Unlike other stresses, the physiological significance and molecular mechanisms involved in the yeast cold response are largely unknown. In the present study, we show that the CWI (cell wall integrity) pathway plays an important role in the growth of Saccharomyces cerevisiae at low temperatures. Cells lacking the Wsc1p (wall integrity and stress response component 1) membrane sensor or the MAPKs (mitogen-activated protein kinases) Bck1p (bypass of C kinase 1), Mkk (Mapk kinase) 1p/Mkk2p or Slt2p (suppressor of lyt2) exhibited cold sensitivity. However, there was no evidence of either a cold-provoked perturbation of the cell wall or a differential cold expression program mediated by Slt2p. The results of the present study suggest that Slt2p is activated by different inputs in response to nutrient signals and mediates growth control through TORC1 (target of rapamycin 1 complex)–Sch9p (suppressor of cdc25) and PKA (protein kinase A) at low temperatures. We found that absence of TOR1 (target of rapamycin 1) causes cold sensitivity, whereas a ras2Δ mutant shows increased cold growth. Lack of Sch9p alleviates the phenotype of slt2Δ and bck1Δ mutant cells, as well as attenuation of PKA activity by overexpression of BCY1 (bypass of cyclase mutations 1). Interestingly, swi4Δ mutant cells display cold sensitivity, but the phenotype is neither mediated by the Slt2p-regulated induction of Swi4p (switching deficient 4)-responsive promoters nor influenced by osmotic stabilization. Hence, cold signalling through the CWI pathway has distinct features and might mediate still unknown effectors and targets.
All eukaryotic cells employ MAPKs (mitogen-activated protein kinases) to relay extracellular signals and to activate the appropriate cell response . Specifically, in Saccharomyces cerevisiae there are five central MAPKs, Fus3p Fus3 (cell fusion 3), Kss1p [kinase suppressor of Sst2 (supersensitive 2) mutations], Hog (high osmolarity glycerol response) 1p, Slt2p (suppressor of lyt2) and Smk1p (sporulation-specific MAPK), which control mating, filamentous growth, HOG, CWI (cell wall integrity) and sporulation pathways respectively [2,3]. From them, there is no signal transduction pathway or transcription factor known to respond exclusively to low temperatures. Although the existence of such a pathway cannot be ruled out, the available data show that known signal transduction mechanisms, operating in response to other kinds of stimuli, may also be triggered by cold stress .
Traditionally, the HOG pathway has been considered to be involved only in the genetic response to hyperosmotic stress . However, it is now clear that the HOG pathway responds to various extracellular stimuli, and that it is required to activate a wide range of cellular responses [5–7]. In particular, cold activation of Hog1p triggers a specific transcriptional program at low temperatures, increases the synthesis of glycerol and determines freeze survival in yeast cells . Nevertheless, Hog1-mediated signalling is required only for the expression of a subset of cold-induced genes. Typical cold marker genes such as OLE1 (oleic acid requiring 1), TIP1 (temperature shock-inducible protein 1) and NSR1 (nuclear localization sequence-binding protein), are fully induced at low temperatures independently of MAPK Hog1p activity , thus supporting the notion that additional cold-signal transduction pathways are operative in S. cerevisiae.
The CWI pathway provides a means to fortify and repair damage to the cell wall in order to withstand stressful environments . Perturbations on the cell surface and environmental signals are generally detected by a small set of plasma membrane-spanning sensors, the Wsc (wall integrity and stress response component) family members Mid2p (mating pheromone-induced death 2) and Mtl1p (mid-two like 1) , which transmit signals to Rom2p (Rho1 multicopy suppressor 2), a GEF (guanine-nucleotide-exchange factor) of the GTP-binding protein Rho1. Rho1p then activates the Pkc1 (protein kinase C1) protein kinase which in turn, activates an MAPK module ; Pkc1p phosphorylates Bck1p (bypass of C kinase 1), a Mkk (MAPK kinase) kinase, which transmits the signal to both Mkk1p and Mkk2p. These finally activate the last member of the cascade, Slt2p/Mpk1p. Despite there being slight differences, the CWI function of maintaining cell integrity is conserved between fungi, and CWI pathway activation is not restricted to an individual stimulus, but can be elicited in response to cell wall stress compounds, oxidative stress or heat shock, among others . Indeed, heat stress triggers the increased phosphorylation of the MAPK Slt2p , and null mutants in many pathway components display cell lysis defects when cultivated at high temperatures . In addition, loss of PKC1 results in osmoremedial cell lysis at all growth temperatures, suggesting a general function of the CWI pathway or alternate CWI pathway branches in thermal stress. However, there is no evidence of this role at low growth temperatures.
The variety of different stimuli that activate the CWI pathway suggests that a common stress is generated by all of the activating conditions. There is accumulating evidence that the plasma membrane stretch is the underlying physical stress that leads to activation of CWI signalling . Nevertheless, Slt2p activation might proceed through additional inputs, and mediate different effectors and parallel MAPK pathways . Recently, the Sho1 branch of the HOG pathway has been identified as being required for the full activation of the CWI pathway in response to zymolyase treatment . There is also evidence that the HOG and CWI pathways are not mutually exclusive [14,15], indicating that, although pathway specificity is necessary, the HOG and CWI MAPK pathways can also be positively co-ordinated.
The CWI pathway might also respond to and be stimulated by other signals. Previous studies have suggested that nutrient starvation might be on the basis of some phenotypic features of cold-shocked cells [16,17]. Consistently with this, mutants in various elements of the TOR (target of rapamycin) and cAMP-dependent PKA (protein kinase A) signalling pathways, the two major pathways transmitting nutrient signals to regulate cell growth [18,19], show a cold-growth defect. Furthermore, a recent work has indicated that the TORC1 (TOR complex 1) activates PKA by preventing the Slt2p-mediated activation of Bcy1p (bypass of cyclase mutations 1) , the PKA regulatory subunit. In turn, PKA in yeast controls the activity of the stress-inducible transcription factors Msn2p/Msn4p, among others .
In the present study, we have investigated the implication of different MAPKs in the cold stress response. Our results provide clear evidence for the first time of the implication of the CWI pathway in the cold-adaptive response in S. cerevisiae and identify Slt2p as an essential mediator of cell growth, probably by modulating the TORC1–cAMP–PKA signalling network. The significance of the present study is further highlighted by the finding that Swi4p (switching deficient 4) is a Slt2p-independent regulator of cold growth.
MATERIALS AND METHODS
Media and culture conditions
Yeast cells were cultured at 30°C in defined media, YPD [1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose] medium, SD medium [0.17% yeast nitrogen base without amino acids (Difco), 0.5% ammonium sulphate and 2% glucose) or SCD [SD medium supplemented with the appropriate amino acid drop-out mixture; Formedium]. S. cerevisiae transformants carrying geneticin- (kanMX4), nourseothricin- (natMX4) or the hygromycin- (hphMX4) resistant module were selected on YPD agar plates containing 200 mg/l of G-418 (Sigma), 50 mg/l of nourseothricin (clonNAT, WERNER Bioagents) or 300 mg/l of hygromycin B (Formedium) respectively [22,23]. Escherichia coli cells was grown in LB (Lurai–Bertani) medium supplemented with ampicillin (50 mg/l).
Stress sensitivity tests
For the cold stress experiments, cells were grown to mid-exponential phase at 30°C, collected, transferred to fresh medium and incubated at 12 or 15°C. In some experiments, NaCl or CFW (calcofluor white)-containing YPD was used for testing growth under osmotic or wall-damage stress conditions respectively. Plate phenotype experiments were done by diluting the cultures to D600=1.0 and spotting (3 μl) 10-fold serial dilutions. Unless indicated, colony growth was inspected after 2–4 days of incubation at 30°C or after 8–10 days at 12°C.
Strains and plasmids
The S. cerevisiae strains and plasmids used in the present study are listed in Supplementary Tables S1 and S2 (at http://www.BiochemJ.org/bj/446/bj4460477add.htm) respectively. The Trp+ prototrophic derivative of the S. cerevisiae EG123 wt (wild-type) strain (trp1-1) was obtained by transforming with the YIplac212 integrative vector . The TRP1, SLT2, BCK1, WSC1, WSC2, WSC3, MID2 and MSN4 deletion strains were constructed by PCR-based gene replacement using the kanMX4, natMX4 and hphMX4 disruption modules contained in plasmids pFA6a-kanMX4 , pAG25 or pAG32  respectively, and synthetic oligonucleotides (Supplementary Table S3 at http://www.BiochemJ.org/bj/446/bj4460477add.htm). Gene disruptions were confirmed by diagnostic PCR (Supplementary Table S3). S. cerevisiae strains were transformed by the lithium acetate method . E. coli cells (DH10B strain) were transformed by electroporation.
Microarray experiments and data analysis
Wt and slt2Δ mutant cells were grown in SCD medium at 25°C to a final D600=0.5 and transferred to 12°C conditions for 3 h. Total RNA was extracted from 100 ml of each culture by mechanical disruption following the instructions for the RNeasy Midi kit manufacturer (Qiagen). RNA concentration was measured at 260 nm and sample quality was checked in a 2100B Bioanalyzer (Agilent Technologies). Under these experimental conditions (wt and slt2Δ strain), three microarray experiments corresponding to three biological replicates were processed and analysed. Total RNA (250 ng) were labelled using the 3′ IVT express kit (Affymetrix) following the manufacturer's instructions. The biotin-labelled cRNA obtained was fragmented and hybridized to the Affymetrix GeneChip® Yeast Genome 2.0 array for 16 h at 45°C. Hybridized microarrays were washed and stained with a streptavidin–phycoerythrin conjugate in a GeneChip® Fluidics Station 450. All of these procedures were carried out as suggested by the manufacturer. The corresponding fluorescence signal of the hybridization was acquired in a GeneChip® 3000 7G scanner. After scanning, numerical data were obtained and processed with the Expression Console software (Affymetrix) and the RMA (robust means analysis) algorithm. Signal ratios were obtained as a quotient between the means of normalized signal data for each biological replicate. P values were obtained with the limma algorithm on the Babelomics site (http://babelomics.bioinfo.cipf.es/). The genes with a signal ratio <0.75 were selected and are displayed in Supplementary Table S4 (at http://www.BiochemJ.org/bj/446/bj4460477add.htm).
Whole-cell extracts for Hog1p and Slt2p detection, protein separation by SDS/PAGE (8% gel) and electroblotting were carried out as described previously . Dual phosphorylated Hog1p was detected by an antibody specific against phosphorylated p38 MAPK (catalogue number 9215, Cell Signaling, Technology). A rabbit polyclonal antibody raised against a recombinant protein corresponding to the C-terminus (residues 221–435) of S. cerevisiae Hog1p was used as a loading control (catalogue number sc-9079, Santa Cruz Biotechnology). Antisera were applied at dilution of 1:1000 (phosphorylated and total Hog1p). The phosphorylated form of Slt2p was detected with an anti-[phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)] antibody (1:1000 dilution; catalogue number 4370, Cell Signaling Technology). A rabbit polyclonal antibody raised against HA (haemagglutinin) protein (1:2000 dilution; catalogue number sc-805, Santa Cruz Biotechnology) was used for the detection of HA-tagged Slt2p. The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit antibody (catalogue number 7074, Cell Signaling Technology) applied at 1:2000 or 1:5000 dilutions (HA). The blots were performed and images captured as described previously .
Cells were stained with rhodamine–phalloidin as described previously . For quantification, we counted 1000 budded cells in which the bud was smaller than the mother cell. We considered that cells were depolarized when no actin cables were microscopically detected. We repeated this experiment three times.
The β-galactosidase activity was determined at 30°C using cell extracts and the substrate ONPG (o-nitrophenyl β-D-galactopyranoside) as described previously . The definition of 1 unit was the amount of enzyme that is able to convert 1 nmol of ONPG per min under the assay conditions. The values provided represent the average of three independent experiments each conducted in triplicate.
Absence of the MAPK Slt2p has a great impact on cell growth at low temperature
We investigated the effects of the absence of FUS3, KSS1, HOG1, SLT2 or SMK1, the genes encoding for the five MAPKs known in yeast, on growth at low temperatures. As expected, hog1Δ mutant cells of the BY4741 wt strain displayed a slight growth defect in SCD medium at 12°C (Figure 1A), in agreement with the previously reported functional role of the HOG pathway under these conditions . No significant effect at 12°C was found by deleting FUS3, KSS1 or SMK1. However, lack of Slt2p, the MAPK of the CWI pathway, caused a severe cold-growth defect, which was less pronounced in rich YPD medium (Figure 1A). This strain also exhibited a slight growth defect at 30°C, which could be associated with its inositol auxotrophy (the Ino− phenotype), a feature previously reported in mutants of the CWI pathway . As seen, the absence of inositol (SCD-INO, Figure 1A) intensified the growth defect of the slt2Δ mutant cells at 30°C, as compared with that observed in standard SCD medium. Conversely, the addition of excess inositol (75 μM final concentration) rescued its growth at 30°C to wt levels. However, the cold-sensitivity phenotype of slt2Δ cells was evident regardless of the absence or the presence of inositol (Figure 1A). Finally, the growth defect at low temperatures was not strain-dependent since a slt2Δ mutant of the S. cerevisiae EG123 wt strain showed a similar behaviour (Figure 1B), and was observed only in a medium of low osmotic strength. Indeed, the cold-sensitive phenotype of slt2Δ cells was prevented in the presence of 0.8 M sorbitol (Supplementary Figure S1 at http://www.BiochemJ.org/bj/446/bj4460477add.htm), a response frequently displayed by cell wall mutants.
Absence of the MAPK Slt2p impairs growth at low temperature
Adaptation to cold requires membrane sensor Wsc1p and a functional MAPK module of the CWI pathway
Attempts were made to dissect the implication of different elements of the yeast CWI pathway of the cold response. Growth of mutant strains lacking elements upstream of Slt2p (see Figure 2A for an overview of the pathway) was tested at 30°C and 12°C (Figure 2B). We first analysed the functional role of membrane sensors Wsc1p–3p, Mid2p and Mtl1p, and GEF Rom2p, all of which act upstream of Pkc1p (Figure 2A). Neither Rom2p (Figure 2B) or its homologue Rom1p (results not shown) displayed a cold-sensitivity phenotype and only Wsc1p appeared to play a role in yeast cell growth at 12°C (Figure 2B). Nevertheless, we investigated the possibility of a partial redundancy among sensors. Nine double disruption mutants from the ten possible combinations and one triple mutant, wsc3Δ wsc2Δ wsc1Δ, were constructed and tested for growth at 12°C (Supplementary Figure S2 at http://www.BiochemJ.org/bj/446/bj4460477add.htm). We were unable to obtain a mid2Δ wsc1Δ double mutant, suggesting that the simultaneous absence of these sensors in the BY4741 yeast background is lethal, which is in agreement with previous reports . It was observed that the WSC1 deletion mutants showed a cold-growth defect phenotype in all of the cases, thus confirming the major role of this sensor at low temperatures. Nevertheless, Wsc2p and Wsc3p played a particular function as shown by the more severe cold-growth defect of the double and triple mutants as compared with the single wsc1Δ mutant (Supplementary Figure S2).
Membrane sensor Wsc1p and the MAPK module of the CWI pathway are required to provide cold-growth ability in S. cerevisiae
We then checked if a MAPK module was necessary (Figure 2B). Growth at low temperatures was clearly diminished by the deletion of BCK1, the MAPKKK (Mkk kinase) of the pathway. Lack of the redundant MAPKKs Mkk1p and Mkk2p had no apparent effect, although the double mkk1Δ mkk2Δ mutant displayed a clear cold-growth defect (Figure 2B). As expected, these phenotypes, as well as those found for the wsc1Δ mutants, were rescued either fully or partly by the use of the osmotic stabiliser sorbitol (Supplementary Figures S1 and S2). Hence, yeast growth at low temperatures requires the activity of proteins that sense and respond to cell wall and membrane damage.
Cold is unable to trigger the signalling activity of Pkc1p and its overproduction provides no advantages for cold growth
The MAPK cascade for the CWI pathway is initiated by Pkc1p which, in turn, is stimulated by the action of the GEFs Rho1p, Rom1p and Rom2p . However, we were unable to show a cold-growth defect in single mutants of the tested GEFs (Figure 2B). Nevertheless, different Rho1p effectors may be active in response to different types of cell-wall stress, including low temperature. Consequently, we attempted to obtain further evidence of cold-instigated signalling through the CWI pathway. Since loss of PKC1 is lethal, we measured the activation of the protein kinase in cold-shocked cells. In response to several stresses that perturb the cell wall, the actin cytoskeleton undergoes a transient depolarization followed by repolarization , a dynamic process that depends on the GTPase Rho1 and Pkc1p, the latter controlling the actin depolarization, and Slt2p, which is responsible for actin repolarization . As shown in Figure 3(A), the actin cytoskeleton remained insensitive to a downward shift in temperature. Similar results were observed after 6 h at 12°C (Figure 3A) and even at 8°C (results not shown).
Pkc1 plays no apparent role in the cold response
We then analysed the effects on growth at 12°C of a high copy number expression of PKC1. As Figure 3(B) shows, the overexpression of PKC1 in wt strain cells caused no effects on growth at either 30°C or 12°C. We then checked whether a high expression level of PKC1 could overcome the cold phenotype of slt2Δ mutant cells. Again, the overexpression of PKC1 had no effect on yeast growth at 12°C, and was even clearly detrimental at 30°C (Figure 3B). Thus it seems that Slt2p exerts an inhibitor effect on Pkc1p activity, as indicated under other conditions , and that an excess of protein kinase encoded by PKC1 is negative for yeast growth.
Cell exposure at low temperature results in Slt2p phosphorylation, which depends on the upstream elements of the CWI pathway
The above results question the signalling through the CWI pathway in response to a downward shift in temperature. To clarify this issue, we monitored the activation by phosphorylation of Slt2p at 12°C. Exponentially growing cells of pSLT2-HA transformants of the slt2Δ mutant were transferred from 25°C to 12°C and at different times samples were taken for Western blot analysis. A downward shift in temperature was shown to increase the signal of phospho-Slt2p, which peaked after 2 h of cold exposure (Figure 4A). Later the level of phospho-Slt2p started to decrease, although the phosphorylation of MAPK was still evident even after 3 h at 12°C (Figure 4A). These changes were not the result of increased abundance of Slt2p, which scarcely varied as judged by the HA signal (Figure 4A). Furthermore, increased phosphorylation of Slt2p at low temperatures was absolutely dependent on the presence of a functional Bck1 protein (Figure 4A).
Slt2p is activated by phosphorylation and exerts its function through a catalytic mechanism
Deletion of WSC1 decreased the increased phosphorylation of Slt2p at 12°C, whereas the combined deletion of all the WSC members abrogated the response (Figure 4A). The ratio of the Slt2p phosphorylation signal after 120 min at 12°C to the corresponding signal at 25°C (time 0) was approximately 5, 2 and 1 for the wt, the single wsc1Δ mutant and the triple mutant respectively (Figure 4A), thus confirming the role of WSC family sensors in the activation of the MAPK at low temperatures. Consistently with this, the cold-growth defect of wsc1Δ mutants, either the single wsc1Δ or the triple wsc3Δ wsc2Δ wsc1Δ mutant, was alleviated by the expression of a hyperactive allele of BCK1, BCK1-20 . Quite remarkably, the effect of BCK1-20 was specific as it was unable to suppress the phenotype of the slt2Δ mutant strain (Figure 4B). We also noted that lack of WSC1 increased the basal level of phospho-Slt2p in cells grown at 25°C (time 0, Figure 4A), an effect that was abolished when mutant cells were transformed with a plasmid containing the WSC1 gene (results not shown). We can offer an obvious explanation for this result, but it could indicate a role of Wsc1p in a down-regulation mechanism of CWI signalling.
Cold growth requires the kinase activity of Slt2p
We wondered if Slt2p requires its protein kinase activity in providing cold adaptation. To address this issue, slt2Δ mutant cells were transformed with the plasmid pSLT2-km, which contains a mutant form of Slt2p (K54R) lacking catalytic activity . As a control, the cells were transformed with plasmid pSLT2 carrying a wt form of the MAPK-encoding gene . As shown, the mutant allele failed to complement the cold-growth defect of the slt2Δ mutant, whereas the allele encoding a native Slt2 protein completely restored the wt phenotype at 12°C (Figure 4C). We then analysed the overexpression of SLT2 to see if it could alleviate the growth defect of a bck1Δ mutant strain at low temperatures. However, this was not the case (Figure 4C). Hence, our results indicate that Slt2p is activated by increased phosphorylation and exerts its function through a catalytic mechanism.
Cold does not induce an Slt2p-dependent transcriptional response, but a functional MAPK is necessary to ensure the full expression of a subset of genes
The above results confirmed the existence of cold-instigated signalling through the MAPK module of the CWI pathway and the need for a catalytically active Slt2 protein. We therefore examined whether this event induces the transcription mediated by known Slt2p-dependent transcription factors (Figure 5A shows a schematic representation). We first checked the Rlm1p-induced expression at 12°C by using a plasmid (p2XRlm1-lacZ) containing two Rlm1p-binding sites fused to the E. coli lacZ gene. As shown in Figure 5(B), the short promoter with the Rlm1p-binding boxes was able to promote β-galactosidase production in the presence of 40 μg/ml of CFW. However, the up-regulation of the reporter was missing at low temperatures, a result that well matches the absence of a cold-growth defect in the rlm1Δ mutant (Figure 5A). We also noted that Slt2p activity was required for the basal expression of the reporter at either 25°C or 12°C (Figure 5B). We then tested if the increased phosphorylation of Slt2p at 12°C induced the expression of FKS2 (FK506 sensitivity protein 2; pFKS2-lacZ, Figure 5B), which encodes one of two alternative catalytic subunits of 1,3-β-glucan synthase. A previous study showed that the expression of this target is regulated through SBF [SCB (Swi4/6 cell cycle box)-binding factor] in response to cell wall damage , and we had observed that the deletion of SWI4 or SWI6 results in cold sensitivity (Figure 5A). However, we were unable to detect the cold-provoked up-regulation of FKS2 (Figure 5B), although, once again, the absence of Slt2p decreased the reporter's basal expression.
Epistatic effects on the cold growth of SLT2, SWI4 and SWI6, and Slt2p-mediated transcriptional activity at low temperatures
We then compared the transcriptome of the wt and slt2Δ mutant cells at 12°C. The data obtained from the microarray assays revealed few differences in the mRNA levels under this condition (Supplementary Table S4). Nevertheless, 55 genes were found to be less expressed in the mutant strain, of which, 27% encoded cell wall proteins (GO accession number GO:0071554, P value=2.44E−10). Thus Slt2p is required to maintain the basal expression of a subset of genes at low temperatures, a function in which Rlm1p, Swi4p or Swi6p could play a role. However, the transcription factors do not seem to be targets of the cold-provoked phosphorylation of Slt2p, and this activation does not appear to trigger a cold-specific and Slt2p-dependent transcriptional program.
SWI4 plays a specific role in the growth of yeast cells at low temperatures
The experiments shown in Figure 5(A) revealed that the lack of Swi4p was inhibitory for the cold growth of S. cerevisiae. We also observed that the Swi6 was required for growth; however, this appeared to be independent of the ambient temperature. To further clarify the functional connection of these proteins with the MAPK module of the CWI pathway, BCK1 or SLT2 were deleted in the swi4Δ- or swi6Δ-null mutant backgrounds and the resulting double mutants were tested for growth at 30°C and 12°C. As Figure 5(C) shows, all of the mutants displayed an evident growth defect at 30°C and especially so at 12°C. Furthermore, the phenotype appeared to be the sum of the phenotypes observed for the single mutants, which suggests the absence of genetic interactions. We also noted that the growth defect of swi4Δ bck1Δ and swi4Δ slt2Δ mutants at 30°C was rescued by the presence of 75 μM inositol, whereas the phenotype at 12°C was maintained under these conditions (Supplementary Figure S3 at http://www.BiochemJ.org/bj/446/bj4460477add.htm). In contrast, the phenotype of the swi6Δ mutants was insensitive to the presence of larger amounts of inositol in the culture medium, or even proved detrimental (Supplementary Figure S3).
The effect of Slt2p at low temperatures is independent of Hog1p
We checked whether the growth defect of slt2Δ mutant cells at 12°C could be influenced by the activity of the HOG pathway. Previous work has shown that MAPK Hog1p is phosphorylated in cold-exposed cells , and there is evidence of a cross-talk between the HOG and the CWI pathways [6,14]. Accordingly, we first analysed the phosphorylation state of Hog1p at 12°C in a Slt2p-deficient strain. Exposure of yeast cells to either low temperatures  or NaCl  triggers a quick and transient phosphorylation of the MAPK. However, absence of Slt2p extended the time window of Hog1p increased phosphorylation upon cold or osmotic stress (Figure 6A). This result fits well with a model in which Slt2p inhibits the cold-instigated phosphorylation of Hog1p. Similarly, the lack of Hog1p altered the phosphorylation kinetics of Slt2p in response to a lowering of the temperature, with the CWI pathway MAPK being activated earlier, an effect which was observed more clearly in CFW-exposed cells (Figure 6B).
Interaction between the CWI and the HOG pathways
We then analysed the epistatic effects of HOG1 and SLT2 on cold growth. As shown in Figure 6(C), disruption of HOG1 in a slt2Δ strain had no major effect on the cold growth of the single mutant. Likewise, the double slt2Δ hog1Δ mutant displayed the same growth defect on NaCl as the single hog1Δ strain. Conversely, absence of Hog1p in a slt2Δ background provided cell protection against CFW (Figure 6C). Altogether, our results demonstrate the existence of a cross communication between the CWI and HOG pathways. This phenomenon appears to be important in the adaptive response of yeast cells to CFW. However, our data are inconsistent with a simple model in which cold-activated Slt2p mediates yeast growth at low temperatures by modulating the activity of the HOG pathway.
The PKA pathway is a downstream effector of the cold-activated CWI MAPK module
As mentioned above, recent work has demonstrated the existence of a molecular link between TORC1 and PKA via the CWI pathway . In particular, TORC1 inhibits Bcy1p phosphorylation, the regulatory subunit of PKA, via Sch9p (suppressor of cdc25) and Slt2p . As shown in Figure 7(A), the overexpression of BCY1 or TPK1 (Takashi's protein kinase 1), the latter encoding a PKA catalytic subunit, had no effect on the growth of the wt BY4741 strain. The slt2Δ strain also remained insensitive to high expression levels of TPK1. However, an extra copy of BCY1 partially alleviated the growth defect of the MAPK mutant at 12°C (Figure 7A), a result that was not observed in a swi4Δ strain, thus confirming, the specific role of this protein at low temperatures.
Implication of PKA effectors and targets on cold growth
We then analysed the roles of the transcription factors MSN2 and MSN4. The overexpression of MSN2 has been found to reverse the sensitivity phenotype to oxidative stress of a CWI pathway mutant lacking the sensor MTL1 . The deletion of MSN2, MSN4 or the knockout of both genes, had very small or no effect on cold growth (Figure 7B). Furthermore, the overexpression of MSN2 provided only a slight cold-growth improvement of both the bck1Δ and slt2Δ mutant strains (Figure 7C). Finally, we measured the activation of a STRE (stress-response element)–lacZ reporter in both the wt and slt2Δ mutant cells at 12°C. As shown in Supplementary Figure S4 (at http://www.BiochemJ.org/bj/446/bj4460477add.htm), no significant increase in β-galactosidase activity was detected upon cold shock. In contrast, clear inductions were observed in glucose-starved cells (Supplementary Figure S4). Altogether, our results suggest that the Slt2p-mediated attenuation of PKA activity might be relevant in yeast cells' adaptation to low temperatures. However, Msn2p/Msn4p play a minor role, if any, in this response, suggesting that PKA might control unknown targets of relevance in the adaptive cold response.
The TORC1 and the RAS–cAMP signalling cascade
We further investigated the role of regulatory proteins upstream of PKA. The deletion of either TOR1 or SCH9 did not alter the growth of wt BY4741 at low temperatures. Nevertheless, the absence of the latter alleviated the phenotype of slt2Δ and bck1Δ mutant cells (Figure 8A). SCH9 deletion has been reported to diminish the activity of PKA  and the PKA-dependent phosphorylation of several targets, including MSN2 and MAF1 [36–38]. Furthermore, the lack of a functional Tor1p caused a growth defect at 12°C in cells of the CML128 strain (Figure 8B). Unlike BY4741, this yeast strain is auxotrophic for tryptophan which increases its cold sensitivity. In good agreement with this, a ras2Δ mutant strain showed a cold-resistant phenotype in a Trp− background (Figure 8B), which was reversed to a wt behaviour by integration of plasmid YIplac204 (TRP1). RAS2 encodes a positive regulator of adenylate cyclase and, consequently, cells lacking this gene displayed low PKA activity . Moreover, cells lacking IRA2 (inhibitory regulator of the RAS–cAMP pathway 2), a negative regulator of the pathway , exhibited a cold-growth defect even in the BY4741 wt strain (results not shown). Hence, the proteins that indirectly regulate the activity of the PKA pathway influence, in turn, growth at low temperatures.
Genetic and biochemical evidence of the interactions among Sch9p, Tor1p and Slt2p in regulating growth at low temperatures
Lastly, we analysed if cold-induced Slt2p phosphorylation was altered by the absence of Sch9p. Previous work has shown that the MAPK is phosphorylated and activated by rapamycin treatment upon the deletion of SCH9, suggesting an indirect molecular link between both proteins . Accordingly, Slt2p was phosphorylated in cold-shocked cells of the sch9Δ mutant strain (Figure 8C).
The present study has uncovered the functional role of the CWI pathway in the response of yeast cells to cold stress. The results show the existence of signalling through the CWI pathway in response to a downward shift in temperature and the need for some cell surface sensors, elements of the CWI–MAPK module and transcription factors for cold growth. However, there is no evidence for either a cold-provoked perturbation of the cell wall or a differential cold-expression program mediated by MAPK Slt2p. Hence, the cold activation of this pathway may respond to specific mechanisms and mediate certain outputs.
CWI signalling is triggered in response to a variety of chemicals and environmental conditions that induce cell wall damage [9–11]. Thus Slt2p activation at 12°C suggests a cold-provoked deficiency in cell wall integrity and/or functionality. However, we were unable to detect a cold-stimulated up-regulation of the lacZ reporters driven by Rlm1p- and Swi4/Swi6-responsive promoters, the main known output of the CWI pathway . Moreover, Slt2p activity was required for basal expression at low temperatures, but the list of down-regulated genes did not include the key genes required for cell wall remodelling . To support this view, we found that cold shock has no effect on the sensitivity of yeast cells to zymolyase treatment, whereas long-term cold exposure increases resistance to this agent in either wt or slt2Δ mutant cells (results not shown).
Alternatively, the cold activation of the CWI pathway might be triggered by perturbations in the plasma membrane, as suggested for other sources of stress [10,41]. Indeed, a downward shift in temperature causes a significant drop in membrane fluidity  and this change appears to be detected by the Sln1p cell surface cold sensor as the primary signal for HOG pathway activation . However, the cold-stimulated phosphorylation of MAPK Hog1p peaks at around 5 min after the onset of stress, whereas Slt2p activation peaks after 120–180 min. Thus this result suggests that the CWI pathway does not directly sense a drop in temperature, but a secondary effect provoked by this change, a mechanism that has already been suggested to operate under heat-stress conditions .
The most widely recognised change in cell membranes at low temperatures is the unsaturation of lipid acyl chains . Phospholipids with unsaturated fatty acids have a lower melting point and exhibit more flexibility than phospholipids with saturated acyl chains. In S. cerevisiae, this adaptation involves the induction of OLE1 , the yeast gene for fatty acid desaturase, which raises the unsaturation index and fluidity of the plasma membrane . A low ambient temperature may also alter the repertoire of membrane proteins, as recently shown for transporters like Gap1 (general amino acid permease 1), Tat (tyrosine and tryptophan amino acid transporter) 1p or Tat2p . Consistent with this, several of the 106 mutants displaying growth defects at low temperatures  are affected in ribosomal proteins and translation, pre-rRNA processing for ribosome biogenesis or assembly, and protein folding. Interestingly, unfolded protein stress activates CWI signalling and Slt2p is an important determinant of ER (endoplasmic reticulum) stress survival . Thus low temperatures are expected to cause a drastic change in the microenvironment of plasma membrane sensors, which could ultimately induce CWI signalling.
Like heat stress, Wsc1p is seen to be the most important sensor for the response of the CWI pathway to cold. A wsc1Δ mutant shows reduced Slt2p phosphorylation at 12°C and a cold-growth defect, which is exacerbated by the simultaneous loss of WSC2 and WSC3. Thus Wsc family sensors appear to play a key role in activating the CWI pathway in response to changes in ambient temperature. Nevertheless, activation by cold and heat stress appears to differ in terms of the signalling route. As we found, cold has no major effect on the growth of yeast cells lacking Rom1p or Rom2p, two well-known upstream effectors of PKC, which controls the CWI–MAPK module . In addition, we were unable to observe depolarization of the actin cytoskeleton in cold-exposed cells, a change promoted by heat stress, among other factors , or cold growth effects through the overexpression of PKC1. We also found that phosphorylation of Slt2p was absolutely dependent on Bck1p, whereas the strains lacking MAPKKK were still able to activate Slt2p in heat-shocked cells . Hence, how the cold signal inputs the MAPK module from the Wsc1p–3p membrane sensors to Bck1p and the exact need of Pkc1p in cold-instigated signalling remain unclear. In agreement with this, cold signalling could mediate the activity of the GTPase Rho1p, which controls Pkc1p-independent functions in response to stress. Accordingly, the lack of SAC7, a GAP for Rho1p GTPase, results in a cold-sensitivity phenotype .
Unusual activation mechanisms have been proposed for the CWI pathway signalling promoted by zymolyase and caffeine treatment. The former requires the Sho1p branch of the HOG pathway, Pkc1p and the CWI–MAPK module [6,13]. The existence of shared components and a cross-talk between the HOG and the CWI pathways is well documented , as is the activation of Hog1p in response to cold [4,8]. Caffeine signalling to Slt2p involves the inhibition of Tor1p and is mediated through Rom2p . Although, we found evidence that the HOG and CWI pathways are mutually regulated in response to a downward shift in temperature, the results of the present study do not support the view of a Hog1p-dependent signal being responsible for Slt2p activation. However, our observations suggest that, as for caffeine exposure, the cold-instigated phenotype might be partially dependent on the inhibition of Tor1p-regulated cellular functions.
Previous reports have demonstrated that the inactivation of TORC1 by rapamycin treatment  or nitrogen starvation  triggers Slt2p activation. TORC1, together with the cAMP–PKA pathway, is the main controller of yeast cell growth in response to nutrients [18,19]. TORC1 inhibition activates Slt2p via an indirect mechanism mediated by Sch9p , a downstream target of TORC1 signalling . Slt2p then phosphorylates and activates Bcy1p directly, which results in reduced PKA activity . We found that the absence of TOR1 causes cold sensitivity, whereas a ras2Δ mutant shows increased cold growth. We also observed that the knockout of IRA2, which is a negative regulator of the cAMP–PKA pathway , results in cold sensitivity (results not shown). Moreover, a lack of Sch9p alleviates the phenotype of slt2Δ and bck1Δ mutant cells, as well as attenuation of PKA activity by the overexpression of BCY1. The simplest interpretation of these results is that low temperatures inhibit the activity of membrane-associated proteins, including amino acid transporters , and cause nutrient starvation, which then triggers the inactivation of TORC1–PKA. Thus Slt2p appears to play a role in the co-ordinated regulation of these pathways, and this event is important in the adaptive response of yeast cells to cold.
This result led us to hypothesize the role of Msn2p/Msn4p at low temperatures. Active PKA is thought to regulate multiple processes, including transcription, energy metabolism and cell-cycle progression, by the phosphorylation of key substrates . Regulated by PKA, the transcription factors Msn2p/Msn4p, which control the general stress response , play a fundamental role in the induction of a significant subset of cold-induced genes . Moreover, yeast has been show to exhibit an Msn2p/Msn4p-dependent adaptive response, which sustains viability at near-freezing temperatures . However, our results are inconsistent with a role of Msn2p/Msn4p at low, but still permissive, temperatures.
Cold stress causes a growth defect in those cells lacking Swi4p or Swi6p. This is somewhat surprising since, as we discussed, the cold activation of Slt2p does not induce the expression regulated by SBF. Cells of the mutant swi6Δ strain also displayed a growth defect at 30°C. However, the growth defect of swi4Δ cells was observed specifically at 12°C and was not rescued by osmotic stabilization. Swi4p is a key transcriptional regulator of the yeast cell cycle [9–11]. Nevertheless, its function might provide a link among nutrient acquisition, nutrient assimilation, energy metabolism and cell-cycle control, as proposed previously . For example, previous results have shown that the cell-cycle transcriptional regulation of genes is not directly linked to cell cycle functions, like PMA1 (plasma membrane ATPase 1), MET (methionine requiring) or PHO (phosphate metabolism) regulon genes . Thus a Swi4-dependent transcriptional program might be critical to help cope with metabolic restrictions induced at low temperatures. How this function is regulated and the exact targets controlled by Swi4p in response to cold still await further analysis.
Known signalling pathways appear to mediate the genetic response to cold. Nevertheless, cold sensitivity is often associated with defects in protein–protein interactions  and, consequently, cold stress may be exploited to uncover specific signalling circuits and novel interactions.
bypass of C kinase 1
bypass of cyclase mutations 1
cell wall integrity
FK506 sensitivity protein 2
cell fusion 3
high osmolarity glycerol response
inhibitory regulator of the RAS–cAMP pathway 2
kinase suppressor of supersensitive 2
mitogen-activated protein kinase
mating pheromone-induced death 2
mid-two like 1
oleic acid requiring 1
protein kinase A
protein kinase C1
Rho1 multicopy suppressor 2
Swi4/6 cell cycle box-binding factor
suppressor of cdc25
suppressor of lyt2
switching deficient 4
tyrosine and tryptophan amino acid transporter
target of rapamycin
TOR complex 1
Takashi's protein kinase 1
wall integrity and stress response component
1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose
Isaac Córcoles-Sáez performed the main research and analysed and interpreted data; Lídia Ballester-Tomas performed some of the stress sensitivity tests and analysed data; Maria de la Torre-Ruiz performed the actin staining experiments; Jose A. Prieto constructed some of the mutants used, wrote the paper, contributed to discussion and reviewed/edited the paper prior to submission; and Francisca Randez-Gil designed the research, interpreted data and wrote the paper.
We thank Dr David Engelberg (Hebrew University of Jerusalem, Jerusalem, Israel), Dr Carl Mann (Saclay CEA, Paris France), Dr François Doignon (University of Bordeaux, Bordeaux, France), Dr Kevin Morano (University of Texas Medical School, Houston, TX, U.S.A.), Dr Michael Hall (University of Basel, Basel, Switzerland), Dr Yu Jiang (University of Pittsburgh School of Medicine, Pittsburgh, PA, U.S.A.), Dr David Levin (Boston University, Boston, MA, U.S.A.), Dr Francisco Estruch (University of Valencia, Valencia, Spain) and Dr Matthias Rose (Frankfurt University, Frankfurt, U.S.A.) for providing plasmids and yeast strains.
This work was supported by the Comisión Interministerial de Ciencia y Tecnología project (AGL2010-17516) from the Ministry of Economy and Competitiveness of Spain (MINECO). I.C.-S. and L.B.-T. were supported by a predoctoral fellowship within the ‘FPI’ program.