Role of Hog1 and Yaf9 in the transcriptional response of Saccharomyces cerevisiae to cesium chloride

Valerio Del Vescovo, Viviana Casagrande, Michele M. Bianchi, Eugenia Piccinni, Laura Frontali, Cristina Militti, Vivienne Fardeau, Frédéric Devaux, Claudio Di Sanza, Carlo Presutti, Rodolfo Negri


We analyzed the global transcriptional response of Saccharomyces cerevisiae cells exposed to different concentrations of CsCl in the growth medium and at different times after addition. Early responsive genes were mainly involved in cell wall structure and biosynthesis. About half of the induced genes were previously shown to respond to other alkali metal cations in a Hog1-dependent fashion. Western blot analysis confirmed that cesium concentrations as low as 100 mM activate Hog1 phosphorylation. Another important fraction of the cesium-modulated genes requires Yaf9p for full responsiveness as shown by the transcriptome of a yaf9-deleted strain in the presence of cesium. We showed that a cell wall-restructuring process promptly occurs in response to cesium addition, which is dependent on the presence of both Hog1 and Yaf9 proteins. Moreover, the sensitivity to low concentration of cesium of the yaf9-deleted strain is not observed in a strain carrying the hog1/yaf9 double deletion. We conclude that the observed early transcriptional modulation of cell wall genes has a crucial role in S. cerevisiae adaptation to cesium.

  • caesium
  • alkali metal cations
  • cell wall
  • YAF9
  • HOG1

cation homeostasis is accurately regulated in all organisms. For instance, alkali metal cations stimulate osmotic responses in essentially the same way as sugar or sugar alcohols at concentrations causing similar water activity (9). Additionally, monovalent cations can be toxic for the cell and can trigger cation-specific adaptation responses. These regulations involve modulation of the cell transcriptome. Saccharomyces cerevisiae is the most advanced model organism for the study of the global transcriptional response to salt stress. Despite the large amount of data available on the global transcriptional response of S. cerevisiae to osmotic changes and high Na+ concentrations (4, 7, 19, 20, 22), very little is known about its response to other monovalent cations like lithium, whose effects have been studied only at very low concentrations in galactose-containing medium (3), rubidium, or cesium. A comparison between the transcriptome changes induced by different cations would distinguish between different components of the response to salt stress: osmoadaptation, general response to ionic strength, ion-specific damage response. The osmotic stress signaling in S. cerevisiae is mainly controlled by the high osmolarity glycerol (HOG)/MAP kinase pathway, which is well conserved in all eukaryotes. It is characterized by a sensor system located in the plasma membrane, an upstream control system (G protein, phosphorelay) and a cascade of kinase activities (9). The HOG pathway is activated within few minutes by osmotic upshift and activates several transcription factors that control the response to osmotic stress. Additionally, the protein kinase A, the Snf1 AMP-dependent kinase pathways, and the calcineurin/Crz1 signaling pathway are involved in transcriptional response to osmotic and salt stress (17, 21, 23).

We analyzed the transcriptional response of S. cerevisiae to cesium chloride as a function of time and concentration. We were able to identify a component of this response, which is largely dependent on the HOG pathway and common to sodium, cesium, lithium, and potassium, and a second one, which is Hog1 independent and specific for cesium or shared by a subset of alkali metal cations. We and others previously showed that Yaf9, a component of the NuA4 and SWR chromatin modifying complexes (11, 12), is involved in cesium resistance (14, 1). This role could be based on a specific targeting of the chromatin complexes to specific cesium-responsive genes (1).

We provide here evidence that the HOG-dependent, HOG-independent, and Yaf9-mediated responses to cesium are functionally interdependent.


Strain and culture conditions.

S. cerevisiae strain BY4741 (Matα, His3Δ1, leuΔ0, lys2Δ0, ura3Δ0) and the corresponding hog1Δ, srl1Δ, yaf9Δ, fit2Δ, cwp1Δ, and cwp2Δ strains were from Euroscarf. The double-deleted yaf9Δ/hog1Δ strains were obtained by crossing the BY4741 (hog1Δ) strain with the FY1679 (yaf9Δ) strain. After sporulation of the diploid progeny and asci dissection, the double-deleted strains were selected by segregation analysis of G418 resistance and verification by PCR of the presence of the kanMX4 module in both the YAF9 and HOG1 loci. Growth curves were performed by culturing strains in YPD (1% yeast extract, 2% bactopeptone, 2% glucose) in Erlenmeyer flasks (28°C, 175 rpm) to the midlog phase [optical density at 600 nm (OD600) = 0.3]. Then cultures were divided into aliquots, concentrated salt solutions or identical volumes of sterile water were added, and incubation was continued to the stationary phase. The CsCl titration experiments were performed growing the cells to the midlog phase (OD600 = 0.4–0.5) in YPD medium at 28°C. Cells were divided in seven samples, and CsCl was added to a final concentration of 30, 50, 100, 150, 200, and 250 mM, respectively. Twenty minutes after CsCl addition, the cells were collected by centrifugation and frozen on dry ice before RNA extraction.

The time-course experiments were performed under the same growth conditions. Cultures were divided in eight samples. Four were treated with 100 mM of CsCl, and four were untreated controls. CsCl-treated samples and untreated control samples were collected 5, 10, 20, and 40 min after salt addition. The cells were harvested by centrifugation and frozen on dry ice before RNA extraction.

RNA extraction, analysis, and Northern blot.

Cells were suspended in 1 ml of AE buffer (50 mM sodium acetate pH 5, 10 mM EDTA), centrifuged, and suspended in 0.4 ml of AE buffer plus 1% (wt/vol) SDS. Cells were lysed with phenol-chloroform (5:1, pH 4.7), heated at 65°C for 4 min, and transferred at −40°C for 10 min, and the aqueous phase was separated by centrifugation. After a second extraction with phenol-chloroform (24:1 pH 5.2), RNA was precipitated with ethanol, dried, and suspended in sterile water. Aliquots of RNAs (10 μg) were suspended in 50% (vol/vol) formamide, 2.2 M formaldehyde, and MOPS buffer (0.56% MOPS, 5 mM sodium citrate, 1 mM EDTA), heated at 65°C for 15 min, and loaded onto a formaldehyde/agarose gel (1% wt/vol agarose, 50 mM NaCl, 4 mM EDTA). After electrophoresis, RNAs were transferred to nylon filters (BioBondTM-Plus) following the Northern blot procedure.

Filters were hybridized with 32P-labeled probes obtained from PCR amplification of the following open reading frames: YAF9, RPL30, HOG1, CMK2, SED1, HOR2, CWP1, CWP2, FIT2, and SRL1. The hybridized filters were exposed to Amersham Biosciences Storage Phosphor Screen and images were subsequently acquired by Typhoon 9200 phosphoimager for signal quantification.

cDNA labeling.

RNA (total of 100 μg) of from each sample was further purified using QIAGEN RNeasy Mini Kit. For cDNA synthesis, 20 μg of total RNA was mixed with 5 μg of random examers and 2 μg of 16mers oligo dT and incubated at 70°C for 10 min. cDNA was synthesized in a final volume of 40 μl with 125 μM each of dATP, dCTP, and dGTP; 75 μM of dTTP; 250 μM of Cy(3 or 5)-dUTP; 10 mM DTT; and 400 U SuperscriptII reverse transcriptase (Invitrogen) in 1× reaction buffer. The samples were incubated for 2 h at 42°C. We hydrolyzed RNA by the addition of 15 μl of 0.1 M NaOH, incubation for 10 min at 70°C, and subsequent neutralization by the addition of 15 μl of 0.1 M HCl. The neutralized cDNA was purified with QIAGEN QIAquick PCR Purification Kit and eluted with 30 μl of ddH2O. Typhoon 9200 phosphoroimager was used to quantify Cy3 and Cy5 incorporation.

Hybridization and image acquisition.

Slides were prehybridized at 42°C for at least 45 min in a solution containing, 5× SSC, 0.1% SDS, and 1% BSA. The labeled cDNAs (Cy3 sample and Cy5 sample mixed) were added to 35 μl of hybridization buffer containing 50% formamide, 10× SSC, and 0.2% SDS and preheated at 70°C for 3 min. Hybridization was carried out for 16 h at 42°C, and unbound DNA was washed off in three steps with solutions containing: 1) 1× SSC 0.2% SDS preheated at 42°C, 2) 0.1× SSC 0.2% SDS, and 3) two times 0.1× SSC.

A ScanArray Lite Microarray Scanner (Packard Bioscience) was used to acquire images, and GenePix Pro 6 software and ScanArray Express software were used to quantify hybridization signals. Absent and marginal spots were flagged automatically by software and subsequently each slide was inspected manually.

Data analysis.

We filtered the data to exclude artifacts, saturated spots, and low signal spots. Assuming that most of the genes have unchanged expression, we normalized the Cy3/Cy5 ratios using the VARAN online tool, selecting the following parameters: log2 ratio type; windows range, fixed; windows parameter, 60; consider log-ratios as normal; confidence interval, 0.9999; normalization method, Lowess fit normalization ( Since each gene is represented by two replicas spots on the array, data were treated with GEPAS on-line tool (, which averaged the two replica spots. Titration, time course, and wild-type vs. yaf9 deleted strain comparison experiments were performed twice from independent cultures. Gene ontology analysis was performed using the Babelomics platform (

The complete dataset has been deposited in the EMBL-EBI ArrayExpress repository (E-MEXP-1292 = The transcriptional response of S. cerevisiae to cesium titration; E-MEXP-1293 = Transcription profiling of S. cerevisiae wild-type and yaf9-mutant strains after cesium chloride salt stress) where other experimental details can be found.

Analysis of hog1 phosphorylation.

We prepared cell extracts by growing the cells to OD600 = 1 in YPD. NaCl or CsCl was added in concentrations of 50, 100, 200, and 300 mM. At the indicated times (2, 5, 10, and 20 min) after salt addition, 1.5 ml samples were taken out, collected by centrifugation, suspended in 75 μl of loading buffer (100 mM Tris·HCl, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and boiled for 10 min at 100°C. After 5 min of centrifugation at 14,000 rpm, the supernatants were collected and supplemented with phosphatase inhibitors (2 mM orthovanadate, 10 mM Na pyrophosphate) and stored at −20°C.

Aliquots of 15 μl of cell extracts/lane were loaded on a 7.5% polyacrylamide-SDS gel and run at 60 V. After electrophoresis, the proteins were transferred to nitrocellulose or polyvinylidene difluoride PVDF filter by electroblotting at 300 mA and 4°C for 1 h. Transfer efficiency was monitored by red Ponceau staining. After destaining, the filter was washed twice with TBS (50 mM Tris, 150 mM NaCl; pH 7.5), once with TBST (TBS + 0.1% Tween 20), and finally incubated with blocking solution (TBST + 5% nonfat milk, Bio-Rad).

Hog1 phosphorylation was detected by hybridization with anti-phospho-p38 MAPK (Thr180/Tyr182) (28B10) mouse MAb (Cell Signaling), diluted 1:1,200 in TBST + 5% nonfat milk, and incubated overnight with the filter at 4°C.

The filter was subsequently washed in TBST and incubated with goat anti-mouse IgG, conjugated to horseradish peroxidase (HRP) from Pierce, diluted 1:10,000, and incubated 1 h at room temperature (RT).

After reaction with Lumi-Light Western Blotting (Roche) the filter was exposed to autoradiography. The filter was subsequently stripped with 0.1 M glycine, pH 2.5, for 1 h at RT and hybridized, for loading control, with anti-COOH-terminal Hog1 (Santa Cruz Biotechnology), diluted 1:1,000, and incubated 1 h at RT. The filter was then washed and incubated with rabbit anti-goat IgG HRP (Santa Cruz Biotechnology, diluted 1:10,000) and incubated 1 h at RT. The signals were revealed as described above.

Cytohelicase sensitivity.

Cells were grown on YPD medium in Erlenmeyer flask at 28°C with shaking (175 rpm) to the midlog phase (OD600 = 0.3). Cultures were then divided, and concentrated salt solutions or equal volumes of water were added. Final concentrations of salts were 50, 100, and 300 mM CsCl. Cultures were further incubated for 1 h with salt, collected by centrifugation, washed with water, and suspended in 1 M sorbitol, 10 mM EDTA, 0.1 M Na citrate, pH 5.8, to a density of 2 × 108 cells per ml. We added 50 μl of 40 mg/ml cytohelicase (Sigma) per ml of cell suspension, and samples were incubated at 37°C for 1.5 h. Cell lysis was monitored by suspension in water aliquots from the reaction mixtures, thorough vortexing, and OD600 measurement. OD values at 90 min are reported as percentages of the starting value.

Secreted glucoamylase activity.

The wild-type strain and the yaf9Δ and hog1Δ strains were transformed with vector YEpG-GAAC6 (a gift of G. Kunze) that contained the Arxula adeninivorans glucoamylase (GAM) gene under the control of the constitutive GAP promoter of S. cerevisiae. Transformed clones were grown in flasks in 25 ml of YPD and YPD plus 50 mM CsCl for about 24 h at 28°C with shaking until they reached ∼2 OD600. Cultures were then centrifuged, and 15 μl/ml of 3 M Na acetate pH 5.2 and 20 μl/ml of 1% potato soluble starch (Sigma) were added to supernatants. Starch hydrolysis by the GAM enzyme was monitored by drawing a 1 ml sample from the reaction mixture each 10 min, adding 50 μl of 0.1 NI2 in 0.12 M KI, and measuring absorbance at 580 nm (16). The unit of GAM activity is defined as the amount of enzyme that reduce 1 OD580 unit in 1 min. The total activity per ml has been corrected by the cell concentration (OD600) of the cultures.


Genes modulated by different concentrations of CsCl.

S. cerevisiae strain BY4741 could grow in YPD medium in the presence of CsCl up to 250 mM (Supplemental Fig. S1A),1 although doubling time increased significantly and biomass yield was reduced. RNA samples for transcriptome analysis were prepared by growing the cells in YPD medium: when the culture reached the midlog phase (OD = 0.3), medium was supplemented with 30, 50, 100, 150, 200 and 250 mM CsCl. Cells were collected 20 min after CsCl addition, and total RNA was analyzed by DNA microarrays containing the entire coding repertoire (a summary is reported in Supplemental Table S1). Tables 1 and 2 report a list of 104 induced and repressed genes, respectively, that appear significantly modulated (log2 ratio ≥1 or ≤−1) at least at three salt concentrations. Gene ontology analysis (cellular component) showed that this list is particularly enriched in structural constituent of cell wall (13%, 4.5-fold enrichment, P < 0.0004). For what concerns the biological process classification, the list is significantly enriched in “cell wall organization and biogenesis” (12%, 2.8-fold enrichment, P < 0.005) and “electron transport” (9.5%, 3.1-fold enrichment, P < 0.008).

View this table:
Table 1.

Genes significantly induced by at least three concentrations of CsCl

View this table:
Table 2.

Genes significantly repressed by at least three concentrations of CsCl

General response to alkali metal cations and specific response to cesium.

Most (44/60) of the genes significantly induced by cesium were previously shown to be induced by at least another alkali metal cation (Table 1); 29 of these genes (reported in bold) show a general response to alkali metal cations, being activated by moderate and/or high concentrations of potassium (18), relatively low concentrations of lithium (3), and moderate or high concentrations of sodium (4, 19). Most of the genes belonging to this group were shown to require a functional Hog1 to be induced by potassium (18) or by sodium (19). A second group of 31 cesium-induced genes reported in Table 1 shows a less general response to alkali cations, being not induced by potassium; 15 of them were induced also by sodium and/or lithium, while 16 genes were induced only by cesium. We performed Northern blot analysis of some genes representative of the two groups of genes in the presence of different concentrations of CsCl, NaCl, and LiCl. We tested three genes of the first group (HOR2, CWP1 and SED1), three of group 2 (FIT2, CWP2 and SRL1), plus four genes not included in the list of significantly modulated genes (HOG1, YAF9 and CMK2 and RPL30) at CsCl concentrations ranging from 30 to 250 mM, NaCl concentrations from 50 to 400 mM, and LiCl concentrations from 50 to 200 mM (Fig. 1 and data not shown). HOR2, SED1, and CWP1 responded to cesium, lithium, and sodium; CWP2 responded to cesium and lithium; FIT2 and SRL1 were found to be induced by cesium and sodium but repressed by lithium; YAF9 and HOG1 were slightly induced by all three cations; RPL30 and CMK2 were not consistently modulated. The activation of group 1 genes was roughly linear in terms of salt concentration for all three cations tested at least up to 0.4 M (Supplemental Fig. S3A), while the three genes of group 2 did not show any linearity (Supplemental Fig. S3B). Northern blots of RNA extracted from hog1Δ strain (Fig. 2) confirm that activation of HOR2 and SED1 by cesium and sodium is totally Hog1-dependent, while CWP1 and SRL1 partially respond to both cations in the hog1-deleted strain.

Fig. 1.

Northern blot showing mRNA accumulation at 20 min from exposure of strain BY4741 to the indicated mM concentrations of CsCl, NaCl, and LiCl. The hybridizations were performed in series on the same filter; the EtBr fluorescence intensity of the rRNA band(s) is shown at the bottom. A: CWP1, SED1, HOR2, and RPL30. B: CMK2, YAF9, and CWP2. C: HOG1, SRL1, and FIT2.

Fig. 2.

Northern blot showing mRNA accumulation 20 min after exposure of hog1Δ strain BY4741 to the indicated mM concentrations of CsCl, NaCl, and LiCl. The hybridizations were performed in series on the same filter; EtBr fluorescence image of the 25S rRNA is shown at the bottom for each gel. A: CWP1, SED1, SRL1, and HOR2 in the hog1Δ strain BY4741. B: quantification of data from A; CWP1 (▴), SED1(▪), SRL1 (⧫) and HOR2 (×).

Hog1 is phosphorylated at low concentrations of cesium.

To verify the role of Hog1 in the response to cesium, we analyzed the extent of its phosphorylation in the presence of increasing concentrations of CsCl by Western blot analysis. Cell extracts were prepared at different CsCl concentrations and times from CsCl addition and were analyzed using an antibody specific for the phosphorylated form of Hog1 (15). As shown in Fig. 3A, CsCl concentrations as low as 200 mM stimulate Hog1 phosphorylation within 5 min. Moreover, the extent of phosphorylation increases at higher CsCl concentrations. Interestingly, cesium appears to be less effective than sodium in triggering Hog1 phosphorylation. Hog1 phosphorylation in response to low concentrations of sodium is very fast with a peak between 2 and 5 min (9, 15). Figure 3B and our results not shown indicated that CsCl also elicits a fast response of the HOG pathway: a phosphorylation peak is observed 5 min after 200 mM CsCl addition.

Fig. 3.

Western blot analysis of Hog1 phosphorylation in response to cesium. A: Hog1 phosphorylation in wild-type strain. Cell extracts were prepared at 5 min from the addition of the indicated concentrations of CsCl or NaCl. Extracts from the hog1-deleted strain were used as a control. B: time course of Hog1 phosphorylation in response to 200 mM cesium in wild-type strain. Cell extracts were prepared at the indicated times from the addition of 0.2 M CsCl as described in materials and methods. Extracts from the hog1-deleted strain were used as a control.

Role for Yaf9 in the response to cesium.

We previously showed that Yaf9, a component of NuA4 and SWR chromatin modifying complexes, is involved in cesium resistance (1).

We therefore directly compared transcriptomes of wild-type BY4741 and yaf9-deleted strains in YPD medium 20 min after the addition of 100 and 150 mM CsCl. The full set of data is reported in Supplemental Table S3.

Figure 4 shows that 28 genes (27% of the total) belonging to the lists of Tables 1 and 2 are deregulated in the yaf9 deleted strain. The larger group (cluster A in Fig. 4) encompasses 18 genes induced at several cesium concentrations and downregulated in the yaf9-deleted strain compared with the wild-type strain. For most of them, this downregulation is observed only in the presence of cesium, indicating that Yaf9 is involved in their cesium-dependent induction, but not in their basal expression. An opposite regulation (Yaf9-dependent repression by CsCl) was observed for genes of cluster B. On the contrary, genes of clusters C and D showed an enhancement of CsCl regulation (induction or repression) in the absence of Yaf9.

Fig. 4.

Expression of cesium-modulated genes in the yaf9-deleted strain in absence of presence of cesium. Columns 1–3: log2 ratio between yaf9Δ and wild type in 0, 100, and 150 mM CsCl, respectively (data from Supplemental Table S3). Columns 4–9: log2 ratio between the indicated CsCl concentrations and the untreated control (data from Supplemental Table S1). Color scale: red Log2 = 2; Green Log2 = −2.

A confirmatory Northern blot reported in Fig. 5A showed that SRL1 was poorly expressed and not induced by CsCl and NaCl in the yaf9-deleted strain; CWP2 was even slightly repressed in the presence of both cations, while, as a control, HOR2 still appears perfectly responsive to both cesium and sodium (see quantification in Fig. 5B).

Fig. 5.

Northern blot showing mRNA accumulation 20 min after exposure of yafΔ strain BY4741 to the indicated mM concentrations of CsCl and NaCl. The hybridizations were performed in series on the same filter; EtBr fluorescence image of the 25S rRNA is shown at the bottom for each gel. A: CWP2, SRL1, and HOR2 in the yafΔ strain BY4741. B: quantification of data from A; CWP2 (•), SRL1 (⧫), and HOR2 (×).

Seven genes listed in Table 1 (indicated with an asterisk) were previously found to be modulated by Yaf9 in a different genetic background at 1 h after the addition of 300 mM CsCl (1). We were unable to confirm these data, but it is possible that the presence of Yaf9 could influence the responsiveness of an even larger set, up to one-third of the total of cesium-modulated genes.

Since our microarray data showed a slight transcriptional induction of the YAF9 gene at four different CsCl concentrations (Supplemental Table S1), we confirmed its modulation by Northern blot in presence of cesium, lithium, and sodium (Fig. 1).

Effects of CsCl on cell wall structure.

We showed that an important fraction of the genes that were found significantly modulated 20 min from the addition of cesium code for structural components of the cell wall or for proteins involved in cell wall structure and organization. If their transcriptional modulation really reflected a change in protein expression, we would expect cesium addition to have an effect on the cell wall structure. We therefore tested the cytohelicase (β-glucanase) sensitivity, which can be considered diagnostic of cell wall restructuring, of cells grown for 1 h on YPD with different CsCl concentrations. The lytic action of cytohelicase weakens the cell wall and, after suspension in ipotonic medium (water), provokes cell lysis and a decrease of OD600, which is approximately a linear function of the digestion time (Supplemental Fig. S4). In the wild type, incubation with CsCl confers a resistance to cytohelicase increasing with salt concentration (Fig. 6). Interestingly, the hog1Δ strain is more sensitive than the wild type to cytohelicase and does not show any increase in resistance after incubation in CsCl (Fig. 6). We also tested cytohelicase with strains deleted in the cell wall genes (CWP1, CWP2, FIT2, SED1, and SRL1) modulated by cesium. Strikingly, we found a strong resistance induced by cesium for the strain carrying a deletion in SRL1 (Fig. 6), a gene regulated both by Hog1 and Yaf9, but no differences with all the other single gene-deleted strains, compared with the wild-type strain (data not shown).

Fig. 6.

Cytohelicase resistance of the wild-type strain BY4741 and of hog1Δ-, yaf9Δ-, and srl1Δ-deleted strains after growth on YPD and treatment with different concentrations of CsCl: white, no CsCl added; light grey, 50 mM CsCl; dark grey, 100 mM CsCl; black, 300 mM CsCl. Resistance is reported as OD600 values observed after 90 min of digestion in percent over the initial values. Values are average of 3 independent experiments; bars indicate SD.

To further characterize cell wall restructuring in response to cesium, we tested the efficiency of the secretory pathway by measuring the production of an heterologous extracellular enzyme, the GAM, in the medium. Strains transformed with a plasmid harboring the A. adeninivorans GAM gene were used. Supplemental Figure S5 shows that incubation in CsCl led to a consistent (∼1.8-fold) reduction of the secreted activity in the wild-type strain. While hog1 deletion leads to a limited reduction of GAM secreted activity (∼1.4-fold), the reduction was much more pronounced in the yaf9-deleted strain (∼3.2-fold).

HOG1 deletion rescues the sensitivity to cesium of the yaf9-deleted strain.

Our results indicate that YAF9 and HOG1 control a significant fraction of cesium-responsive genes. We constructed strains carrying double deletions in the YAF9 and HOG1 genes by genetic crossing of single-deleted genes, as reported in materials and methods, and we tested whether the HOG1 deletion had any effects on the sensitivity to cesium of the yaf9-deleted strain. Results, illustrated in Fig. 7, indicate that sensitivity to high NaCl concentration (0.8 M), and hence to high osmolarity, correlates with deletion of HOG1 also in double-deleted strains. On the contrary, sensitivity to a concentration of CsCl as low as 30 mM was only found with the single-deleted strain of YAF9 gene and not in the double-deleted strains. The YAF9 deletion did not generate an osmosensitive strain, as shown by growth on 0.8M NaCl.

Fig. 7.

Cesium and sodium resistance on plate. Wild type, hog1Δ, yaf9Δ, and hog1Δ/yaf9Δ double-deleted strains (ΔΔ2A and ΔΔ4C are 2 independent segregant strains) were cultured on YPD (1% yeast extract, 2% bactopeptone, 2% glucose) and then inoculated on YPD plates containing cesium and sodium chlorides at the indicated concentrations.


A hog1 signature on the general response to monovalent cations.

We analyzed S. cerevisiae transcriptome changes 20 min after adding different concentrations of CsCl. This early time window was chosen because of the fast response to salt to optimize the number of potentially modulated genes. A time-course experiment performed at an intermediate CsCl concentration (100 mM) confirmed that, also in the case of cesium, most of the modulations are detectable between 5 and 30 min after salt addition (Supplemental Table S2). Our supplementary data reported in Supplemental Fig. S2 show that cesium did not have an effect on RNA stability, as was demonstrated for lithium (6): the observed changes in mRNA accumulation are therefore likely to be due to regulation at the level of de novo transcription. The restricted list of genes induced at least at three concentrations of CsCl contained several genes previously known to respond to alkali metal cations. Some of these genes are responsive also to moderate and/or high concentrations of potassium and most of them to relatively low concentrations of lithium and to moderate concentrations of sodium. Most of the genes belonging to this group were previously shown to require a functional HOG pathway to be induced by potassium or by sodium, a requirement that we confirmed by Northern analysis for three of them, in the case of cesium. A second group of cesium-induced genes shows a less general response to alkali cations, being not induced by potassium but responding to sodium and/or lithium or only to cesium. Most of them appear to be stimulated in a Hog1-independent fashion. This group is more likely to respond specifically to toxic cations effects rather than to the generic cation sensor systems. It is therefore very likely that for the genes of the first group, all alkali cations, even at relatively low concentrations, can induce transcription by activating at least one branch of the HOG/MAP kinase pathway. As a matter of fact, we observed a detectable Hog1 phosphorylation in response to 200 mM cesium concentration in within 2 min from salt addition, by Western blot (Fig. 3). However, the strength of the osmotic response through the HOG pathway might depend on the nature of the single cation. In fact the amount of Hog1 phosphorylation was lower with cesium than with sodium and slightly delayed.

Transcription regulation.

A database search for transcription factors that bind to the promoters of the 104 genes modulated by cesium ( identified different combinations of transcription activators (Tables 1 and 2). Some of these factors are direct target of the HOG pathway, whose activation seems to be required for at least one-half of the induced genes. As previously suggested, neither the ability to bind these transcription factors nor the necessary Hog1 activation is sufficient to confer to those promoters a general responsiveness to salt; some other mechanism of selection must be operative, possibly at the level of chromatin structure and dynamics. It has previously been shown that phosphorylated Hog1 tightly binds chromatin in response to osmostress and stimulates gene transcription by mechanisms other than the simple modification of activators, for example recruiting chromatin modification complexes (5). We showed here that many of the genes modulated by cesium, including several Hog1-dependent genes, showed altered transcription regulation in yaf9Δ mutants. We also showed that the gene coding for this protein, which is a component of NuA4 and SWR chromatin modifying complexes, is induced at different concentrations of lithium, cesium, and sodium at a relatively low concentration (Fig. 1). Since YAF9 deletion increases sensitivity to cesium and lithium (Ref. 1 and Supplemental Fig. S1) and induces changes in the cell wall structure and composition (Refs. 1, 14 and Supplemental Fig. S7), we propose a role for Yaf9 in targeting one or both chromatin modifying complexes NuA4 and SWR to some of the cesium- and lithium-responsive promoters. Gene targeting by Yaf9 might occur independently or in coordination with Hog1 phosphorylation. A genome-wide analysis of Yaf9 DNA binding in vivo in presence or absence of cesium could verify this hypothesis.

Structural changes in the cell wall in response to cesium.

Since the mRNA abundance of at least 14 genes coding for structural components of cell wall and of other genes involved in its biosynthesis and organization are significantly modulated at 20 min from cesium addition, it was reasonable to expect a major change in the cell wall structure after exposure to this cation. Our data confirm a fast and dose-dependent change of cell wall (cytohelicase sensitivity) that seems to be controlled by Hog1. On the other hand, cell wall changes affecting the secretory pathway (GAM secretion) might rely more specifically on Yaf9. The individual absence of proteins transcriptionally regulated by Yaf9 does not have detectable effect on cell wall structure when cells are exposed to cesium, except for Srl1, that amplifies the effect of cesium on cell wall resistance to cytohelicase. HOG1-dependent changes in the cell wall organization were previously documented in response to low external pH (10), and altered chitin deposition was previously observed in a yaf9-deleted strain (1). We conclude that an important aspect of yeast adaptation to cesium is represented by transcriptional modulation of cell wall genes, a process whose failure could explain the slower growth of the yaf9-deleted strain in the presence of cesium salt (Supplemental Fig. S1) and its sensitivity to low concentration of cesium on plates (Refs. 1, 2 and Fig. 7). Interestingly, introduction of HOG1 deletion in a yaf9-deleted strain completely rescues sensitivity to CsCl. This result is not surprising: we showed here that both proteins are involved in the modulation of the cell wall transcriptome in response to cesium, but their effects are only partially overlapping. Changes in the abundance of cell wall components might be interdependent (13), and some Yaf9-regulated but Hog1-independent proteins, like Srl1, have been shown to have a key role in cell wall stability (8). However, when strains are grown on solid medium in the presence of very low concentration (30 mM) of CsCl, we are specifically observing the toxic effects of cesium, while general osmotic effects should be not relevant. The Hog1-dependent changes, which could be protective at high osmotic pressure, could instead be deleterious for the cell wall proteome changes induced by cesium and affected by the deletion of YAF9. Proteomic analysis of the different strains exposed to cesium could confirm this hypothesis. Finally, the existence of parallel and compensative pathways, which could be more active in the absence of both YAF9 and HOG1, cannot be excluded, if we consider that signaling pathways are usually interconnected, redundant or even conflicting. Indeed, HOG1 seems to play a role in downregulating and resetting transcriptional levels of genes induced by the general stress response (including cesium- induced genes like DDR48, ALD3, and GLK1) after the cells have adapted to new osmotic conditions (18).


This work was supported by the Centre of Excellence in Molecular Biology and Medicine (BEMM) and the Ministry of University and Research (FIRB, Cofin and Ateneo 'La Sapienza').


  • * V. Del Vescovo and V. Casagrande contributed equally to this work.

  • 1 The online version of this article contains supplemental material.

  • Address for reprint requests and other correspondence: R. Negri, Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Roma, La Sapienza, Piazzale A. Moro 5-00185-Roma, Italy (e-mail: Rodolfo.negri{at}

    Article published online before print. See web site for date of publication (


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