transcriptional activator proteins to be an additional control mechanism inducing the stress response during zinc deficiency. Promoter sequence analysis identified the stress-response element (STRE) motif, recognized by Msn2/4p, and was significantly enriched in the promoters of genes induced by zinc deficiency. An investigation using genome-wide analyses revealed a distinct regulon consisting of STRE-containing genes whose zinc-responsive expression was abolished in an msn2 msn4 double mutant. An STRE-driven lacZ reporter construct confirmed that expression of the genes within this regulon was perturbed by the deletion of MSN2 and MSN4 and also implicated Hog1p as a contributing factor. This research provides a better understanding of the molecular mechanisms involved in the yeast response to zinc deficiency during fermentation.

Introduction

Zinc is an essential micronutrient for cellular function. It is required by many proteins for structural stability (Magonet, 1992), as a catalytic or a cocatalytic factor (Ohtsu, 2000; Auld, 2001; Ohtsu & Fukuzumi, 2001), and has an effect on cell mechanisms (Francis, 1994; Truong-Tran, 2001). Zinc also plays a vital role in beer fermentation, stimulating early ethanol production, amino utilization and the generation of aroma compounds (Vecseri-Hegyes, 2006). However, the crop quality (Kenbaev & Sade, 2002) and the processing involved in wort production (Vecseri-Hegyes, 2005) have a major influence on the zinc content available for fermentation. Therefore, it is important that suitable levels of zinc are maintained to avoid ‘sluggish’ and incomplete fermentations (Stehlik-Tomas, 1994; Bromberg, 1997; Rees & Stewart, 1998).

In Saccharomyces cerevisiae, expression of the zinc transporters ZRT1, ZRT2 and ZRT3 is induced during zinc deficiency and mediated by the transcriptional activator, Zap1p (Zhao & Eide, 1997; Zhao, 1998). Using these genes, a conserved 11-bp consensus sequence, 5′-ACCYYNAAGGT-3′, known as the zinc-responsive element (ZRE), was identified and shown to be necessary for zinc-responsive transcriptional regulation via Zap1p (Zhao, 1998). An elegant study by Lyons (2000) used genome-wide expression analysis to determine an extended set of genes that form part of the zinc regulon in yeast controlled by Zap1p. A high proportion of the genes, 458, were induced during zinc deficiency, with 24% of these containing the ZRE or ZRE-like binding motifs (Lyons, 2000). A relatively small proportion, 46, of the induced genes was shown to be regulated by Zap1p, indicating that the remaining genes induced during zinc deficiency may be controlled by unknown transcriptional regulators. Recently, the zinc-responsive regulon for Zap1p has been further characterized by examining the differential regulation of Zap1p targets, but no other regulatory proteins were identified (De Nicola, 2007; Wu, 2008).

Expression analysis in an industrial strain of S. cerevisiae under zinc-deficient growth conditions with maltose as the main carbon source (Higgins, 2003) identified a significant overlap with genes induced in the Lyons (2000) study, but the presence of genes that were known to be regulated by the homologous Msn2p and Msn4p (Msn2/4p) transcription factors was observed. Msn2/4p induces the expression of genes under conditions of stress via interaction with the consensus sequence, 5′-AGGGG-3′, known as the stress-response element (STRE) (Marchler, 1993; Martínez-Pastor, 1996). Under stress conditions, it has been shown that a number of factors such as Hog1p, Whi2p/Psr1p and Glc7p/Bud14p activate Msn2/4p, which results in the increased expression of its target genes (Schüller, 1994; Kaida, 2002; Lenssen, 2005). Under nonstress conditions, Msn2/4p activity is downregulated by the Ras-cAMP-PKA pathway (Bissinger, 1989; Smith, 1998). To determine whether zinc deficiency includes a stress response mediated by Msn2/4p, interrogation of the promoter regions of zinc-responsive genes, microarray analysis of mutant strains and functional protein assays were carried out in this study.

Materials and methods

Yeast strains, media and culture conditions

The yeast strains used were W303-1A (MATaade2-1 leu2-3,112 ura3-1 trp1-1 his3-11,15 can1-100) and Δmsn2,4 (W303-1A msn2-3HIS3 msn4-1TRP1). W303-1A-STRE-lacZ (W303-1A URA3STRE-lacZ), Δmsn2,4-STRE-lacZmsn2,msn4 URA3∷STRE-lacZ), Δbud14 (W303-1A-STRE-lacZ bud14kanMX4), Δras2 (W303-1A-STRE-lacZ ras2kanMX4) and Δwhi2 (W303-1A-STRE-lacZ whi2kanMX4) were created in this study. Δhog1 (W303-1A hog1TRP1) was obtained from Schüller (1994). Limiting zinc medium (LZM) was prepared in the same manner as the limiting lron medium prepared by Eide & Guarente (1992), except that zinc (not iron) was the metal excluded from the metal stock. Under non-zinc-limiting conditions, zinc was added to a final concentration of 12 mg L−1. Strains were grown overnight to the mid-log phase (OD600 nm=0.5) at 30 °C, cells were harvested at 6000 g for 5 min and washed three times with distilled water. Cells were then transferred to LZM with zinc (LZM+Zn) and LZM with an initial OD600 nm=0.1, grown to the mid-log phase and harvested before RNA isolation or β-galactosidase (β-gal) assays were performed.

DNA manipulations

Plasmid CTT1-18 (CTT1-18/7x-lacZ), described previously by Marchler (1993), was digested with NcoI (Promega) and transformed into W303-1A [wild type (WT)], Δmsn2,4 and Δhog1, yielding strains W303-1A-STRE-lacZ, Δmsn2,4-STRE-lacZ and Δhog1-STRE-lacZ, respectively. Transformants were selected for uracil prototrophy.

In strain W303-1A-STRE-lacZ, deletions for BUD14, RAS2 and WHI2 were achieved using the primers: 5′-CGCAAGAGTCAGACTGACTCG-3′, 5′-ACTACCTCCTCAACCCCAGTT-3′; 5′-TGACATTTAGGACGGTGAAGC-3′, 5′-TACGTTCTCTTCTGTGAGGCG-3′; and 5′-TTTCTTTTCTCCCCCCAAAG-3′, 5′-TGTACGACTTTATTATGCGGG-3′, respectively, to amplify the specific gene deletion in BY4743 mutants (Euroscarf, Frankfurt, Germany). The PCR fragments generated were transformed into W303-1A-STRE-lacZ and selected for geneticin resistance. Mutants were then confirmed by PCR using the primers 5′-TTTCCAAGCAGATCCGGTGAT-3′, 5′-TGTTGGGATTCCATTGTTGA-3′; 5′-TCTTGAGTGACGATCGTTTGT-3′, 5′-AAATAGCTCTCGGGCGAATA-3′; and 5′-ATAGTGCGAAGAACGGCAAA-3′, 5′-AT-CGAATGCATACAGGCCTA-3′ for BUD14, RAS2 and WHI2, respectively, and NcoI (Promega) digestion.

Regulatory sequence analysis

Gene data sets from De Nicola (2007), Higgins (2003), Lyons (2000) and Wu (2008) and this study were analysed for significant regulatory motifs using regulatory sequence analysis tools (rsat) (Van Helden, 1998). When searching for motifs, five to eight oligonucleotide sizes were chosen for analysis. The P-value represents the probability that the motif could occur in the set of genes by chance.

Microarray description, RNA labelling and hybridization

Saccharomyces cerevisiae microarray slides were obtained from the Ramaciotti Centre for Gene Function Analysis (Sydney, NSW, Australia). Slides were Schott Nexterion® Slide A+ with an amino-link coating (Schott, Mainz, Germany) and spotted with 40-mer oligonucleotide probes for 6250 yeast ORFs (Version MWGSc6K; MWG Biotech, Ebersburg, Germany) in duplicate. Slides were preprocessed by baking at 120 °C and blocking with 5% (v/v) ethanol as per the manufacturer's instructions (Ramaciotti Centre for Gene Function Analysis).

Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA) as outlined previously (Alic, 2004). The integrity of the RNA was analysed using an RNA 6000 Nano LabChip® on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA (20 μg) was reverse transcribed, labelled and hybridized as outlined previously (Alic, 2004) for the slides labelled with cyanine dyes, except that yeast tRNA was used instead of Escherichia coli tRNA. After hybridization, the slides were washed in 2 × saline sodium citrate (SSC), 0.2% sodium dodecyl sulphate (SDS) for 10 min, 2 × SSC for 10 min and 0.2 × SSC for 10 min before drying the slides by centrifugation. Biological duplicates were analysed in technical duplicate using a dye swap and slides were scanned using an Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA).

Data acquisition

Image analysis of the microarray slides was performed using genepix pro 6.0 (Molecular Devices). A signal for each gene was determined to be ‘present’ if there were no artefacts associated with the spot and the program could identify the spot intensity above the background intensity. Normalization was performed on the data using the LOWESS method in the genespring gx 7.3.1 (Agilent Technologies) analysis software package. The genes whose expression ratio (WT/double mutant) was significantly different from unity were identified based on Welch's analysis of one-way anova, where the variances were not assumed to be equal and a level of significance of 0.05 was set. The complete raw microarray data set is available at the gene expression omnibus database (accession number: GSE11878).

β-Gal assays

β-Gal activity was measured as described (Rose & Botstein, 1983; Beckhouse, 2008), and activity units were calculated as follows: [A420 nm× total reaction volume (mL)]/[0.0045 × incubation time (min) × extract volume (mL) × protein concentration (mg mL−1)]. The concentration of protein was determined using the Bradford method (Bradford, 1976).

Fold induction for each strain tested was determined by dividing LZM β-gal activity by LZM+Zn β-gal activity. Statistical analysis on these fold induction levels for each strain was performed using a one-way anova with Tukey's comparison method and was used to highlight where the fold induction levels differ if they do (Montgomery, 2001). The anova determines whether the mean fold induction levels of at least one pair of WT or mutants were different. Using Tukey's comparison method, we were able to determine for each pair of fold induction levels whether their differences were statistically and significantly different from zero. Such a test was carried out using a 0.05 level of significance. All statistical computation was carried out using minitab (version 15) (Minitab Inc., PA).

Results

Promoter analysis reveals an Msn2/4p consensus sequence in genes responsive to zinc deficiency

The analysis of the promoter regions of genes induced during the exposure of yeast to zinc-deficient culture conditions was performed using promoter analysis software, rsat (Van Helden, 1998). Two core consensus sequences for known transcriptional activator proteins were identified from this analysis (Table 1). The sequence 5′-AGGGG-3′ was significantly overrepresented in the promoters of induced genes under zinc-deficient conditions in industrial (Higgins, 2003) and laboratory (Lyons, 2000; Wu, 2008) yeast strains, but was not identified from the results presented by De Nicola (2007). Identification of the STRE strongly indicated that the Msn2/4p transcriptional activators may play a role in regulating gene expression under zinc-deficient conditions. Additionally, the promoter analysis identified a number of other promising motifs to which no known transcriptional activator binds; however, it seems likely that these result from variations of the STRE motif. As expected, the Zap1p consensus core sequence, 5′-ACCYYNAAGGT-3′, was also overrepresented in upregulated genes.

1

Promoter analysis of genes induced during zinc deficiency

Gene cluster Promoter element Putative-binding protein P-value 
Forward Reverse 
All upregulated genes in an industrial strain of S. cerevisiae (Higgins, 2003AGGGG CCCCT Msn2/4p 1.7 × 10−7 
ATGGG CCCAT – 8.7 × 10−6 
AAGGG AAGGG – 2.8 × 10−4 
CCCCC GGGGG – 1.89 × 10−3 
GGGGA TCCCC – 1.89 × 10−3 
CCCTTA TAAGGG – 8.6 × 10−9 
CCCATA TATGGG – 3.3 × 10−6 
CCCCTA TAGGGG – 3.3 × 10−5 
AGGGAG CTCCCT – 6.0 × 10−5 
CCCCTTA TAAGGGG – 1.0 × 10−4 
CCCTAAAG CTTTAGGG Zap1p 1.6 × 10−5 
All upregulated genes in a laboratory strain of S. cerevisiae (Lyons, 2000AGGGG CCCCT Msn2/4p 2.0 × 10−9 
AAGGG CCCTT – 8.8 × 10−9 
ACCCC GGGGT – 1.8 × 10−5 
CCCCC CCCCC – 1.5 × 10−4 
GGGGA TCCCC – 2.2 × 10−4 
CCCTTA TAAGGG – 3.1 × 10−7 
AGGGAG CTCCCT – 2.8 × 10−6 
AAGGGG CCCCTT – 3.9 × 10−6 
CCCCTA TAGGGG – 9.3 × 10−6 
AAGGGAG CTCCCTT – 3.4 × 10−6 
CCCCTAA TTAGGGG – 4.4 × 10−6 
CCTTGAA TTCAAGG – 9.9 × 10−6 
CCTTGAAG* CTTCAAGG Zap1p 5.4 × 10−7 
CCCTTGAA TTCAAGGG Zap1p 2.6 × 10−6 
Microarray results for Zap1p targets from time-course and dose–response studies (Wu, 2008AGGGG CCCCT Msn2/4p 4.6 × 10−4 
AAGGG CCCTT – 5.3 × 10−4 
AGGGGG CCCCCT – 2.8 × 10−4 
CCCTAAAG CTTTAGGG* Zap1p 2.1 × 10−5 
Gene cluster Promoter element Putative-binding protein P-value 
Forward Reverse 
All upregulated genes in an industrial strain of S. cerevisiae (Higgins, 2003AGGGG CCCCT Msn2/4p 1.7 × 10−7 
ATGGG CCCAT – 8.7 × 10−6 
AAGGG AAGGG – 2.8 × 10−4 
CCCCC GGGGG – 1.89 × 10−3 
GGGGA TCCCC – 1.89 × 10−3 
CCCTTA TAAGGG – 8.6 × 10−9 
CCCATA TATGGG – 3.3 × 10−6 
CCCCTA TAGGGG – 3.3 × 10−5 
AGGGAG CTCCCT – 6.0 × 10−5 
CCCCTTA TAAGGGG – 1.0 × 10−4 
CCCTAAAG CTTTAGGG Zap1p 1.6 × 10−5 
All upregulated genes in a laboratory strain of S. cerevisiae (Lyons, 2000AGGGG CCCCT Msn2/4p 2.0 × 10−9 
AAGGG CCCTT – 8.8 × 10−9 
ACCCC GGGGT – 1.8 × 10−5 
CCCCC CCCCC – 1.5 × 10−4 
GGGGA TCCCC – 2.2 × 10−4 
CCCTTA TAAGGG – 3.1 × 10−7 
AGGGAG CTCCCT – 2.8 × 10−6 
AAGGGG CCCCTT – 3.9 × 10−6 
CCCCTA TAGGGG – 9.3 × 10−6 
AAGGGAG CTCCCTT – 3.4 × 10−6 
CCCCTAA TTAGGGG – 4.4 × 10−6 
CCTTGAA TTCAAGG – 9.9 × 10−6 
CCTTGAAG* CTTCAAGG Zap1p 5.4 × 10−7 
CCCTTGAA TTCAAGGG Zap1p 2.6 × 10−6 
Microarray results for Zap1p targets from time-course and dose–response studies (Wu, 2008AGGGG CCCCT Msn2/4p 4.6 × 10−4 
AAGGG CCCTT – 5.3 × 10−4 
AGGGGG CCCCCT – 2.8 × 10−4 
CCCTAAAG CTTTAGGG* Zap1p 2.1 × 10−5 

The sequences are shown significantly over-represented as possible promoter motifs in industrial and laboratory strains of Saccharomyces cerevisiae.

*

Based on the consensus ZRE sequence, 5′-ACCYYNAAGGT-3′ (Lyons, 2000).

Sequence derived by rsat analysis of putative Zap1p-regulated genes in an industrial strain of S. cerevisiae (Higgins, 2003).

Microarray analysis of the msn2 msn4 double mutant during zinc deficiency

To identify whether Msn2/4p are active inducers of gene expression during zinc deficiency, we performed a genome-wide expression analysis on zinc-deficient WT cells in comparison with zinc-deficient cells of the msn2,4 double mutant. During zinc deficiency, 141 genes were differentially expressed between the WT and the msn2,4 double mutant strains. Of these genes, 73 were significantly reduced in the msn2,4 double mutant compared with the WT (Table 2 and Supporting Information, Table S1). It was then of interest to determine whether these 73 differentially expressed genes could be regulated by Msn2/4p. This was performed by analysing the promoter regions of the gene set using rsat (Van Helden, 1998). The analysis identified that the STRE motif is a highly significant sequence enriched in this regulon with a significance index twofold higher than the entire gene set (141 genes). This greater value of significance indicates that the STRE is likely to be a regulatory element. Of the 73 genes, 72% contained the consensus sequence for the STRE within their promoter region (Table 2). Further rsat analysis of these genes revealed that they contained no consensus sequence for Zap1p and that many of the possible significant sequences derived contained the STRE motif.

2

Potential Msn2/4p target genes

Gene name Description Number of STREs Significance index* Expression fold change P-value* 
Genes with consensus STREs (AGGGG) 
YNR034W-A Protein of unknown function 30.97 240.4 5.18 × 10−3 
DDR2 DNA damage-responsive 2 protein 4 30.97 147.3 3.18 × 10−3 
HXK1 Hexokinase isoenzyme I 5 30.97 138.7 1.26 × 10−4 
GPH1 Glycogen phosphorylase 3 30.97 61.3 1.27 × 10−4 
YER067W Protein of unknown function, high similarity to uncharacterized S. cerevisiae Yil057p 30.97 54.9 5.58 × 10−4 
HSP12 Heat-shock protein of 12 kDa 7 30.97 46.5 1.01 × 10−2 
RTC3 Protein of unknown function involved in RNA metabolism 30.97 44.8 1.78 × 10−2 
STF2 Protein of unknown function, high similarity to uncharacterized C. glabrata Cagl0f08745gp 30.97 38.8 7.34 × 10−3 
PGM2 Phosphoglucomutase 2 5 30.97 38.6 1.39 × 10−3 
HXT7 High-affinity glucose transporter nearly identical to Hxt6p 30.97 27.9 1.56 × 10−3 
HXT6 High-affinity glucose transporter nearly identical to Hxt7p 30.97 22.1 2.50 × 10−3 
GLC3 Glycogen-branching enzyme 30.97 19.7 9.86 × 10−3 
CTT1 Catalase T 1 4 30.97 17.1 4.26 × 10−2 
HSP26 Heat-shock protein of 26 kDa 4 30.97 13.1 6.41 × 10−4 
MSC1 Protein that affects meiotic homologous chromatid recombination 2 30.97 11.1 4.40 × 10−2 
ARG1 Argininosuccinate synthetase 30.97 8.2 4.10 × 10−4 
DCS2 Nonessential, stress-induced regulatory protein 30.97 7.8 4.24 × 10−2 
HOR7 Protein of unknown function 30.97 7.4 6.03 × 10−3 
YIL169C Protein of unknown function 30.97 6.8 8.45 × 10−3 
GPX1 Phospholipid hydroperoxide glutathione peroxidase 30.97 6.8 2.20 × 10−2 
USV1 Putative transcription factor containing a C2H2 zinc finger 30.97 6.8 1.36 × 10−2 
SPI1 Stationary phase-induced 1 protein 3 30.97 6.7 5.65 × 10−5 
URA10 Orotate phosphoribosyltransferase 2 30.97 6.1 9.59 × 10−3 
YGP1 Cell wall-related secretory glycoprotein 30.97 5.2 7.31 × 10−3 
NCE102 Protein of unknown function 30.97 5.1 1.98 × 10−3 
HSP31 Heat-shock protein 31 30.97 4.5 7.00 × 10−4 
CRS5 Copper-binding metallothionein 30.97 3.9 2.64 × 10−3 
TFS1 Carboxypeptidase Y inhibitor 30.97 3.7 9.89 × 10−3 
COX5B Subunit Vb of cytochrome c oxidase 30.97 3.4 1.51 × 10−2 
YNL300W Glycosylphosphatidylinositol-dependent cell-wall protein 30.97 3.4 3.04 × 10−4 
TMA10 Protein of unknown function that associates with ribosomes 30.97 3.4 8.25 × 10−3 
PBI2 Cytosolic inhibitor of vacuolar proteinase B 30.97 3.3 2.79 × 10−3 
ALD4 Mitochondrial aldehyde dehydrogenase 30.97 3.3 7.78 × 10−3 
YJR096W Putative xylose and arabinose reductase 30.97 3.1 1.69 × 10−3 
MCR1 Mitochondrial NADH-cytochrome b5 reductase, involved in ergosterol biosynthesis 30.97 3.0 1.86 × 10−2 
SOD2 Mitochondrial superoxide dismutase 30.97 3.0 9.00 × 10−3 
YJR008W Putative protein of unknown function 30.97 3.0 3.36 × 10−3 
QNQ1 Protein of unknown function 30.97 2.9 2.91 × 10−2 
OM45 Protein of unknown function, major constituent of the mitochondrial outer membrane 30.97 2.9 3.70 × 10−2 
DDR48 Stress protein induced by heat shock, DNA damage, or osmotic stress 2 30.97 2.9 2.08 × 10−2 
TDH1 Glyceraldehyde-3-phosphate dehydrogenase, isozyme 1 30.97 2.8 6.27 × 10−3 
TPS2 Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex 30.97 2.8 2.03 × 10−3 
MMF1 Mitochondrial protein involved in maintenance of the mitochondrial genome 30.97 2.7 5.26 × 10−3 
FMP16 Putative protein of unknown function 30.97 2.7 3.96 × 10−2 
FMP46 Putative redox protein 30.97 2.6 2.82 × 10−3 
YER053C-A Putative protein of unknown function 30.97 2.6 6.07 × 10−3 
YMR291W Putative kinase of unknown function 30.97 2.6 5.05 × 10−3 
MDH1 Mitochondrial malate dehydrogenase 30.97 2.5 3.83 × 10−2 
ICY1 Protein of unknown function 30.97 2.5 1.55 × 10−2 
GSP2 GTP-binding protein 30.97 2.3 2.03 × 10−2 
COS8 Nuclear membrane protein 30.97 2.3 3.12 × 10−3 
PRB1 Vacuolar protease B 30.97 2.2 2.64 × 10−2 
RIB5 Riboflavin synthase 30.97 2.1 4.95 × 10−2 
FMP10 Protein of unknown function 30.97 2.1 5.79 × 10−4 
Gene name Description Number of STREs Significance index* Expression fold change P-value* 
Genes with consensus STREs (AGGGG) 
YNR034W-A Protein of unknown function 30.97 240.4 5.18 × 10−3 
DDR2 DNA damage-responsive 2 protein 4 30.97 147.3 3.18 × 10−3 
HXK1 Hexokinase isoenzyme I 5 30.97 138.7 1.26 × 10−4 
GPH1 Glycogen phosphorylase 3 30.97 61.3 1.27 × 10−4 
YER067W Protein of unknown function, high similarity to uncharacterized S. cerevisiae Yil057p 30.97 54.9 5.58 × 10−4 
HSP12 Heat-shock protein of 12 kDa 7 30.97 46.5 1.01 × 10−2 
RTC3 Protein of unknown function involved in RNA metabolism 30.97 44.8 1.78 × 10−2 
STF2 Protein of unknown function, high similarity to uncharacterized C. glabrata Cagl0f08745gp 30.97 38.8 7.34 × 10−3 
PGM2 Phosphoglucomutase 2 5 30.97 38.6 1.39 × 10−3 
HXT7 High-affinity glucose transporter nearly identical to Hxt6p 30.97 27.9 1.56 × 10−3 
HXT6 High-affinity glucose transporter nearly identical to Hxt7p 30.97 22.1 2.50 × 10−3 
GLC3 Glycogen-branching enzyme 30.97 19.7 9.86 × 10−3 
CTT1 Catalase T 1 4 30.97 17.1 4.26 × 10−2 
HSP26 Heat-shock protein of 26 kDa 4 30.97 13.1 6.41 × 10−4 
MSC1 Protein that affects meiotic homologous chromatid recombination 2 30.97 11.1 4.40 × 10−2 
ARG1 Argininosuccinate synthetase 30.97 8.2 4.10 × 10−4 
DCS2 Nonessential, stress-induced regulatory protein 30.97 7.8 4.24 × 10−2 
HOR7 Protein of unknown function 30.97 7.4 6.03 × 10−3 
YIL169C Protein of unknown function 30.97 6.8 8.45 × 10−3 
GPX1 Phospholipid hydroperoxide glutathione peroxidase 30.97 6.8 2.20 × 10−2 
USV1 Putative transcription factor containing a C2H2 zinc finger 30.97 6.8 1.36 × 10−2 
SPI1 Stationary phase-induced 1 protein 3 30.97 6.7 5.65 × 10−5 
URA10 Orotate phosphoribosyltransferase 2 30.97 6.1 9.59 × 10−3 
YGP1 Cell wall-related secretory glycoprotein 30.97 5.2 7.31 × 10−3 
NCE102 Protein of unknown function 30.97 5.1 1.98 × 10−3 
HSP31 Heat-shock protein 31 30.97 4.5 7.00 × 10−4 
CRS5 Copper-binding metallothionein 30.97 3.9 2.64 × 10−3 
TFS1 Carboxypeptidase Y inhibitor 30.97 3.7 9.89 × 10−3 
COX5B Subunit Vb of cytochrome c oxidase 30.97 3.4 1.51 × 10−2 
YNL300W Glycosylphosphatidylinositol-dependent cell-wall protein 30.97 3.4 3.04 × 10−4 
TMA10 Protein of unknown function that associates with ribosomes 30.97 3.4 8.25 × 10−3 
PBI2 Cytosolic inhibitor of vacuolar proteinase B 30.97 3.3 2.79 × 10−3 
ALD4 Mitochondrial aldehyde dehydrogenase 30.97 3.3 7.78 × 10−3 
YJR096W Putative xylose and arabinose reductase 30.97 3.1 1.69 × 10−3 
MCR1 Mitochondrial NADH-cytochrome b5 reductase, involved in ergosterol biosynthesis 30.97 3.0 1.86 × 10−2 
SOD2 Mitochondrial superoxide dismutase 30.97 3.0 9.00 × 10−3 
YJR008W Putative protein of unknown function 30.97 3.0 3.36 × 10−3 
QNQ1 Protein of unknown function 30.97 2.9 2.91 × 10−2 
OM45 Protein of unknown function, major constituent of the mitochondrial outer membrane 30.97 2.9 3.70 × 10−2 
DDR48 Stress protein induced by heat shock, DNA damage, or osmotic stress 2 30.97 2.9 2.08 × 10−2 
TDH1 Glyceraldehyde-3-phosphate dehydrogenase, isozyme 1 30.97 2.8 6.27 × 10−3 
TPS2 Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex 30.97 2.8 2.03 × 10−3 
MMF1 Mitochondrial protein involved in maintenance of the mitochondrial genome 30.97 2.7 5.26 × 10−3 
FMP16 Putative protein of unknown function 30.97 2.7 3.96 × 10−2 
FMP46 Putative redox protein 30.97 2.6 2.82 × 10−3 
YER053C-A Putative protein of unknown function 30.97 2.6 6.07 × 10−3 
YMR291W Putative kinase of unknown function 30.97 2.6 5.05 × 10−3 
MDH1 Mitochondrial malate dehydrogenase 30.97 2.5 3.83 × 10−2 
ICY1 Protein of unknown function 30.97 2.5 1.55 × 10−2 
GSP2 GTP-binding protein 30.97 2.3 2.03 × 10−2 
COS8 Nuclear membrane protein 30.97 2.3 3.12 × 10−3 
PRB1 Vacuolar protease B 30.97 2.2 2.64 × 10−2 
RIB5 Riboflavin synthase 30.97 2.1 4.95 × 10−2 
FMP10 Protein of unknown function 30.97 2.1 5.79 × 10−4 

The genes listed showed Msn2/4p-dependent regulation pattern whose promoters contain sequences that match the previously published STRE-consensus sequence, 5′-AGGGG-3′.

Msn2/4p-dependent regulation of genes in bold has been confirmed independently either in this study or in other reports by lacZ-reporter fusions and/or Northern blotting.

*

Calculated for each sequence by rsat program. The significance index is the minus log transform of the E-value.

Probability that the genes by chance alone are regulated by Msn2/4p. The P-values obtained from the raw microarray data and normalized using the LOWESS method and Welch's parametric anova. Each value is the average of three biological arrays with dye swaps.

Of the remaining genes that did not contain the consensus STRE sequence, 62% contained STRE-like sequences and 38% contained no STRE or STRE-like sequences, and yet their expression was considerably affected by the absence of Msn2/4p (Table S1). Although these 16 genes show no direct link to Msn2/4p due to the absence of the consensus STRE sequence, it may be possible that Msn2/4p recognizes a highly similar sequence for gene induction or plays a role with other factors that affects the expression of these genes.

These results revealed a general trend for the induction of genes containing STREs within their promoters. Genes that have a fold increase >10 contain, on average, four STRE motifs and genes with lower induction levels have on average two to three STRE motifs (Table 2). However, as with most motifs, there were exceptions. Some highly expressed genes such as RTC3 and others were found to contain relatively few STREs whereas HSP12, which contains seven STREs, was found to show a fold change lower than expected. The results obtained from genome-wide and promoter analyses show strong evidence to indicate that the Msn2/4p complex plays a role in regulating gene expression under zinc-deficient growth conditions.

Msn2/4p-dependent regulation of gene expression during zinc deficiency

A strain harbouring mutations in both the MSN2 and the MSN4 genes in this study was shown to impair the expression of STRE-regulated genes during zinc deficiency. To provide further evidence of Msn2/4p-dependent regulation, the level of protein activity was examined using a reporter construct consisting of the STRE-containing promoter of the CTT1 gene fused in-frame with the bacterial lacZ gene (STRE-lacZ) (Marchler, 1993). WT and the msn2,4 double mutant strains were transformed with the STRE-lacZ reporter construct and were grown to the logarithmic phase. Cells were then transferred to zinc-replete (LZM+Zn) and zinc-deficient (LZM) media and the level of β-gal activity was assayed (Fig. 1a). The WT strain showed increased β-gal activity during zinc deficiency, further validating the zinc-dependent transcription of STRE-containing genes identified in the microarray studies. In contrast, β-gal activity in the msn2,4 double mutant was severely reduced in the absence of zinc, confirming that gene induction during zinc deficiency was Msn2/4p dependent.

1

Zinc responsiveness of the STRE driven-lacZ reporter construct. (a) Yeast strains, W303 WT, Δmsn2,4, Δbud14, Δhog1, Δras2 and Δwhi2 were grown and shifted to conditions with normal zinc (LZM+Zn) and limited zinc (LZM) until samples in the logarithmic phase (≈OD 0.5) were collected and β-gal activity was measured. (b) The fold induction for each strain represents the ratio of induction in zinc limitation to normal zinc. The asterisk above Δhog1 denotes Tukey's significance. Error bars in both figures represent SD from mean.

1

Zinc responsiveness of the STRE driven-lacZ reporter construct. (a) Yeast strains, W303 WT, Δmsn2,4, Δbud14, Δhog1, Δras2 and Δwhi2 were grown and shifted to conditions with normal zinc (LZM+Zn) and limited zinc (LZM) until samples in the logarithmic phase (≈OD 0.5) were collected and β-gal activity was measured. (b) The fold induction for each strain represents the ratio of induction in zinc limitation to normal zinc. The asterisk above Δhog1 denotes Tukey's significance. Error bars in both figures represent SD from mean.

Numerous upstream factors, such as Bud14p, Hog1p, Whi2p and Ras2p, have previously been associated with Msn2/4p activation of transcription (Schüller, 1994; Kaida, 2002; Lenssen, 2005). Therefore, we chose to examine the effect of the deletions of each of the upstream factors on Msn2/4p activity during zinc deficiency (Fig. 1a). In the strain lacking HOG1, β-gal activity was reduced approximately fivefold under zinc-deficient conditions when compared with the WT strain, which resulted in a decrease in the overall extent of induction to about half that of the WT (Fig. 1b). Using Tukey's comparison method (Montgomery, 2001), the mean fold induction level for Δhog1 was significantly dissimilar to WT, but significantly similar to the msn2,4 double mutant (95% confidence), indicating that Hog1p may play a role in positively regulating Msn2/4p function. Under zinc-replete and zinc-deficient conditions, it was observed that the deletion of RAS2 induced large amounts of β-gal activity in comparison with the WT. Although β-gal activity was increased dramatically, the degree of induction shown in Fig. 1b was statistically similar to that of the WT as was the response in the bud14 and whi2 mutant strains. The whi2 mutation exhibited low levels of Msn2/4p activation according to the reporter assay in comparison with the WT (Fig. 1a), indicating that Msn2/4p activity may be affected by its absence. However, the extent of change reported for Δwhi2 under replete and deficient conditions (Fig. 1b) was similar, as indicated by Tukey's comparison, to that of the WT, indicating that the reduced reporter activity was due to other mechanisms not involved with zinc metabolism.

Discussion

Environmental niches introduce cells to dynamic conditions under which nutrients can be limiting or abundant. Cells must adjust their genomic expression program rapidly to adapt to these new conditions. Industrial fermentations can present the yeast with conditions requiring such an adaptation. Zinc availability is of major concern during the fermentation process because the deficiency results in sluggish and incomplete fermentation. When yeast cells are exposed to zinc-deficient conditions, Zap1p induces the expression of genes involved in increasing cellular zinc levels (Zhao & Eide, 1997; Lyons, 2000). Interestingly, a large subset of genes that were regulated in a Zap1p-independent manner was also induced under zinc-deficient growth conditions. The presence of an overrepresented STRE motif in these genes supported the hypothesis proposed by Higgins (2003) that Msn2/4p may play a role in inducing the expression of genes during zinc deficiency in addition to the induction of STRE-regulated genes in the presence of a variety of other stresses such as osmotic, oxidative, heat, pressure and nitrogen starvation (Kobayashi & McEntee, 1993; Martínez-Pastor, 1996; Gasch, 2000; Causton, 2001; Hasan, 2002; Domitrovic, 2006). These upregulated genes harbouring the STRE motif were also identified in zinc limitation studies (Lyons, 2000; Higgins, 2003; Wu, 2008), adding further weight to the findings that Msn2/4p plays a role in the stress of zinc deficiency. Interestingly, the STRE motif was not identified in the upregulated gene set from the data presented by De Nicola (2007). The De Nicola (2007) study used continuously fed chemostat cultures instead of batch cultures and added enough zinc to the medium to sustain yeast growth. This is in contrast to the present and other zinc-deficiency studies where zinc availability was decreased to a level that does not support long-term yeast growth, and thus better reflects the conditions found in industrial ‘stuck’ fermentations where yeast growth and fermentation cease. Additionally, this also suggests that the induction of the Msn2/4p regulon is a stress response as a result of zinc starvation rather than a cellular mechanism to overcome zinc deficiency.

A number of motifs with high homology to the STRE, but not identical, were also identified in the promoters of genes affected by the msn2,4 mutation. This suggests that they may function as activating sequences for Msn2/4p in a manner similar to the results of Lyons (2000), where ZRE-like sequences were present in genes that have been confirmed to be regulated by Zap1p. An example of this is the HSP30 gene, which was highly induced during zinc deficiency, but does not harbour the consensus STRE or ZRE motif. However, it does contain an STRE-like motif, 5′-AAGGG-3′, which may be functional. Mutational analysis of the core element of the consensus STRE (Martínez-Pastor, 1996) did not indicate that this predicted STRE-like motif would not be functional, and since that report, it has been shown that Msn2/4p does affect HSP30 induction (Hahn & Thiele, 2004; Schüller, 2004). Therefore, it may be possible that Msn2/4p can induce gene expression through this STRE-like element.

The transcriptional activation of the reporter gene contained within the CTT1-18/7x plasmid is regulated by STREs derived from the CTT1 gene (Marchler, 1993). The STRE-lacZ reporter showed that STRE-dependent expression in the msn2,4 double mutant was markedly affected and basal activity for the reporter gene was undetected. Induction was completely abolished when zinc was absent, providing compelling evidence to support Msn2/4p as an additional inducer of gene expression during this stress. Since we had provided evidence at the molecular level that Msn2/4p directs the expression of a specific regulon during zinc starvation, we sought to identify the molecular mechanism accountable for Msn2/4p activation for this condition. Screening of a number of mutants involved in controlling Msn2/4p activity revealed that its activation due to zinc starvation may be linked to HOG1. This mitogen-activated protein kinase has also been shown to regulate the expression of STRE-driven genes during osmotic stress (Schüller, 1994; Rep, 2000). Recent research has shown that the role of Hog1p extends more widely than just in response to osmotic stress to include other stress conditions such as exposure to heat, oxidants, citric acid, cesium chloride and arsenite (Winkler, 2002; Bilsland, 2004; Lawrence, 2004; Thorsen, 2006; Del Vescovo, 2008). Screening of the RAS2 mutant showed that Ras2p and hence the protein kinase A (PKA) pathway negatively regulate Msn2/4p activity during zinc deficiency. The PKA signal-transduction pathway regulates the nucleocytoplasmic shuttling of this transcription factor complex, and hence gene transcription, under conditions of stress (Jacquet, 2003; Garmendia-Torres, 2007). It is widely known that the PKA signal-transduction pathway negatively regulates Msn2/4p activity, resulting in its retention in the cytoplasm to prevent the activation of STRE-responsive genes under conditions of no stress (Bissinger, 1989; Görner, 1998; Smith, 1998), and it is therefore very interesting that this pathway also affects Msn2/4p activity during zinc starvation.

These results characterize a larger proportion of yeast genes that are upregulated in response to zinc deficiency and confirm that they are part of the Msn2/4p regulon. The molecular mechanisms controlling this regulon are similar to those for other stress conditions, which suggests that this is the cells’ defence against zinc starvation. This research provides fermentation scientists with an improved understanding of the yeast response to zinc limitation and starvation, which may provide opportunities to overcome this challenge.

Authors' contribution

V.J.H, P.J.R. and I.W.D. generated hypotheses and the experimental outline; V.J.G., V.J.H. and V.L. conducted the experiments; V.J.G., A.G.B., E.J.B. and V.J.H. analysed the data; and V.J.G., A.G.B., I.W.D. and V.J.H. prepared the manuscript.

Acknowledgements

Vincent Higgins gratefully acknowledges support from the Australian Research Council's Linkage Projects funding scheme (project number LP0775238), Foster's Group Limited and The University of Western Sydney. The authors are also grateful for the technical support of Kellie McNamara. The authors are grateful to G. Marchler for providing the CTT1-18/7x plasmid and C. Schüller for providing the HOG1 mutant strain for use in this study.

References

Alic
N
Felder
T
Temple
MD
Gloeckner
C
Higgins
VJ
Briza
P
Dawes
IW
(
2004
)
Genome-wide transcriptional responses to a lipid hydroperoxide: adaptation occurs without induction of oxidant defenses
.
Free Radical Bio Med
 
37
:
23
35
.
Auld
DS
(
2001
)
Zinc coordination sphere in biochemical zinc sites
.
Biometals
 
14
:
271
313
.
Beckhouse
AG
Grant
CM
Rogers
PJ
Dawes
IW
Higgins
VJ
(
2008
)
The adaptive response of anaerobically grown Saccharomyces cerevisiae to hydrogen peroxide is mediated by the yap1 and Skn7 transcription factors
.
FEMS Yeast Res
 
8
:
1214
1222
.
Bilsland
E
Molin
C
Swaminathan
S
Ramne
A
Sunnerhagen
P
(
2004
)
Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance
.
Mol Microbiol
 
53
:
1743
1756
.
Bissinger
PH
Wieser
R
Hamilton
B
Ruis
H
(
1989
)
Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via RAS-Cyclic AMP pathway
.
Mol Cell Biol
 
9
:
1309
1315
.
Bradford
MM
(
1976
)
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding
.
Anal Biochem
 
72
:
248
254
.
Bromberg
SK
Bower
PA
Duncombe
GR
Fehring
J
Gerber
L
Lau
VK
Tata
M
(
1997
)
Requirements for zinc, manganese, calcium, and magnesium in wort
.
J Am Soc Brew Chem
 
55
:
123
128
.
Causton
HC
Ren
B
Koh
SS
Harbison
CT
Kanin
E
Jennings
EG
Lee
TI
Tru
HL
Lander
ES
Lander
RA
(
2001
)
Remodeling of yeast genome expression in response to environmental changes
.
Mol Biol Cell
 
12
:
323
337
.
Del Vescovo
V
Casagrande
V
Bianchi
MM
et al
(
2008
)
Role of Hog1 and Yaf9 in the transcriptional response of Saccharomyces cerevisiae to cesium chloride
.
Physiol Genomics
 
33
:
110
120
.
De Nicola
R
Hazelwood
LA
De Hulster
EAF
Walsh
MC
Knijnenburg
TA
Reinders
MJT
Walker
GM
Pronk
JT
Daran
JM
Daran-Lapujade
P
(
2007
)
Physiological and transcriptional responses of Saccharomyces cerevisiae to zinc limitation in chemostat cultures
.
Appl Environ Microb
 
73
:
7680
7692
.
Domitrovic
T
Fernandes
CM
Boy-Marcotte
E
Kurtenbach
E
(
2006
)
High hydrostatic pressure activates gene expression through Msn2/4 stress transcription factors which are involved in the acquired tolerance by mild pressure precondition in Saccharomyces cerevisiae
.
FEBS Lett
 
580
:
6033
6038
.
Eide
D
Guarente
L
(
1992
)
Increased dosage of a transcriptional activator gene enhances iron-limited growth of Saccharomyces cerevisiae
.
J Gen Microbiol
 
138
:
347
354
.
Francis
SH
Colbran
JL
McAllister-Lucas
LM
Corbin
JD
(
1994
)
Zinc interactions and conserved motifs of the cGMP-binding cGMP-specific phosphodiesterase suggest that it is a zinc hydrolase
.
J Biol Chem
 
269
:
22477
22480
.
Garmendia-Torres
C
Goldbeter
A
Jacquet
M
(
2007
)
Nucleocytoplasmic oscillations of the yeast transcription factor Msn2: evidence for periodic PKA activation
.
Curr Biol
 
17
:
1044
1049
.
Gasch
AP
Spellman
PT
Kao
CM
Carmel-Harel
O
Eisen
MB
Storz
G
Botstein
D
Brown
PO
(
2000
)
Genomic expression programs in the response of yeast cells to environmental changes
.
Mol Biol Cell
 
11
:
4241
4257
.
Görner
W
Durchschlag
E
Martinez-Pastor
MT
Estruch
F
Ammerer
G
Hamilton
B
Ruis
H
Schüller
C
(
1998
)
Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity
.
Gene Dev
 
12
:
586
597
.
Hahn
JS
Thiele
DJ
(
2004
)
Activation of the Saccharomyces cerevisiae heat shock transcription factor under glucose starvation conditions by Snf1 protein kinase
.
J Biol Chem
 
279
:
5169
5176
.
Hasan
R
Leroy
C
Isnard
AD
Labarre
J
Boy-Marcotte
E
Toledano
MB
(
2002
)
The control of the yeast H2O2 response by the Msn2/4 transcription factors
.
Mol Microbiol
 
45
:
233
241
.
Higgins
VJ
Rogers
PJ
Dawes
IW
(
2003
)
Application of genome-wide expression analysis to identify molecular markers useful in monitoring industrial fermentations
.
Appl Environ Microb
 
69
:
7535
7540
.
Jacquet
M
Renault
G
Lallet
S
De Mey
J
Goldbeter
A
(
2003
)
Oscillatory nucleocytoplasmic shuttling of the general stress response transcriptional activators Msn2 and Msn4 in Saccharomyces cerevisiae
.
J Cell Biol
 
161
:
497
505
.
Kaida
D
Yashiroda
H
Toh-e
A
Kikuchi
Y
(
2002
)
Yeast Whi2 and Psr1-phosphatase form a complex and regulate STRE-mediated gene expression
.
Genes Cells
 
7
:
543
552
.
Kenbaev
B
Sade
B
(
2002
)
Response of field-grown barley cultivars grown on zinc-deficient soil to zinc application
.
Commun Soil Sci Plan
 
33
:
533
544
.
Kobayashi
N
McEntee
K
(
1993
)
Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae
.
Mol Cell Biol
 
13
:
248
256
.
Lawrence
CL
Botting
CH
Antrobus
R
Coote
PJ
(
2004
)
Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid
.
Mol Cell Biol
 
24
:
3307
3323
.
Lenssen
E
James
N
Pedruzzi
I
et al
(
2005
)
The Ccr4-Not complex independently controls both Msn2-dependent transcriptional activation – via a newly identified Glc7/Bud14 type I protein phosphatase module – and TFIID promoter distribution
.
Mol Cell Biol
 
25
:
488
498
.
Lyons
TJ
Gasch
AP
Gaither
LA
Botstein
D
Brown
PO
Eide
DJ
(
2000
)
Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast
.
P Natl Acad Sci USA
 
97
:
7957
7962
.
Magonet
E
Hayen
P
Delforge
D
Delaive
E
Remacle
J
(
1992
)
Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase
.
Biochem J
 
287
:
361
365
.
Marchler
G
Schüller
C
Adam
G
Ruis
H
(
1993
)
A Saccharomyces cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions
.
EMBO J
 
12
:
1997
2003
.
Martínez-Pastor
MT
Marchler
G
Schüller
C
Marchler-Bauer
A
Ruis
H
Estruch
F
(
1996
)
The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress-response element (STRE)
.
EMBO J
 
15
:
2227
2235
.
Montgomery
DC
(
2001
)
Design and Analysis of Experiments
 , pp.
96
99
.
Wiley
,
New York
.
Ohtsu
H
Fukuzumi
S
(
2001
)
Coordination of semiquinone and superoxide radical anions to the zinc ion in SOD model complexes that act as the key step in disproportionation of the radical anions
.
Chem Eur J
 
7
:
4947
4953
.
Ohtsu
H
Shimazaki
Y
Odani
A
Yamauchi
O
Mori
W
Itoh
S
Fukuzumi
S
(
2000
)
Synthesis and characterization of imidazolate-bridged dinuclear complexes as active site models of Cu,Zn-SOD
.
J Am Chem Soc
 
122
:
5733
5741
.
Rees
EMR
Stewart
GG
(
1998
)
Strain specific response of Brewer's yeast strains to zinc concentrations in conventional and high gravity worts
.
J Inst Brew
 
104
:
221
228
.
Rep
M
Krantz
M
Thevelein
JM
Hohmann
S
(
2000
)
The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes
.
J Biol Chem
 
275
:
8290
8300
.
Rose
M
Botstein
A
(
1983
)
Construction and use of gene fusions to lacZ (beta-galactosidase) that are expressed in yeast
.
Method Enzymol
 
101
:
167
180
.
Schüller
C
Brewster
JL
Alexander
MR
Gustin
MC
Ruis
H
(
1994
)
The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene
.
EMBO J
 
13
:
4382
4389
.
Schüller
C
Mamnun
YM
Mollapour
M
Krapf
G
Schuster
M
Bauer
BE
Piper
PW
Kuchler
K
(
2004
)
Global phenotypic analysis and transcriptional profiling defines the weak acid response regulon in Saccharomyces cerevisiae
.
Mol Biol Cell
 
15
:
706
720
.
Smith
A
Ward
MP
Garrett
S
(
1998
)
Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation
.
EMBO J
 
17
:
3556
3564
.
Stehlik-Tomas
V
Zetic
VG
Stanzer
D
Grba
S
Vahcic
N
(
1994
)
Zinc, copper and manganese enrichment in yeast Saccharomyces cerevisiae
.
Food Technol Biotech
 
42
:
115
120
.
Thorsen
M
Di
Y
Tängemo
C
et al
(
2006
)
The MAPK Hog1p modulates Fps1p-dependent arsenite uptake and tolerance in yeast
.
Mol Biol Cell
 
17
:
4400
4410
.
Truong-Tran
AQ
Carter
J
Ruffin
RE
Zalewski
PD
(
2001
)
The role of zinc in caspase activation and apoptotic cell death
.
Biometals
 
14
:
315
330
.
Van Helden
J
Andre
B
Collado-Vides
J
(
1998
)
Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies
.
J Mol Biol
 
281
:
827
842
.
Vecseri-Hegyes
B
Fodor
P
Hoschke
À
(
2005
)
The role of zinc in beer production, I. Wort production
.
Acta Aliment Hung
 
34
:
17
24
.
Vecseri-Hegyes
B
Fodor
P
Hoschke
À
(
2006
)
The role of zinc in beer production, II. Fermentation
.
Acta Aliment Hung
 
35
:
373
380
.
Winkler
A
Arkind
C
Mattison
CP
Burkholder
A
Knoche
K
Ota
I
(
2002
)
Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress
.
Eukaryot Cell
 
1
:
163
173
.
Wu
CY
Bird
AJ
Chung
LM
Newton
MA
Winge
DR
Eide
DJ
(
2008
)
Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae
.
BMC Genomics
 
9
:
370
.
Zhao
H
Eide
D
(
1997
)
Zap1p, a metalloregulatory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae
.
Mol Cell Biol
 
17
:
5044
5052
.
Zhao
H
Butler
E
Rodgers
J
Spizzo
T
Duesterhoeff
S
Eide
D
(
1998
)
Regulation of zinc homeostasis in yeast by binding of the ZAP1 transcriptional activator to zinc-responsive promoter elements
.
J Biol Chem
 
273
:
28713
28720
.

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