The response of yeast cells to sudden temperature downshifts has received little attention compared with other stress conditions. Like other organisms, both prokaryotes and eukaryotes, in Saccharomyces cerevisiae a decrease in temperature induces the expression of many genes involved in transcription and translation, some of which display a cold-sensitivity phenotype. However, little is known about the role played by many cold-responsive genes, the sensing and regulatory mechanisms that control this response or the biochemical adaptations at or near 0°C. This review focuses on the physiological significance of cold-shock responses, emphasizing the molecular mechanisms that generate and transmit cold signals. There is now enough experimental evidence to conclude that exposure to low temperature protects yeast cells against freeze injury through the cold-induced accumulation of trehalose, glycerol and heat-shock proteins. Recent results also show that changes in membrane fluidity are the primary signal triggering the cold-shock response. Notably, this signal is transduced and regulated through classical stress pathways and transcriptional factors, the high-osmolarity glycerol mitogen-activated protein kinase pathway and Msn2/4p. Alternative cold-stress generators and transducers will also be presented and discussed.
signal transduction pathways
Decreases in ambient temperature are common in almost every ecological niche. A drop in temperature may take place seasonally, daily or just unexpectedly, depending on the region, climate and environment. The effects of low temperature on life have been studied thoroughly, as cold influences the structural and functional properties of cellular components negatively, both physically and chemically. A decrease in membrane fluidity and diffusion rates, alterations in molecular topology or modifications in enzyme kinetics reportedly occur as a consequence of low temperatures (Gast et al., 1993; Thieringer et al., 1998; Inouye et al., 1999). To withstand such temperature downshifts, organisms have developed mechanisms that enable them to adapt and survive. These mechanisms (commonly referred to as the ‘cold-shock response’) have been extensively studied in bacteria and plants. In Escherichia coli, Bacillus subtilis and other prokaryotes, a certain group of proteins is specifically (but not uniquely) induced upon a cold-shock (Thieringer et al., 1998; Gualerzi et al., 2003). These cold-shock proteins are involved in transcription, translation and other fundamental functions that play a role in maintaining nucleic acid structure. However, little is known about the mechanisms underlying the transcriptional activation of cold-responding genes. In plants, cold-inducible genes cover a wider range of functions, and some of them also respond to drought or high salinity (Shinozaki & Yamaguchi-Shinozaki, 1996; Seki et al., 2002b). Moreover, it is thought that there is some cross-communication between the signal transduction pathways responding to these stresses (Seki, 2002a, 2004), although an exclusive pathway for cold-induced gene expression has also been proposed (Shinozaki & Yamaguchi-Shinozaki, 1996).
Although the cold-shock response has been widely studied in bacteria and plants, relatively little attention has been paid to fungi, and specifically to yeast. The budding yeast Saccharomyces cerevisiae has colonized different natural and artificial environments, all of which can be subject to low temperatures. Yeast growing on the ground or on the surface of grapes and other fruits are exposed to temperature downshifts like those described above. On the other hand, when these yeasts are used in certain brewing and wine fermentations, they are exposed to temperatures of around 10–12°C, far below their natural physiological temperature (25–30°C). Moreover, yeasts, as well as other microorganisms, are stored at very low temperature (4°C) in industrial and laboratory conditions. This temperature is chosen because it is generally accepted that although it is growth-restrictive, viability is fully maintained over long periods of time.
All these features indicate that S. cerevisiae must have the necessary molecular machinery to survive and adapt to cold, and that this is probably one of the reasons why this species is so widespread. Therefore, the study of the cold-shock response in S. cerevisiae is of crucial interest for both basic and technological reasons (Randez-Gil et al., 2003). The growth in high-throughput analytical techniques has led to a number of publications on this subject in recent years. Our knowledge of the cold response in yeast, although limited, is expanding and we are gaining new insight into other molecular mechanisms, signalling pathways and regulators that, until now, were apparently unrelated to low temperature. The state of the art and some possible models for cold-signal generation, signal transduction and function of cold-responding genes, as well as the prospects for future research, are discussed in this review.
Time and temperature-dependent and -independent genetic responses to cold
Apart from these characteristic cold-stress markers, part of the genetic response to low temperature seems to be time-dependent. Examination of different microarray-based studies reveals sequential changes in the transcriptional profile during the time-course of exposure to 10°C. Researchers have noted two (Schade et al., 2004) or even three (Sahara et al., 2002) stages, depending on the stress duration. Initial responses (0–2 h) include the enhanced expression of key genes involved in phospholipid synthesis, such as INO1 and OPI3, and fatty-acid desaturation (OLE1) (Sahara et al., 2002; Schade et al., 2004). Other genes that are induced within this period are those related to transcription (RNA helicases such as DBP2, RNA polymerase subunits such as RPA49 and RNA processing proteins such NSR1, among others), and an important number of ribosomal protein genes. Thus, 94 of the 323 genes found to be up-regulated after yeast was incubated for 2 h at 10°C encode ribosomal proteins (Sahara et al., 2002).
Remarkably, most of these transcription-related and ribosomal genes are abruptly repressed after longer incubation periods (4–24 h) at 10°C (Sahara et al., 2002; Schade et al., 2004) and also at 4°C (Homma et al., 2003; Murata et al., 2006). This phase is characterized to some degree by the transcriptional activation of typical stress-marker genes. Indeed, some members of the main gene family involved in cellular protection, the heat shock protein (HSP) genes, are induced at this stage. Genes encoding HSPs, namely HSP12, HSP26, HSP42, HSP104, SSA4, SSE2 and YRO2, are up-regulated after 4–12 h of transfer to 10°C (Sahara et al., 2002; Schade et al., 2004), as well as at similar times at 4°C (Homma et al., 2003; Murata et al., 2006), 0°C (Kandror et al., 2004) and −80°C (Odani et al., 2003). Other members of the HSP family (i.e. CIS3, HMS2, HSC82, HSP30, HSP60, HSP78, HSP82, HSP150, SSA1, SSA2) are repressed at 10°C (Sahara et al., 2002), whereas at lower temperatures their mRNA content increases (Murata et al., 2006). This discrepancy indicates that different members of the HSP gene family are differentially regulated in both a time- and a temperature-dependent manner. The inventory of genes that are induced after 4–6 h of cold incubation and beyond also includes genes involved in the metabolism of reserve carbohydrate glycogen (GLG1, GSY1, GLC3, GAC1, GPH1 and GDB1), and trehalose (TPS1, TPS2 and TSL1), and genes for detoxifying reactive oxygen species (ROS) and defence against oxidative stress, such as catalase (CTT1), glutaredoxin (TTR1), thioredoxin (PRX1) and glutathione transferase (GTT2).
Cold-sensitive mutants and the cold ribosome adaptation hypothesis
In view of the available data, it appears that yeasts adapt to temperature decreases in terms of the duration and the extent of the stress. This is especially evident if we consider the opposite regulation displayed by ribosomal and transcription-related genes between short and long incubation periods at 10 or 4°C (Sahara et al., 2002; Schade et al., 2004; Murata et al., 2006), which is consistent with previous reports about cold growth defective mutants. Several of the 106 mutants displaying growth defects at low temperatures (Hampsey, 1997) (catalogued in http://mips.gsf.de/proj/yeast/CYGD/db/index.html) are affected in ribosomal proteins and translation (Winzeler et al., 1999; Zhang et al., 2001), or in proteins involved in pre-rRNA processing for ribosome biogenesis or assembly (Lee & Baserga, 1997), protein folding (Craig & Jacobsen, 1985), exocytosis (Lehman et al., 1999) and nucleus–cytosol exchange (Noguchi, 1997, 1999). These observations indicate the need for remodelling the translational machinery and secondary structure of nucleic acids compromised by cold-shock; moreover, this could support a model for cold ribosome adaptation in S. cerevisiae that is similar to that proposed for E. coli (Jones & Inouye, 1996). At temperatures that are low, but still permit growth, there is an up-regulation of genes that are essential for cold growth (Fig. 1). By contrast, such genes are expressed minimally or repressed after prolonged periods of stress (when all the transcriptional and translational machinery has already been remodelled) or under severe cold conditions as at 4°C (generally considered to be a growth-restrictive temperature, although minimal growth still occurs), where low or no de novo protein synthesis is required (Fig. 1).
Main responses of Saccharomyces cerevisiae to decreasing temperatures. At mid- to low temperatures, the specific transcriptional and translational machinery is up-regulated in order to enable cold-adapted growth. These mechanisms are switched off when the temperature is low enough to be growth-restrictive. By contrast, freeze-protective mechanisms, such as fatty acid desaturation and synthesis of osmolytes, are triggered even at permissive temperatures, and intensified at near-freezing conditions, a situation in which further protection systems, such as HSPs, are also activated.
Even so, results from transcriptomic experiments reveal that several genes respond to cold conditions regardless of exposure time and temperature. This finding suggests that not all the cold-regulated genes are involved in functions related to growth.
The freeze-protective function of the cold response
Although some of the cold-responsive proteins might be essential for the cells to adapt and resume growth under the new unfavourable environmental conditions, some evidence suggests that the main aim of other common cold-shock responses is to protect cells against freeze injury (Fig. 1). For example, the stability of Ole1p, the only fatty-acid desaturase known in S. cerevisiae (Stukey, 1989, 1990), seems to be important for cold growth (Loertscher et al., 2006); however, overexpression of OLE1 does not confer growth advantages at low temperatures (Kajiwara et al., 2000). By contrast, production of recombinant desaturases increased the unsaturation index and fluidity of the yeast membrane, and positively influenced freeze tolerance of baker's yeast cells (Rodríguez-Vargas, 2007). These observations suggest that changes in membrane composition after a decrease in temperature might be important in influencing cell survival upon freezing. Membrane organization and dynamic properties are the main targets of freeze injury (Wolfe & Bryant, 1999). Hence, the cold-instigated induction of fatty acid desaturases, cell-wall mannoproteins and seripauperins are consistent with this idea. Similarly, it has been shown that, in addition to temperature downshifts (Zhang et al., 2003), freezing and thawing generate superoxide anions and free radicals (Park et al., 1997; Du & Takagi, 2005), which is consistent with the cold-provoked up-regulation of genes for ROS detoxification and defence against oxidative stress, and with genes encoding HSPs. In this sense, HSPs are molecular chaperones involved in the response to several kinds of stress, preventing protein denaturation and misfolding (Papp et al., 2003; Young et al., 2003).
A downshift in temperature also activates the metabolism of reserve carbohydrates. It has been suggested that glycogen turnover occurs in response to different kinds of stress, such as heat-shock or oxidative damage, because the genes for both its synthesis and its degradation are simultaneously induced, with no net accumulation of this polysaccharide (Parrou et al., 1997). In spite of the similar transcriptional profile observed at low temperatures (Sahara et al., 2002; Schade et al., 2004; Murata et al., 2006), such recycling does not appear to be the case for cold stress, because glycogen is significantly accumulated in yeast after 12 h of treatment at 10°C (Schade et al., 2004). Like glycogen, trehalose starts to accumulate after 12 h of incubation at 10, 4 or 0°C regardless of whether there is growth or not (Kandror et al., 2004; Schade et al., 2004). Consistent with this, the genes involved in the biosynthesis of this disaccharide are cold-induced after 4–8 h of incubation at 10°C. The same activation occurs at lower temperatures, showing maximum induction levels at 0°C (Sahara et al., 2002; Kandror et al., 2004; Schade et al., 2004; Murata et al., 2006). Trehalose is known to accumulate at high levels in response to different stress conditions (Blomberg et al., 2000; Estruch et al., 2000), and several protecting roles have been proposed for this disaccharide, such as membrane and protein stabilization (Singer & Lindquist, 1998; Elbein et al., 2003; Gancedo & Flores, 2004). Moreover, high levels of trehalose have also been correlated with freezing resistance (Kim et al., 1996). Although trehalose is not needed for growth at 10°C (Schade et al., 2004), the results obtained by Kandror (2004) revealed that this compound protects cells against very cold conditions. In fact, viability of cells incubated at 0°C for 5–20 days correlated with the intracellular trehalose content, and cold-instigated accumulation of the disaccharide protected the cells from viability losses due to freezing. Similar behaviour has been observed with glycerol. GPD1, the gene encoding the main enzyme involved in glycerol synthesis (Albertyn et al., 1994), is activated upon a shift to low temperature, and cells begin to accumulate this osmolyte, displaying higher values at 4°C than at 12°C (Panadero et al., 2006). Like trehalose, glycerol accumulation provides freeze-protection, although it is not needed for growth at 12°C (Panadero et al., 2006). Glycerol is the only osmoprotectant solute accumulated by yeast upon hyperosmotic stress, and is also a by-product of redox homeostasis. The protective role of glycerol against freezing is well known and has been explained in terms of the osmotic shrinkage resulting from freezing/thawing processes (Wolfe & Bryant, 1999; Panadero et al., 2006).
In view of the above results, there is a clear link between exposure to low temperatures and resistance to freezing. In natural environments, frosts are often preceded by periods of very low temperatures, so this hypothesis is meaningful from an evolutionary point of view. However, there are still some aspects that need to be clarified. For example, there is no evidence of the possible function of glycogen in cold response, and a better explanation is needed for the protective role of trehalose at 0°C, a temperature at which cells are not frozen. On the other hand, the function of DAN/TIR and PAU family proteins remains unclear. Future work will contribute to clarifying the dual aspects of cold-shock responses, as a way to survive, adapt and grow at low temperatures, as well as a protective mechanism for subsequent freezing.
The cold signal and cold-sensing mechanism
For transcriptional activation to be triggered, yeast cells must sense temperature downshifts. Evidence indicates that changes in the physical state of the membrane could be perceived as a primary signal of a variation in temperature (Vigh et al., 1998). This concept was first put forward by Carratù (1996), who demonstrated that a membrane lipid perturbation causes a signal to be triggered, inducing the transcription of heat-shock genes in yeast. Based on this finding and additional evidence, the group of Murata demonstrated that, in the cyanobacteria Synechocystis, a drop in temperature causes a reduction of the membrane fluidity, and this plays a role in the perception of low temperatures and the subsequent signal transduction (Inaba et al., 2003; Los & Murata, 2004). Genetically engineered Synechocystis cells that lacked genes encoding fatty acid desaturases displayed higher membrane rigidity than the wild-type at both low and physiological temperatures, showing enhanced cold inducibility of gene activation (Inaba et al., 2003). In Synechocystis, expression of these desaturase genes upon cold-shock is controlled by the histidine kinase Hik33 (Suzuki, 2000, 2001), an integral membrane protein that functions as a cold sensor. Similarly, DesK in B. subtilis (Aguilar et al., 2001) and TRP-channels in the mammalian nervous system (McKemy et al., 2002; Peier et al., 2002), which are integral membrane proteins, act as cold-sensors. Furthermore, the extent to which the DesK protein senses temperature is regulated by the rigidity of the membrane lipid bilayer (Mansilla & de Mendoza, 2005). Overall, these observations indicate that changes in the physical state of the membrane caused by a temperature downshift are recognized by cold-sensors anchored in the membrane, generating the cold-signal.
Recently, Hik33 has been shown to regulate the expression of osmostress-inducible genes in Synechocystis (Mikami et al., 2002), suggesting that both osmotic- and cold-stress could be perceived in this organism by common mechanisms and sensing elements. Like Hik33, Sln1p, the only known yeast histidine kinase sensor, has recently been reported in the cold-sensing mechanism (Panadero et al., 2006). Sln1p, together with Ypd1p and Ssk1p, forms a phosphorelay system, which transmits the osmostress signal through different elements of the high-osmolarity glycerol (HOG) pathway, the most important osmotic stress-responding cascade in S. cerevisiae (Hohmann et al., 2002; Westfall et al., 2004). Activation of the HOG pathway was also found to be induced by the membrane-rigidifier agent dimethylsulfoxide (DMSO) (Panadero et al., 2006). Several lines of research in plants and cyanobacteria have shown that this chemical indeed mimics the changes in membrane fluidity caused by a sharp decrease in temperature, and triggers the activation of several cold-induced mitogen-activated protein kinases (MAPKs) (Örvar, 2000; Sangwan et al., 2002). Thus, Sln1p appears to function under either of these stressful conditions (Hayashi & Maeda, 2006; Panadero et al., 2006) and it is likely, therefore, that the basic mechanism of its activation by low temperature and osmotic pressure could be similar or even identical.
The fluidity state of the cell membrane might be a key factor in integrating the sensing mechanism of cold and hyperosmolarity. There is evidence that different osmosensors, such as EnvZ in E. coli or OpuA, a transmembrane transporter of Lactobacilllus lactis with osmosensor and osmoregulator properties, are stimulated by changes in the fluidity of membrane lipids (Tokishita & Mizuno, 1994; van der Heide & Poolman, 2000; van der Heide, 2001). Moreover, studies in B. subtilis and S. cerevisiae indicate that osmotic stress reduces cell-membrane fluidity (López, 2000; Laroche et al., 2001). Altogether, these observations suggest a model in which Sln1p monitors the changes in membrane fluidity caused by different stressors. Moreover, the fact that Sln1p responds to DMSO supports this view. However, more work is required to confirm this idea and to understand the exact molecular mechanism of cold-signal perception.
Cold-induced transduction pathways and transcription factors
Signal transduction pathways are the link between the sensing mechanism and the genetic response. As is the case for the cold signalling mechanism, to date there is no signal transduction pathway or transcription factor that is known to respond exclusively to low temperatures. Although the existence of such a pathway cannot be ruled out, available data point to known signal transduction mechanisms, operating under other kinds of stimuli, which may also be triggered by cold stress. We shall now go on to describe putative and known regulatory mechanisms controlling cold-instigated gene expression.
The MOX factors
The coordinated expression of the DAN/TIR genes under anaerobic conditions reportedly depends on the transcriptional activator MOX4, whereas their repression, in conditions of oxygen availability, is mediated by the two repression factors MOX1 and MOX2 in a heme-dependent fashion (Abramova et al., 2001a). All of these factors act through the consensus sequence AR1 (Cohen et al., 2001). Mox4p, Mox1p and Mox2p (or some factors dependent upon them) are believed to form a heme-sensitive complex similar to the galactose-sensitive Gal4p-Gal80p (Zenke et al., 1996; Platt & Reece, 1998). Like GAL4 regulation, MOX4 expression is autoregulated in anaerobic cells via its AR1 sequence. Interestingly, expression of TIR genes under cold-shock was eliminated in a mox4 mutant strain (Abramova et al., 2001b). Furthermore, MOX4 mRNA was also more abundant in cold-shocked cells (Abramova et al., 2001a), suggesting that induction of the activator contributes to up-regulation of TIR genes under this condition (Fig. 2).
Cold-instigated activation of the MOX factors and RUP processing. (1) When the repressor complex Mox1p–Mox2p is released, the Mox4p factor promotes the transcription of TIP/TIR genes. The cold-provoked induction of these genes is eliminated in a mox4 mutant, suggesting that low temperatures are able to release the repressor complex. (2) The transcriptional induction of OLE1 under cold-shock is dependent on the transcription factor Mga2p, which is released from the nuclear envelope by proteasome-dependent deubiquitination provoked by the action of the Rsp5p factor. This action could therefore be triggered by cold stress (see text for details).
Regulation of OLE1 expression and RUP processing
Unlike DAN/TIR genes, the promoter of OLE1 does not contain AR1 sequences. Unsaturated fatty acids (UFAs) mediate OLE1 repression via the cis transactivation element FAR (fatty acid regulated). Two genes for fatty acid synthesis (FAA1 and FAA4), the transcriptional activator HAP1 and the acyl-coenzyme A (CoA)-binding protein Acb1p have also been reported to regulate OLE1 expression (Choi et al., 1996). In addition, Spt23p and Mga2p, two functionally and structurally related factors, are also necessary for OLE1 transcription (Zhang et al., 1999) (Fig. 2). Spt23p appears to play a major role in fatty acid regulation, whereas Mga2p is known to be essential for the hypoxic induction of OLE1 through a consensus sequence named LORE (for low-oxygen response element) (Jiang et al., 2001). This sequence is also found in several genes induced by hypoxia, including TIR1. All these genes could, therefore, share regulation via LORE (Vasconcelles et al., 2001). The transcriptional induction of OLE1 under cold-shock is also dependent on Mga2p (Nakagawa et al., 2002), suggesting that the LORE-binding complex could drive the expression of a subset, at least, of cold-responsive genes (Fig. 2).
Moreover, Mga2p could also play a role as a cold-sensor, as previously postulated (Nakagawa et al., 2002). This hypothesis was constructed on the basis that a novel pathway has been described in S. cerevisiae, the so-called ‘RUP (regulated ubiquitin/proteasome-dependent) processing’ pathway, which regulates the activation of Spt23p and Mga2p by UFAs (Hoppe et al., 2000, 2001). Spt23p and Mga2p are initially created as dormant precursors, anchored in the endoplasmic reticulum or nuclear envelope membranes by their C-terminal tails. The shortage of UFAs leads to the ubiquitination of Spt23p and Mga2p, releasing the N-terminal transcription factor domain into the cytosol by the action of the 26S proteasome in a dual way: first, processing the transcriptional factor into its active form and second, destroying it after the activation of genes such OLE1 (Auld et al., 2006). In the processing of Mga2p, UFAs exert a modest influence, with hypoxia being the main signal that triggers its processing (Jiang et al., 2002). In this scenario, a change in the physical state of the membrane due to a downshift in temperature or to hypoxia conditions (Nakagawa et al., 2003) could activate Mga2p by starting off the RUP processing (Fig. 2).
The cAMP-PKA and general stress response
Thirty-two protein phosphatase genes have been identified in the S. cerevisiae genome (Sakumoto et al., 1999). From all of them, only YVH1 was found to be induced by cold-shock (Hampsey et al., 1997) and to play a role in growth at low temperatures (Sakumoto et al., 1999). YVH1 encodes a dual-specificity protein phosphatase (DSP), which exhibits a catalytic domain displaying protein-tyrosine phosphatase activity and a C-terminal cysteine-rich motif (Fauman & Saper, 1996; Muda et al., 1999). Yvh1p shares the control of some cellular processes such as sporulation, growth and glycogen accumulation with protein kinase A (PKA). The cAMP-PKA pathway in S. cerevisiae plays a major role in the control of the genetic response to a wide variety of stresses (Thevelein & de Winde, 1999; Estruch et al., 2000; Wilson & Roach, 2002; Santangelo et al., 2006), through the phosphorylation of the Msn2p and Msn4p transcription factors (Griffioen & Thevelein, 2002) (Fig. 3, pathway 1). Upon stressful conditions, Msn2/4p migrates to the nucleus and promotes the transcription of an important number of stress-responding genes, which have the recognition sequence called STRE (stress response element) in their promoter (Martínez-Pastor, 1996). It has been suggested that Yvh1p could negatively modulate the cAMP level or a downstream component of the PKA signalling cascade. In fact, deletion of YVH1 resulted in decreased expression of a multi-STRE reporter construction (Beeser & Cooper, 2000). Like Yvh1p, Mck1p, a Ser/Thr/Tyr protein kinase, might also be involved in the cold-shock signalling via PKA. Genetic data indicate that Yvh1p acts upstream of Mck1p, at least in sporulation signalling (Beeser & Cooper, 1999). Moreover, Mck1p is involved in the phosphorylation of Bcy1p (Griffioen et al., 2003), the negative regulator of PKA. This modification occurs under environmental stress conditions such as heat-shock, salt or oxidative stress and determines the distribution of Bcy1p between the nucleus and cytoplasm. The specific relocation of Bcy1p could help to drive the PKA activity towards certain substrates under these conditions. In addition, Mck1p is necessary for the correct stress response, owing to its ability to promote indirectly a DNA-Msn2p complex (Hirata et al., 2003), without the need of Msn2p phosphorylation. Deletion of MCK1 leads to a growth defect at low temperature (de Jesus Ferreira, 2001) and inhibition of STRE-dependent transcription (Hirata et al., 2003), similar to that found in the yvh1 mutant (Sakumoto et al., 1999).
cAMP-PKA and TOR pathways. (1) Mutants in the Tpk1/2/3p (PKA)-negative regulators Yvh1p and Mck1p show growth defects at low temperatures, indicating the involvement of the cAMP-PKA pathway in the cold response. This pathway starts with the formation of the GDP-binding complex Ras1/2p/Ira1/2p/Cdc25p, which activates Cyr1p for cAMP synthesis. Inhibition of this synthesis by cold, in addition to Yvh1p and Mck1p activation, would prevent Tpk1/2/3p-mediated phosphorylation of the transcription factors Msn2/4p, avoiding their nuclear export. (2) Disruption of either TPD3 or CDC55 causes cold sensitivity. Inhibition of Tap42p phosphorylation caused by cold would prevent the formation of the complex Tap42p/Php21/22p//Tpd3p/Cdc55p, also blocking the exportation of Msn2/4p from the nucleus, with the subsequent induction of genes encoding trehalose, glycerol and other STRE genes. By contrast, localization of Msn2/4p is regulated by PKA in a TORC1-dependent manner, whereas the TORC2 complex acts on Rom2p activation (see text for details).
The cold signal transduction via the cAMP-PKA signalling pathway would, as anticipated, be essential in determining the genetic response to cold. Indeed, genes induced at the late phase of a cold-shock, like those encoding trehalose and glycogen metabolism enzymes, as well as HSP12, HSP26, HSP42, SSA4 and the ROS-defensive genes CTT1, PRX1 and TTR1, are known to be strictly dependent on Msn2/4p (Kandror et al., 2004; Schade et al., 2004). Expression of early responding genes such as DPB2, NSR1, OPI3 and RPA49, as well as PAU genes, whose cold-provoked induction is maintained over time, has also been reported to depend on a functional PKA pathway (Wang et al., 2004). Moreover, signalling through the cAMP-PKA pathway would play a major role in providing cold adaptation and tolerance to freezing. Mutants lacking MSN2/MSN4 die more rapidly at 0°C or upon freezing (Kandror et al., 2004). Mutants in RAS2, the gene for a regulatory protein in the synthesis of cAMP, clearly showed a better freeze– thaw tolerance than the wild-type, while mutants in BCY1 were sensitive to this stress (Park et al., 1997). In the same regard, mutants from a commercial baker's yeast strain, showing partial inactivation in adenylate cyclase, were better at maintaining freeze resistance (van Dijck, 2000). These results indicate than any event involving a decrease in the level of PKA activity improves cell tolerance to freeze stress. Consistent with this, deletion of IRA2 encoding the Ras GTPase (an activator of the PKA pathway) led to a freeze-sensitivity phenotype. Similarly, mutants in PDE2, the high-affinity phosphodiesterase encoding gene, also showed impaired freezing tolerance (Park et al., 2005b).
The PKC pathway
Signalling through the so-called PKC pathway (for a review, see Levin et al., 2005) is triggered under conditions that jeopardize cell-wall stability, including heat-shock (Kamada et al., 1995) and hypo-osmotic stress (Davenport et al., 1995). Activation of the G-protein Rho1, the master regulator of the pathway, triggers a linear MAPK signalling cascade that starts with Pkc1p, a homologue to the mammalian protein kinase C (PKC), and finish with the Slt2p/Mpk1p kinase. The PKC pathway controls important cell functions, such as cell-wall maintenance and actin polarization. In addition, Pkc1p regulates additional targets that are separated from the MAPK cascade (Levin, 2005). Activation of Rho1p is stimulated by Rom2p, which has GDP/GTP exchanging activity. Disruption of ROM2 leads to temperature-sensitive growth defects at both high (37°C) and low (11°C) temperatures (Manning et al., 1997). These defects include failure to form normal bud polarization and sensitivity to the microtubule depolymerizing drug benomyl. Although the activation of the PKC pathway by heat stress is well documented (Kamada et al., 1995; Heinisch et al., 1999), the phenotype of the rom2 null mutant suggests a general function of PKC in perceiving and signalling changes in environmental temperature. Some collateral observations support this idea. For example, mutants in several genes encoding proteins related to actin polarization are cold sensitive. Among them, Num1p (Revardel & Aigle, 1993) interacts with Bni1p, a formin that nucleates the assembly of linear acting filaments in response to activation by the PKC pathway (Levin et al., 2005). In addition, some mutants in Rho3p, a homologue of Rho1p that is involved in Bni1p activation during bud growth, also confer sensitivity to low temperatures (Imai et al., 1996).
PKC may also play a role in connection with the cAMP-PKA pathway. Evidence of this interaction has been provided by Park (2005a). These authors demonstrated that Rom2p can negatively regulate the Ras-cAMP pathway by controlling cAMP levels. Thus, a rom2 null mutant showed sensitivity to freeze stress, a phenotype that was suppressed by additional mutation of RAS2 or in a tpk1 tpk2 background. By contrast, the absence of either IRA2 or PDE2 exacerbated the freeze-sensitivity phenotype of the single rom2 mutant.
The TOR pathway
In plants, cold acclimatization is associated with rapid and reversible changes in the phosphorylation status of specific pre-existing proteins that allow the expression of cold-acclimatized specific (CAS) genes (Monroy et al., 1993). The type 2A Ser/Thr protein phosphatase (PP2A) is involved in basic cellular processes, such as metabolism, transcription and signal transduction (Ariño, 1993). One of the signals regulated by PP2A is a downshift in temperature (Monroy et al., 1998). The composition of PP2A subunits determines its specificity, activity and subcellular localization. In plants, TAP46 codes for a PP2A regulatory subunit, and is induced by cold-shock, suggesting that Tap46p targets PP2A under this condition (Harris et al., 1999). In S. cerevisiae, the homologue to plant TAP46 is TAP42. As in plants, Tap42p associates to PP2A catalytic subunits, Sit4p or Pph21/22p (di Como & Arndt, 1996; Jiang & Broach, 1999). This association depends on the Tor proteins, phosphatidylinositol kinase-related kinases, which are members of the target of rapamycin (TOR) signalling cascade, a pathway that controls cellular functions necessary for cell growth and metabolism in response to environmental cues (Schmelzle & Hall, 2000; Wullschleger et al., 2006). By contrast, inhibition of yeast TOR activity leads to transcriptional activation of genes so they can adapt to nutrient depletion (di Como & Arndt, 1996; Jiang & Broach, 1999; Rohde et al., 2004). Recently, it has been shown that inactivation of Tap42p prolongs the up-regulation of several Msn2/4p-dependent genes after heat-shock (Düvel, 2003). Therefore, a functional TOR pathway would be involved in nuclear export of the transcription factors under nonstressing conditions, a fact that has already been demonstrated for Msn2p (Santhanam et al., 2004). Phosphorylated Tap42p competes with Cdc55p/Tpd3p for binding to the catalytic subunit Pph21/22p, increasing protein synthesis under these conditions (Jiang & Broach, 1999). Interestingly, disruption of either TPD3 or CDC55 causes cold sensitivity (Healy et al., 1991). These data strongly suggest that, as in plants, PP2A via Tap42p, Cdc55p and Tpd3p may play a role in cold response, and that the transcription factors Msn2/4p mediate the TOR signalling (Fig. 3, pathway 2). Hence, the cold-provoked induction of Msn2/4p-dependent genes would be signalled through TOR, as well as via the PKA pathway. In this respect, it has recently been proposed that the cAMP-PKA pathway is a TOR effector branch (Schmelzle et al., 2004) involved in Msn2p localization and other readouts that are not controlled by Tap42p/Sit4p.
Further evidence that the TOR pathway is involved in cold response comes from the functional analysis of distinct TOR complexes (Loewith et al., 2002; Wullschleger et al., 2006). As well as its shared role with Tor1p in cell growth, Tor2p displays a unique function in the organization of cytoskeleton. Thus, tor2 mutants exhibit abnormal polarized distribution of the actin cytoskeleton, a phenotype that is rescued by overexpression of the actin-specific TCP-1 chaperone (Schmidt et al., 1996). Interestingly, mutants in genes encoding different subunits of this chaperone (Stoldt et al., 1996), e.g. CCT2, CCT3 and TCP1, or in all the components of its cochaperone, the GimC complex (Siegers et al., 1999), showed clear cold-sensitive phenotypes (Chen et al., 1994; Geissler et al., 1998). This suggests a Tor2p-mediated activation of protein complexes involved in maintaining the cytoskeleton under cold stress. Moreover, the abnormal actin polarization displayed by tor2 mutants was reverted by enhanced expression of PKC1, ROM2 and other effectors of the PKC pathway (Helliwell et al., 1998). Hence, it seems clear that maintenance of an adequate cell polarization upon cold-shock requires interplay between TOR and PKC pathways.
However, we cannot rule out the possibility of an indirect effect of low temperatures in the activity of the TOR pathway. Changes in the physical state of the plasma membrane due to abrupt shifts in temperature could significantly alter the activity of membrane-associated enzymes and transporters, lowering nutrient uptake, and leading to TORC1 complex inactivation. For example, stress conditions, such as heat-shock, have been reported to cause a severe decrease in amino acid import (Schmidt et al., 1998; Chung et al., 2001). This decrease is a consequence of the vacuolar degradation of high-affinity amino acid permeases such as Tat2p or Fur4p (Skrzypek et al., 1998; Bultynck et al., 2006), an event that is also stimulated by a downshift in environmental temperature (Abe & Horikoshi, 2000).
Role of the HOG pathway
As mentioned above, the histidine kinase Sln1p induces signalling through the HOG pathway in response to a downshift in temperature (Hayashi & Maeda, 2006; Panadero et al., 2006). The HOG pathway, one of the five MAP kinase pathways discovered in S. cerevisiae (Gustin et al., 1998; Schwartz & Madhani, 2004), has traditionally been thought to be involved in the genetic response to hyperosmotic stress alone (Hohmann et al., 2002; Westfall et al., 2004). However, recent findings have established novel roles for this pathway, such as adaptation to citric acid stress (Lawrence et al., 2004), heat-shock (Winkler et al., 2002), methylglyoxal resistance (Aguilera et al., 2005), distribution of proteins within the Golgi complex (Reynolds et al., 1998) or cell-wall maintenance (García-Rodriguez, 2000). Recently, results obtained by our group (Panadero et al., 2006) have revealed that downstream elements of the HOG pathway are also involved in cold-signal transduction. The transcription factor Hog1p, which is the last step of the MAPK cascade, is phosphorylated after a downshift in temperature, a fact that is prevented in mutants lacking upstream elements of the pathway. Signalling through HOG causes the activation of GPD1, GLO1 and other Hog1p-dependent osmotically activated genes in response to cold-shock (Panadero et al., 2006) (Fig. 4). These results are in good agreement with previously reported data from other organisms. The Arabidopsis MKK2 pathway mediates signal transduction upon both saline and cold stress (Teige et al., 2004). In the fission yeast Schizosaccharomyces pombe, the homologue pathway to HOG (named SAPK) is also involved in the cold-shock response (Soto et al., 2002). Similarly, activation by hypothermic stress of mammalian MAPK family members, including JNK (c-Jun N-terminal kinase) and p38, the functional homologues to Sty1p and Hog1p (de Nadal, 2002), has been reported in human cells (Gon et al., 1998; Ohsaka et al., 2002; Roberts et al., 2002). Both JNK and p38 are also activated during natural freezing and thawing of the wood frog (Greenway & Storey, 2000). This result strongly suggests that cold-stress responses by MAPKs, particularly p38 and relatives, may be adaptive in dealing with freeze stress, as recently reported (Panadero et al., 2006). In fact, a yeast strain lacking HOG1 showed impaired viability after freezing and frozen storage as compared with the wild-type (Panadero et al., 2006).
The HOG pathway. Sensitivity of the Sln1p/Ypd1p/Ssk1p histidine kinase complex to membrane rigidification caused by low temperature generates a signal that is transduced through the MAPK phosphorylation cascade. Hog1p is phosphorylated and migrates into the nucleus, promoting the transcription of genes for glycerol synthesis, among others (see text for details).
Although S. cerevisiae cold-sensitive mutants were first described over 10 years ago, only very recent studies have started to reveal the existence of cold-shock and freezing-protective responses in this organism, giving some clues to their physiological significance. Many genes with up- or down-regulated expression at low temperatures are known to participate in cellular adaptation to other kinds of stressful situations, such as thermal stress, high osmolarity, oxidation or presence of certain poisons. This phenomenon is very well known in yeast and has been called the general stress response. Hence, it is not surprising that exposure to low or subfreezing temperatures are also included in the scope of this systemic mechanism. Similarly, known sensing molecules, signal transduction machinery and transcription factors that mediate the genetic response to other stressful conditions, appear to be involved in cold response. However, cold activation of several signalling pathways might be indirect. In this way, the loss in membrane fluidity, as a physical consequence of exposure to low temperatures, would diminish the protein mobility of integral membrane proteins, such as amino acid transporters, and be interpreted by the cells as a situation of nutrient starvation.
Diversification of the functional roles of transcription pathways, which were initially believed to respond exclusively to one kind of stress, is an idea supported by an increasing number of new studies, especially in the case of the HOG pathway. Moreover, links between hyperosmotic stress, ratio of unsaturated to saturated fatty acids, and membrane rigidification lead us to speculate about a scenario in which the level of membrane fluidity is the physical perturbation sensed by the cell that triggers the response to different kinds of stresses. However, not all of the cold-induced response is referred to multistress-related genes. This is the case for the TIP/TIR genes, whose function and activation mechanism have yet to be clarified. Hence, the existence of an exclusive mechanism for the cold response in yeast cannot be discarded. Given the increasing interest shown by several groups in this field, we can expect that related works in the near future will shed light on the currently speculative aspects of this response.
We thank in particular all members of the IATA Baker's Yeast Laboratory for their contributions towards several aspects of this work. Our research described in this review was supported by the Comisión Interministerial de Ciencia y Tecnología (projects AGL2000-0524, AGL2001-1203 and AGL2004-462). J.A. is supported by an I3P grant from the Spanish Ministry of Education and Science. The English text was revised by F. Barraclough.
(1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol 14: 4135–4144.
(2000) The dual-specificity protein phosphatase Yvh1p regulates sporulation, growth, and glycogen accumulation independently of catalytic activity in Saccharomyces cerevisiae via the cyclic AMP-dependent protein kinase cascade. J Bacteriol 182: 3517–3528.
(2000) Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions. J Bacteriol 182: 2428–2437.
(1991) CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the β subunit of mammalian type 2A protein phosphatase. Mol Cell Biol 11: 5767–5780.
(2002) Phosphorylation of c-Jun N-terminal kinase in human hepatoblastoma cells is transiently increased by cold exposure and further enhanced by subsequent warm incubation of the cells. Cell Physiol Biochem 12: 111–118.
(1997) The freeze–thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway. Appl Environ Microbiol 63: 3818–3824.
(2005b) The high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae is the major determinant of cAMP levels in stationary phase: involvement of different branches of the Ras-cyclic AMP pathway in stress responses. Biochem Biophys Res Commun 327: 311–319.
(1997) Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose. Microbiology 143: 1891–1900.
(2000) A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth. J Mol Microbiol Biotechnol 2: 521–530.