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Zinc starvation induces a stress response in Saccharomyces cerevisiae that is mediated by the Msn2p and Msn4p transcriptional activators

Victoria J. Gauci, Anthony G. Beckhouse, Victoria Lyons, Eric J. Beh, Peter J. Rogers, Ian W. Dawes, Vincent J. Higgins
DOI: http://dx.doi.org/10.1111/j.1567-1364.2009.00557.x 1187-1195 First published online: 1 December 2009


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.

  • zinc
  • Msn2/4p
  • Zap1p
  • Saccharomyces cerevisiae
  • fermentation


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).


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.

View this table:
Table 1

Promoter analysis of genes induced during zinc deficiency

Gene clusterPromoter elementPutative-binding proteinP-value
All upregulated genes in an industrial strain of S. cerevisiae (Higgins, 2003)AGGGGCCCCTMsn2/4p1.7 × 10−7
ATGGGCCCAT8.7 × 10−6
AAGGGAAGGG2.8 × 10−4
CCCCCGGGGG1.89 × 10−3
GGGGATCCCC1.89 × 10−3
All upregulated genes in a laboratory strain of S. cerevisiae (Lyons, 2000)AGGGGCCCCTMsn2/4p2.0 × 10−9
AAGGGCCCTT8.8 × 10−9
ACCCCGGGGT1.8 × 10−5
CCCCCCCCCC1.5 × 10−4
GGGGATCCCC2.2 × 10−4
Microarray results for Zap1p targets from time-course and dose–response studies (Wu, 2008)AGGGGCCCCTMsn2/4p4.6 × 10−4
AAGGGCCCTT5.3 × 10−4
  • 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.

View this table:
Table 2

Potential Msn2/4p target genes

Gene nameDescriptionNumber of STREsSignificance index*Expression fold changeP-value*
Genes with consensus STREs (AGGGG)
YNR034W-AProtein of unknown function530.97240.45.18 × 10−3
DDR2DNA damage-responsive 2 protein430.97147.33.18 × 10−3
HXK1Hexokinase isoenzyme I530.97138.71.26 × 10−4
GPH1Glycogen phosphorylase330.9761.31.27 × 10−4
YER067WProtein of unknown function, high similarity to uncharacterized S. cerevisiae Yil057p430.9754.95.58 × 10−4
HSP12Heat-shock protein of 12 kDa730.9746.51.01 × 10−2
RTC3Protein of unknown function involved in RNA metabolism230.9744.81.78 × 10−2
STF2Protein of unknown function, high similarity to uncharacterized C. glabrata Cagl0f08745gp230.9738.87.34 × 10−3
PGM2Phosphoglucomutase 2530.9738.61.39 × 10−3
HXT7High-affinity glucose transporter nearly identical to Hxt6p430.9727.91.56 × 10−3
HXT6High-affinity glucose transporter nearly identical to Hxt7p530.9722.12.50 × 10−3
GLC3Glycogen-branching enzyme330.9719.79.86 × 10−3
CTT1Catalase T 1430.9717.14.26 × 10−2
HSP26Heat-shock protein of 26 kDa430.9713.16.41 × 10−4
MSC1Protein that affects meiotic homologous chromatid recombination230.9711.14.40 × 10−2
ARG1Argininosuccinate synthetase230.978.24.10 × 10−4
DCS2Nonessential, stress-induced regulatory protein230.977.84.24 × 10−2
HOR7Protein of unknown function430.977.46.03 × 10−3
YIL169CProtein of unknown function130.976.88.45 × 10−3
GPX1Phospholipid hydroperoxide glutathione peroxidase230.976.82.20 × 10−2
USV1Putative transcription factor containing a C2H2 zinc finger630.976.81.36 × 10−2
SPI1Stationary phase-induced 1 protein330.976.75.65 × 10−5
URA10Orotate phosphoribosyltransferase 2230.976.19.59 × 10−3
YGP1Cell wall-related secretory glycoprotein230.975.27.31 × 10−3
NCE102Protein of unknown function630.975.11.98 × 10−3
HSP31Heat-shock protein 31130.974.57.00 × 10−4
CRS5Copper-binding metallothionein230.973.92.64 × 10−3
TFS1Carboxypeptidase Y inhibitor230.973.79.89 × 10−3
COX5BSubunit Vb of cytochrome c oxidase230.973.41.51 × 10−2
YNL300WGlycosylphosphatidylinositol-dependent cell-wall protein130.973.43.04 × 10−4
TMA10Protein of unknown function that associates with ribosomes330.973.48.25 × 10−3
PBI2Cytosolic inhibitor of vacuolar proteinase B130.973.32.79 × 10−3
ALD4Mitochondrial aldehyde dehydrogenase230.973.37.78 × 10−3
YJR096WPutative xylose and arabinose reductase130.973.11.69 × 10−3
MCR1Mitochondrial NADH-cytochrome b5 reductase, involved in ergosterol biosynthesis230.973.01.86 × 10−2
SOD2Mitochondrial superoxide dismutase130.973.09.00 × 10−3
YJR008WPutative protein of unknown function230.973.03.36 × 10−3
QNQ1Protein of unknown function230.972.92.91 × 10−2
OM45Protein of unknown function, major constituent of the mitochondrial outer membrane330.972.93.70 × 10−2
DDR48Stress protein induced by heat shock, DNA damage, or osmotic stress230.972.92.08 × 10−2
TDH1Glyceraldehyde-3-phosphate dehydrogenase, isozyme 1130.972.86.27 × 10−3
TPS2Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex530.972.82.03 × 10−3
MMF1Mitochondrial protein involved in maintenance of the mitochondrial genome230.972.75.26 × 10−3
FMP16Putative protein of unknown function130.972.73.96 × 10−2
FMP46Putative redox protein230.972.62.82 × 10−3
YER053C-APutative protein of unknown function230.972.66.07 × 10−3
YMR291WPutative kinase of unknown function330.972.65.05 × 10−3
MDH1Mitochondrial malate dehydrogenase130.972.53.83 × 10−2
ICY1Protein of unknown function530.972.51.55 × 10−2
GSP2GTP-binding protein330.972.32.03 × 10−2
COS8Nuclear membrane protein130.972.33.12 × 10−3
PRB1Vacuolar protease B130.972.22.64 × 10−2
RIB5Riboflavin synthase130.972.14.95 × 10−2
FMP10Protein of unknown function230.972.15.79 × 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.

Figure 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.


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.

Supporting Information

Table S1. Potential Msn2/Msn4p target genes.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


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.


  • Editor: Isak Pretorius


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