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Msn4p-activation is essential for the recovery from freezing stress in yeast
BBRC Biochemical and Biophysical Research Communications 352 (2007) 750–755 www.elsevier.com/locate/ybbrc
Msn2p/Msn4p-activation is essential for the recovery from freezing stress in yeast Shingo Izawa *, Kayo Ikeda, Takumi Ohdate, Yoshiharu Inoue Laboratory of Molecular Microbiology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Received 12 November 2006 Available online 27 November 2006
Abstract Since it seems quite difficult for frozen cells to repair the damage caused by freezing, the adequate responses appear to be induced during and/or after the thawing process to recover from the damage due to freezing. In this study, the cellular events happening upon the return from freezing at 30 °C to a growth temperature (28 °C) were investigated. Yap1p, an oxidative stress-responsive transcription factor, was not activated in the thawed cells, indicating that no serious oxidative stress was generated in the frozen-thawed cells. On the other hand, Msn2p and Msn4p, general stress-responsive transcription factors, were activated in the thawed cells and caused the increased expression of a number of Msn2p/Msn4p-target genes including SOD1, SOD2, and several HSP genes. Since almost no expression of Msn2p/Msn4p-target genes was induced before thawing, these results indicate that Msn2p and Msn4p play a role during the recovery process from freezing. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Freeze-thaw stress; Msn2p; Msn4p; Yap1p; Saccharomyces cerevisiae; STRE
Both in the laboratory and in natural habitats, yeast cells probably encounter freezing temperatures much more often than they encounter heat-shock stress. Frozen yeast cells are also utilized for frozen dough technology in the bakery industry. Compared with the responses to heat shock and oxidative stress, the cellular response to freezethaw stress is not well-characterized. Recently, it has been reported that yeast cells induce the synthesis of trehalose and certain heat-shock proteins (HSPs) at near freezing temperatures (0 °C) in an Msn2p/Msn4p-dependent manner [1]. The transcription factors Msn2p and Msn4p recognize the stress-response element (STRE; consensus sequence is 5 0 -CCCCT-3 0 or 5 0 -AGGGG-3 0 ) and induce the activation of STRE-dependent promoters under various stressful conditions [2,3]. Additionally, Panadero et al. also reported that the high osmolarity glycerol (HOG) pathway is activated in response to a downward shift in temperature and that the accumulation of glycerol *
Corresponding author. Fax: +81 774 33 3004. E-mail address: [email protected] (S. Izawa).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.11.100
caused by the activation of Hog1p is essential for tolerance to freeze stress [4]. Since trehalose and glycerol function as cryoprotectants and confer resistance to freeze-thaw stress [5–8], these results clearly indicate that yeast cells undergo changes in transcriptional patterns during the cooling process and prepare the defense against the stress of low temperatures. However, cellular responses induced under such conditions are relatively slow and presumably not continued in frozen cells [1,4]. On the other hand, there is almost no information available about the cellular response in thawed cells. Since it is quite difficult for frozen cells to carry out cellular events such as transcriptional activation and protein synthesis, an adequate recovery response would be required during and/or after thawing to recover from the damage due to freezing. To properly understand the cellular response to freeze-thaw stress, it is necessary to clarify the cellular events happening upon the return to a growth temperature and in the thawed cells. In this study, we investigated the changes in thawed cells, and found that Msn2p and Msn4p, but not Yap1p (an oxidative stress-responsive transcription
S. Izawa et al. / Biochemical and Biophysical Research Communications 352 (2007) 750–755
factor) [9], were activated by thawing which led to the transcriptional activation of Msn2p/Msn4p-target genes.
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Materials and methods
Msn2pGFP
Yeast strains and medium. The Saccharomyces cerevisiae strains used in this study were YPH250 (MATa his3-D200 leu2-D1 trp1-D1 lys2-801 ade2101 ura3-52), msn2Dmsn4D (MATa his3-D200 leu2-D1 trp1-D1 lys2-801 ade2-101 ura3-52 msn2D::HIS3 msn4D::URA3) [10], and yap1D (MATa his3-D200 leu2-D1 trp1-D1 lys2-801 ade2-101 ura3-52 yap1D::HIS3) [11]. Cells were cultured in 50 ml of SD minimal medium (2% glucose and 0.67% yeast nitrogen base w/o amino acids, pH 5.5) with appropriate amino acids and bases at 28 °C with reciprocal shaking (120 rpm) in 300ml Erlenmeyer flasks. Exponential-phase cells were prepared by culturing till an OD610 of 0.5. Plasmids and analytical techniques. pSTRE-lacZ was donated by Dr. D. Engelberg [12]. pAdh1-Msn2-GFP were donated by Dr. C. Schu¨ller [3]. pRS-GFP-Yap1 and pRS-YRE-lacZ were provided by Dr. S. Kuge [9]. pAdh1-Msn4-GFP was constructed as follows: a 1.9-kbp fragment encoding the open-reading frame (ORF) of MSN4 was amplified using 5 0 TTATTGGATCCAAAAATCACCGTGCTTTTTGTGAG-3 0 and 5 0 TCTTTTAAGCTTATTAAAAACAATATAATGCTAGTC-3 0 as primers. The amplicon was digested with HindIII/BamHI and introduced into the HindIII/BamHI sites of pKW430 [13]. Analytical techniques for the bgalactosidase assay and fluorescent microscopic analysis were as described previously [11]. Northern blot analysis. Five milliliters of cell culture was transferred into a thin-wall plastic tube (50 ml volume) and frozen at 30 °C or incubated at 0 °C for 1 h. The cell culture froze up within 10 min at 30 °C. Frozen or chilled cells were incubated in an water bath at 28 °C with shaking (120 rpm). The frozen cell culture was thawed completely within 5 min at 28 °C. Total RNA was extracted from cells by the method of Schmitt et al. [14]. DNA fragments encoding the ORF of each gene were prepared by PCR using yeast genomic DNA as a template, and labeled with a–[32P]dCTP using Random primer labeling kit ver. 2 (Takara Bio, Otsu, Japan). The labeled DNA fragments were used as probes for Northern blotting. Freeze-thaw stress tolerance test. Twenty microliters of cell culture was transferred into a thin-wall plastic tube (1.5 ml volume) and quickly frozen at 30 °C. The cell culture froze up within 3 min. The method of freezethaw stress tolerance test was previously described [7].
Results Msn2p/Msn4p but not Yap1p is activated in frozen-thawed cells First, the intracellular distribution of stress-responsive transcription factors, Msn2p, Msn4p, and Yap1p, in the thawed cells was investigated. These transcription factors are reported to move into the nucleus when they are activated by stress [3,9]. Msn2p and Msn4p respond to various kinds of stress [2,3,15]. On the other hand, Yap1p is responsive to oxidative stress [9,16]. It has been reported that superoxide anions (O2) are caused during freezing and thawing [17]. Thus, we examined whether Msn2p/ Msn4p or Yap1p is activated and accumulates in the nucleus in thawed cells. As shown in Fig. 1A, green fluorescent protein (GFP)tagged Msn2p (Msn2p-GFP) and Msn4p-GFP were concentrated in the nucleus in the thawed cells and the nuclear accumulation of Msn2p-GFP and Msn4p-GFP continued for at least 15 min. Around 25 min after the thawing of
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Fig. 1. Change in the localization of stress-responsive transcription factors in thawed cells. Cells frozen at 30 °C for 1 h were thawed at 28 °C with shaking for the period indicated. Intracellular localization of Msn2p-GFP and Msn4p-GFP (A), and GFP-Yap1p (B) was monitored at the indicated time. BF, before freezing.
cells, most of the Msn2p-GFP and Msn4p-GFP had diffused into the cytoplasm. Additionally, the activity of bgalactosidase derived from STRE-lacZ was gradually increased after thawing (left panel in Fig. 2A). These results indicate the possibility that the transcription of genes containing STRE (Msn2p/Msn4p-target genes) is activated in the thawed cells. Interestingly, the b-galactosidase activity was not increased by just freezing (compare activities of cells before freezing and after 5 min of thawing). This result indicates that Msn2p and Msn4p were not substantially activated during the cooling process under our experimental conditions. We next investigated the expression of various Msn2p/ Msn4p-target genes (STRE-genes) in the thawed cells by conducting a Northern blot analysis. As shown in Fig. 2B, the expression of various STRE-genes was increased in thawed cells of the wild-type strain. Levels of expression of the STRE-genes were increased after 15 min of thawing. Compared with the wild-type cells, the msn2Dmsn4D cells showed little induction of the expression of HSP12, HSP104, CTT1 (encodes catalase), TPS1 (trehalose-6-phosphate synthase), and TPS2 (trehalose-6phosphate phosphatase). These results indicate that the expression of these genes was mainly induced in an Msn2p/Msn4p-dependent manner in the thawed cells. Interestingly, the expression of SOD1 and SOD2 was also
S. Izawa et al. / Biochemical and Biophysical Research Communications 352 (2007) 750–755
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Fig. 2. Activation of Msn2p/Msn4p in the thawed cells. (A) Cells carrying the STRE-lacZ plasmid or YRE-lacZ plasmids were frozen at 30 °C for 1 h and thawed at 28 °C with shaking for the period indicated, and b-galactosidase activity was then assayed. Results represent the means ± SD of three independent experiments. White bars, wild-type cells; black bars, msn2Dmsn4D cells (left panel) or yap1D cells (right panel). (B,C) Expression of target genes of Msn2p/Msn4p (B) and Yap1p (C) was monitored by Northern blot analysis. Cells frozen at 30 °C for 1 h were incubated at 28 °C with shaking for the period indicated. Total RNA was prepared and 15 lg of RNA was applied to each lane. BF, before freezing.
induced by thawing in the wild-type cells but not in the msn2Dmsn4D cells. The SOD1 and SOD2 genes contain the STRE consensus sequence 197 and 141 bp from the initiation codon, respectively, suggesting that they are likely to be transcriptionally regulated in an Msn2p/ Msn4p-dependent manner in the thawed cells. It is noteworthy that the expression of STRE-genes was not increased just after 5 min of thawing in the wild-type cells (Fig. 2B). This result confirmed that Msn2p and Msp4p were not substantially activated before thawing and indicates that activation of Msn2p and Msp4p was induced mainly during the recovery process from freezing. In contrast with Msn2p and Msn4p, GFP-Yap1p did not accumulate in the nucleus in the thawed cells, indicating that Yap1p was not activated after thawing (Fig. 1B). Additionally, results of the YRE-lacZ reporter assay (YRE: Yap1p recognition element) and Northern blot analysis of Yap1p-target genes (TRR1 and GPX2, encode thioredoxin reductase and glutathione peroxidase, respectively) [18,19] clearly indicate that Yap1p was not activated in the thawed cells (Fig. 2A and C ). These results suggest that no serious oxidative stress that causes the activation of Yap1p was generated in the freeze-thawed cells.
Importance of Msn2p/Msn4p-activation in thawed cells for resistance to freeze-thaw stress Next, we tried to evaluate the physiological importance of the activation of Msn2p/Msn4p in the thawed cells. To minimize the activation of Msn2p/Msn4p during the cooling process, small amounts of cells (20 ll per 1.5 ml tube) were rapidly frozen at 30 °C. We compared the resistance to freeze-thaw stress of rapidly frozen cells among the wildtype, the yap1D mutant, and the msn2Dmsn4D mutant (Fig. 3). Susceptibility to freeze-thaw stress of the yap1D cells was almost identical with that of the wild-type cells, indicating that Yap1p has little contribution to the resistance to freeze-thaw stress. On the other hand, the msn2Dmsn4D cells showed greater susceptibility to freezethaw stress than the wild-type cells, indicating that the activated Msn2p and Msn4p in the thawed cells presumably play a crucial role to survive the freeze-thaw stress. Stability of STRE-genes after a shift back to growth temperatures from a near freezing temperature It has been reported that mRNA levels of TPS1, TPS2, and HSP104 were increased in cells kept at a near freezing
S. Izawa et al. / Biochemical and Biophysical Research Communications 352 (2007) 750–755
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Fig. 3. Resistance to freeze-thaw stress was determined by measuring the viability of frozen-thawed cells. Small amounts of cells (20 ll per 1.5 ml tube) were rapidly frozen and incubated for the period indicated at 30 °C. Frozen cells were thawed and plated on YPD medium plates after dilution with 10 mM potassium phosphate buffer. Percent survival was expressed relative to the initial viability prior to freezing. Results are representative of three independent experiments. Circles, wild-type cells; squares, msn2Dmsn4D cells, triangles, yap1D cells.
temperature (0 °C) for 2 days [1], indicating that Msn2p and Msn4p were activated in cells during this period. However, interestingly, these mRNAs were immediately degraded upon the shift from 0 °C to a growth temperature (30 °C) [1]. This finding may imply that Msn2p and Msn4p do not maintain the activated forms during the recovery process from 0 °C. On the other hand, in our experiments, levels of these mRNAs were increased after the shift to 28 °C from a freezing temperature (30 °C) (Fig. 2B). To see whether Msn2p and Msn4p are activated during the recovery process from 30 °C but not from 0 °C, we compared the levels of these mRNAs after the return to a growth temperature (28 °C) from 30 °C or 0 °C (Fig. 4A). In our conditions, cells showed an increase in the levels of TPS1, TPS2, and HSP104 mRNAs upon the return to a growth temperature from both temperatures, and the quick degradation of these mRNAs was not observed upon the shift to 28 °C from 0 °C (Fig. 4A). Additionally, the nuclear accumulation of Msn2p-GFP and Msn4p-GFP was observed for at least 15 min after the shift to 28 °C from 0 °C (Fig. 4B). These results clearly indicate that Msn2p and Msn4p were activated during the recovery process from a near freezing temperature. Such a discrepancy between the previous report [1] and ours is presumably caused by the difference in the period of storage of cells at 0 °C. Whereas cells were stored at 0 °C for 2 days in the study by Kandror et al. [1], cells were incubated at 0 °C for 1 h in our experiment. Under our experimental conditions, chilled cells as well as frozen-thawed cells responded actively to a return to growth temperatures through the induction of STREgenes, suggesting that Msn2p and Msn4p play a role in the recovery from a near freezing temperature as well as a freezing temperature.
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Fig. 4. Changes in the levels of TPS1, TPS2, and HSP104 mRNAs and intracellular localization of Msn2p/Msn4p upon return to a growth temperature. Cells were incubated at 30 °C or 0 °C for 1 h, and subsequently at 28 °C with shaking for the period indicated. A Northern blot analysis (A) and microscopic analysis of Msn2p-GFP and Msn4pGFP (from 0 °C to 28 °C) (B) were carried out. Total RNA was prepared and 15 lg of RNA was applied to each lane. BF, before freezing.
Discussion Importance of the activation of Msn2p/Msn4p after thawing To acquire sufficient resistance to stress, intracellular recovery systems as well as defense systems should function properly. An elevation in levels of intracellular cryoprotectants such as trehalose and glycerol before freezing and during the cooling process presumably function as defense systems in the resistance to freeze-thaw stress [1,4–8]. On the other hand, there have been almost no reports to date about the recovery systems for damage caused by freezethaw stress. We here focused on the cellular responses in the thawed cells, and demonstrated that Msn2p and Msn4p were activated and caused the increased expression of STRE-genes (Figs. 1A and 2B). These findings led us to suppose that Msn2p and Msn4p contribute to the recovery from freezing injury. To verify this idea, we examined the physiological importance of the activation of Msn2p and Msn4p in the thawed cells. As shown in Fig. 2B, increased expression of STRE-genes was not observed in cells kept at 30 °C for 1 h (compare lanes of cells before freezing and after 5 min of thawing). These findings indicate that Msn2p
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and Msn4p were hardly activated at all during the cooling process and the period of freezing under our experimental conditions. This is not surprising since substantial activation of Msn2p and Msn4p is relatively slow and inefficient at near freezing temperatures and during the cooling process [1]. Additionally, we minimized the activation of Msn2p and Msn4p in the cooling process by rapidly freezing a small amount of cells for the freeze-thaw stress tolerance test (Fig. 3). Taking into account these results, the difference in resistance to freeze-thaw stress between the wild-type and msn2Dmsn4D mutant (Fig. 3) is probably due in part to the activation of Msn2p/Msn4p after thawing. At least in our experiments, the activation of Msn2p/ Msn4p during the cooling process has presumably little effect on the resistance to freeze-thaw stress. These results support the idea that Msn2p and Msn4p play a role in the thawed cells and probably contribute to the recovery from the damage due to freezing. Recovery from a near freezing temperature After the shift back from a near freezing temperature (0 °C) to a growth temperature, quick degradation of TPS1, TPS2, and HSP104 mRNAs was not observed in the cells incubated at 0 °C for 1 h (Fig. 4A). However, in the case of cells incubated at 0 °C for 2 days, quick degradation of these mRNAs upon the return to a growth temperature was reported by Kandror et al. [1]. This difference between their results and ours may be due to the difference in the storage period at 0 °C, and probably reflects the provision for chilling injury. As they mentioned, the cellular response to near freezing temperatures is relatively slow and more than 20 h is required for maximal accumulation of trehalose at 0 °C [1]. Indeed, the expression of TPS1, TPS2, and HSP104 was not sufficiently increased in the cells after 1 h at 0 °C (compare lanes of cells before freezing and 0 min after shift from 0 to 28 °C in Fig. 4A). It is likely that cells were not able to prepare sufficiently for the adaptation to low temperatures during the incubation at 0 °C for only 1 h (short-term treatment). In contrast, cells with long-term treatment (2 days) at 0 °C presumably had enough time to get adjusted to the chilled conditions. Thus, cellular responses after the shift back to a growth temperature would be much more important for cells with short-term treatment at 0 °C than cells with long-term treatment, and further induction of the expression of STRE-genes after the return to a growth temperature might be essential for the short-term treated cells to recover from the chilling injury. Msn2p and Msn4p seem to play important roles after chilling (not for a long-term) as well as after freezing during the process of recovery from damage caused by low temperatures. Oxidative stress in the freeze-thawing process Here, we showed that the expression of SOD1, SOD2, and CTT1 genes was induced in the thawed cells
(Fig. 2B). It has been reported that superoxide anions (O2) are generated in yeast cells as a result of freezing and thawing, and that superoxide dismutase (SOD) is required for the resistance to freeze-thaw stress [17]. Taking into account these findings, the induction of SOD in the thawed cells seems quite reasonable. SOD converts superoxide anions to hydrogen peroxide (H2O2), and catalase disproportionates H2O2 to water and O2. Therefore, increased expression of the catalase gene (CTT1) in the thawed cells also seems to be a physiologically reasonable way to detoxify superoxide anions and H2O2. In contrast, the activity of Yap1p and the expression of its target genes such as GPX2 and TRR1 were not affected by freeze-thaw stress (Fig. 1B, 2A and C). Since peroxide stress is critical for the activation of Yap1p [16,19], these results may also suggest that the induced catalase scavenges hydrogen peroxide sufficiently and prevents the activation of Yap1p in the thawed cells. Additionally, Park et al. reported that yap1D cells show the same resistance to freeze-thaw stress as the wild-type cells [17], and our results also imply that Yap1p is not a key factor in the freeze-thaw resistance (Fig. 3). Taken together, these findings indicate that the expression of Yap1p-target genes is less important for the recovery from freezing and acquirement of resistance to freeze-thaw stress. Acknowledgments We are sincerely grateful to Drs. D. Engelberg, S. Kuge, C. Schu¨ller, and K. Weis for providing materials and the constructive discussion. This research was supported by Bio-oriented Technology Research Advancement Institution. References [1] O. Kandror, N. Bretschneider, E. Kreydin, D. Cavalieri, A.L. Goldberg, Yeast adapt to near-freezing temperatures by STRE/ Msn2, 4-dependent induction of trehalose synthesis and certain molecular chaperones, Mol. Cell. 13 (2004) 771–781. [2] M.T. Martinez-Pastor, G. Marchler, C. Schu¨ller, A. Marchler-Bauer, H. Ruis, F. Estruch, The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE), EMBO J. 15 (1996) 2227–2235. [3] W. Go¨rner, E. Durchschlag, M.T. Martinez-Pastor, F. Estruch, G. Ammerer, B. Hamilton, H. Ruis, C. Schu¨ller, Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity, Genes Dev. 12 (1998) 586–597. [4] J. Panadero, C. Pallotti, S. Rodrı´gez-Vargas, F. Randez-Gil, J. Prieto, A downshift in temperature activates the high osmolarity glycerol (HOG) pathway, which determines freeze tolerance in Saccharomyces cerevisiae, J. Biol. Chem. 281 (2006) 4638–4645. [5] C. Coutinho, E. Bernardes, D. Felix, A.D. Panek, Trehalose as cryoprotectant for preservation of yeast strains, J. Biotechnol. 7 (1988) 23–32. [6] R. Hirasawa, K. Yokoigawa, Leavening ability of baker’s yeast exposed to hyperosmotic media, FEMS Microbiol. Lett. 194 (2001) 159–162. [7] S. Izawa, M. Sato, K. Yokoigawa, Y. Inoue, Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and
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glycerol-enriched cells, Appl. Microbiol. Biotechnol. 66 (2004) 108– 114. S. Izawa, K. Ikeda, Y. Inoue, Deficiency in the glycerol channel Fps1p confers increased freeze tolerance to yeast cells: application of the fps1D mutant to frozen dough technology, Appl. Microbiol. Biotechnol. 66 (2004) 303–305. S. Kuge, N. Jones, A. Nomoto, Regulation of yAP-1 nuclear localization in response to oxidative stress, EMBO J. 16 (1997) 1710–1720. Y. Inoue, Y. Tsujimoto, A. Kimura, Expression of the glyoxalase I gene of Saccharomyces cerevisiae is regulated by high osmolarity glycerol mitogen-activated protein kinase pathway in osmotic stress response, J. Biol. Chem. 273 (1998) 2977–2983. S. Izawa, K. Maeda, K. Sugiyama, J. Mano, Y. Inoue, A. Kimura, Thioredoxin deficiency causes the constitutive activation of Yap1, an AP-1-like transcription factor in Saccharomyces cerevisiae, J. Biol. Chem. 274 (1999) 28459–28465. A. Stanhill, N. Schick, D. Engelberg, The yeast Ras/cyclic AMP pathway induces invasive growth by suppressing the cellular stress response, Mol. Cell. Biol. 19 (1999) 7529–7538. K. Stade, C.S. Ford, C. Guthrie, K. Weis, Exportin 1 (Crm1p) is an essential nuclear export factor, Cell 90 (1997) 1041–1050.
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[14] M.E. Schmitt, T.A. Brown, B.L. Trumpower, A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae, Nucleic Acids Res. 18 (1990) 3091–3092. [15] K. Maeta, S. Izawa, Y. Inoue, Methylglyoxal, a metabolite derived from glycolysis, functions as a signal initiator of the high osmolarity glycerol-mitogen-activated protein kinase cascade and calcineurin/ Crz1-mediated pathway in Saccharomyces cerevisiae, J. Biol. Chem. 280 (2005) 253–260. [16] S.T. Coleman, E.A. Epping, S.M. Steggerda, W.S. Moye-Rowley, Yap1p activates gene transcription in an oxidant-specific fashion, Mol. Cell. Biol. 19 (1999) 8302–8313. [17] J.I. Park, C.M. Grant, M.J. Davis, I.W. Dawes, The cytoplasmic Cu, Zn superoxide dismutase of Saccharomyces cerevisiae is required for resistance to freeze-thaw stress, J. Biol. Chem. 273 (1998) 22921– 22928. [18] Y. Inoue, T. Matsuda, K. Sugiyama, S. Izawa, A. Kimura, Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae, J. Biol. Chem. 274 (1999) 27002– 27009. [19] A. Delaunay, D. Pflieger, M.B. Barrault, J. Vinh, M.B. Toledano, A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation, Cell 111 (2002) 471–481.
Msn2p/Msn4p-activation is essential for the recovery from freezing stress in yeast Shingo Izawa *, Kayo Ikeda, Takumi Ohdate, Yoshiharu Inoue Laboratory of Molecular Microbiology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Received 12 November 2006 Available online 27 November 2006
Abstract Since it seems quite difficult for frozen cells to repair the damage caused by freezing, the adequate responses appear to be induced during and/or after the thawing process to recover from the damage due to freezing. In this study, the cellular events happening upon the return from freezing at 30 °C to a growth temperature (28 °C) were investigated. Yap1p, an oxidative stress-responsive transcription factor, was not activated in the thawed cells, indicating that no serious oxidative stress was generated in the frozen-thawed cells. On the other hand, Msn2p and Msn4p, general stress-responsive transcription factors, were activated in the thawed cells and caused the increased expression of a number of Msn2p/Msn4p-target genes including SOD1, SOD2, and several HSP genes. Since almost no expression of Msn2p/Msn4p-target genes was induced before thawing, these results indicate that Msn2p and Msn4p play a role during the recovery process from freezing. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Freeze-thaw stress; Msn2p; Msn4p; Yap1p; Saccharomyces cerevisiae; STRE
Both in the laboratory and in natural habitats, yeast cells probably encounter freezing temperatures much more often than they encounter heat-shock stress. Frozen yeast cells are also utilized for frozen dough technology in the bakery industry. Compared with the responses to heat shock and oxidative stress, the cellular response to freezethaw stress is not well-characterized. Recently, it has been reported that yeast cells induce the synthesis of trehalose and certain heat-shock proteins (HSPs) at near freezing temperatures (0 °C) in an Msn2p/Msn4p-dependent manner [1]. The transcription factors Msn2p and Msn4p recognize the stress-response element (STRE; consensus sequence is 5 0 -CCCCT-3 0 or 5 0 -AGGGG-3 0 ) and induce the activation of STRE-dependent promoters under various stressful conditions [2,3]. Additionally, Panadero et al. also reported that the high osmolarity glycerol (HOG) pathway is activated in response to a downward shift in temperature and that the accumulation of glycerol *
Corresponding author. Fax: +81 774 33 3004. E-mail address: [email protected] (S. Izawa).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.11.100
caused by the activation of Hog1p is essential for tolerance to freeze stress [4]. Since trehalose and glycerol function as cryoprotectants and confer resistance to freeze-thaw stress [5–8], these results clearly indicate that yeast cells undergo changes in transcriptional patterns during the cooling process and prepare the defense against the stress of low temperatures. However, cellular responses induced under such conditions are relatively slow and presumably not continued in frozen cells [1,4]. On the other hand, there is almost no information available about the cellular response in thawed cells. Since it is quite difficult for frozen cells to carry out cellular events such as transcriptional activation and protein synthesis, an adequate recovery response would be required during and/or after thawing to recover from the damage due to freezing. To properly understand the cellular response to freeze-thaw stress, it is necessary to clarify the cellular events happening upon the return to a growth temperature and in the thawed cells. In this study, we investigated the changes in thawed cells, and found that Msn2p and Msn4p, but not Yap1p (an oxidative stress-responsive transcription
S. Izawa et al. / Biochemical and Biophysical Research Communications 352 (2007) 750–755
factor) [9], were activated by thawing which led to the transcriptional activation of Msn2p/Msn4p-target genes.
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Materials and methods
Msn2pGFP
Yeast strains and medium. The Saccharomyces cerevisiae strains used in this study were YPH250 (MATa his3-D200 leu2-D1 trp1-D1 lys2-801 ade2101 ura3-52), msn2Dmsn4D (MATa his3-D200 leu2-D1 trp1-D1 lys2-801 ade2-101 ura3-52 msn2D::HIS3 msn4D::URA3) [10], and yap1D (MATa his3-D200 leu2-D1 trp1-D1 lys2-801 ade2-101 ura3-52 yap1D::HIS3) [11]. Cells were cultured in 50 ml of SD minimal medium (2% glucose and 0.67% yeast nitrogen base w/o amino acids, pH 5.5) with appropriate amino acids and bases at 28 °C with reciprocal shaking (120 rpm) in 300ml Erlenmeyer flasks. Exponential-phase cells were prepared by culturing till an OD610 of 0.5. Plasmids and analytical techniques. pSTRE-lacZ was donated by Dr. D. Engelberg [12]. pAdh1-Msn2-GFP were donated by Dr. C. Schu¨ller [3]. pRS-GFP-Yap1 and pRS-YRE-lacZ were provided by Dr. S. Kuge [9]. pAdh1-Msn4-GFP was constructed as follows: a 1.9-kbp fragment encoding the open-reading frame (ORF) of MSN4 was amplified using 5 0 TTATTGGATCCAAAAATCACCGTGCTTTTTGTGAG-3 0 and 5 0 TCTTTTAAGCTTATTAAAAACAATATAATGCTAGTC-3 0 as primers. The amplicon was digested with HindIII/BamHI and introduced into the HindIII/BamHI sites of pKW430 [13]. Analytical techniques for the bgalactosidase assay and fluorescent microscopic analysis were as described previously [11]. Northern blot analysis. Five milliliters of cell culture was transferred into a thin-wall plastic tube (50 ml volume) and frozen at 30 °C or incubated at 0 °C for 1 h. The cell culture froze up within 10 min at 30 °C. Frozen or chilled cells were incubated in an water bath at 28 °C with shaking (120 rpm). The frozen cell culture was thawed completely within 5 min at 28 °C. Total RNA was extracted from cells by the method of Schmitt et al. [14]. DNA fragments encoding the ORF of each gene were prepared by PCR using yeast genomic DNA as a template, and labeled with a–[32P]dCTP using Random primer labeling kit ver. 2 (Takara Bio, Otsu, Japan). The labeled DNA fragments were used as probes for Northern blotting. Freeze-thaw stress tolerance test. Twenty microliters of cell culture was transferred into a thin-wall plastic tube (1.5 ml volume) and quickly frozen at 30 °C. The cell culture froze up within 3 min. The method of freezethaw stress tolerance test was previously described [7].
Results Msn2p/Msn4p but not Yap1p is activated in frozen-thawed cells First, the intracellular distribution of stress-responsive transcription factors, Msn2p, Msn4p, and Yap1p, in the thawed cells was investigated. These transcription factors are reported to move into the nucleus when they are activated by stress [3,9]. Msn2p and Msn4p respond to various kinds of stress [2,3,15]. On the other hand, Yap1p is responsive to oxidative stress [9,16]. It has been reported that superoxide anions (O2) are caused during freezing and thawing [17]. Thus, we examined whether Msn2p/ Msn4p or Yap1p is activated and accumulates in the nucleus in thawed cells. As shown in Fig. 1A, green fluorescent protein (GFP)tagged Msn2p (Msn2p-GFP) and Msn4p-GFP were concentrated in the nucleus in the thawed cells and the nuclear accumulation of Msn2p-GFP and Msn4p-GFP continued for at least 15 min. Around 25 min after the thawing of
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Fig. 1. Change in the localization of stress-responsive transcription factors in thawed cells. Cells frozen at 30 °C for 1 h were thawed at 28 °C with shaking for the period indicated. Intracellular localization of Msn2p-GFP and Msn4p-GFP (A), and GFP-Yap1p (B) was monitored at the indicated time. BF, before freezing.
cells, most of the Msn2p-GFP and Msn4p-GFP had diffused into the cytoplasm. Additionally, the activity of bgalactosidase derived from STRE-lacZ was gradually increased after thawing (left panel in Fig. 2A). These results indicate the possibility that the transcription of genes containing STRE (Msn2p/Msn4p-target genes) is activated in the thawed cells. Interestingly, the b-galactosidase activity was not increased by just freezing (compare activities of cells before freezing and after 5 min of thawing). This result indicates that Msn2p and Msn4p were not substantially activated during the cooling process under our experimental conditions. We next investigated the expression of various Msn2p/ Msn4p-target genes (STRE-genes) in the thawed cells by conducting a Northern blot analysis. As shown in Fig. 2B, the expression of various STRE-genes was increased in thawed cells of the wild-type strain. Levels of expression of the STRE-genes were increased after 15 min of thawing. Compared with the wild-type cells, the msn2Dmsn4D cells showed little induction of the expression of HSP12, HSP104, CTT1 (encodes catalase), TPS1 (trehalose-6-phosphate synthase), and TPS2 (trehalose-6phosphate phosphatase). These results indicate that the expression of these genes was mainly induced in an Msn2p/Msn4p-dependent manner in the thawed cells. Interestingly, the expression of SOD1 and SOD2 was also
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Fig. 2. Activation of Msn2p/Msn4p in the thawed cells. (A) Cells carrying the STRE-lacZ plasmid or YRE-lacZ plasmids were frozen at 30 °C for 1 h and thawed at 28 °C with shaking for the period indicated, and b-galactosidase activity was then assayed. Results represent the means ± SD of three independent experiments. White bars, wild-type cells; black bars, msn2Dmsn4D cells (left panel) or yap1D cells (right panel). (B,C) Expression of target genes of Msn2p/Msn4p (B) and Yap1p (C) was monitored by Northern blot analysis. Cells frozen at 30 °C for 1 h were incubated at 28 °C with shaking for the period indicated. Total RNA was prepared and 15 lg of RNA was applied to each lane. BF, before freezing.
induced by thawing in the wild-type cells but not in the msn2Dmsn4D cells. The SOD1 and SOD2 genes contain the STRE consensus sequence 197 and 141 bp from the initiation codon, respectively, suggesting that they are likely to be transcriptionally regulated in an Msn2p/ Msn4p-dependent manner in the thawed cells. It is noteworthy that the expression of STRE-genes was not increased just after 5 min of thawing in the wild-type cells (Fig. 2B). This result confirmed that Msn2p and Msp4p were not substantially activated before thawing and indicates that activation of Msn2p and Msp4p was induced mainly during the recovery process from freezing. In contrast with Msn2p and Msn4p, GFP-Yap1p did not accumulate in the nucleus in the thawed cells, indicating that Yap1p was not activated after thawing (Fig. 1B). Additionally, results of the YRE-lacZ reporter assay (YRE: Yap1p recognition element) and Northern blot analysis of Yap1p-target genes (TRR1 and GPX2, encode thioredoxin reductase and glutathione peroxidase, respectively) [18,19] clearly indicate that Yap1p was not activated in the thawed cells (Fig. 2A and C ). These results suggest that no serious oxidative stress that causes the activation of Yap1p was generated in the freeze-thawed cells.
Importance of Msn2p/Msn4p-activation in thawed cells for resistance to freeze-thaw stress Next, we tried to evaluate the physiological importance of the activation of Msn2p/Msn4p in the thawed cells. To minimize the activation of Msn2p/Msn4p during the cooling process, small amounts of cells (20 ll per 1.5 ml tube) were rapidly frozen at 30 °C. We compared the resistance to freeze-thaw stress of rapidly frozen cells among the wildtype, the yap1D mutant, and the msn2Dmsn4D mutant (Fig. 3). Susceptibility to freeze-thaw stress of the yap1D cells was almost identical with that of the wild-type cells, indicating that Yap1p has little contribution to the resistance to freeze-thaw stress. On the other hand, the msn2Dmsn4D cells showed greater susceptibility to freezethaw stress than the wild-type cells, indicating that the activated Msn2p and Msn4p in the thawed cells presumably play a crucial role to survive the freeze-thaw stress. Stability of STRE-genes after a shift back to growth temperatures from a near freezing temperature It has been reported that mRNA levels of TPS1, TPS2, and HSP104 were increased in cells kept at a near freezing
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Fig. 3. Resistance to freeze-thaw stress was determined by measuring the viability of frozen-thawed cells. Small amounts of cells (20 ll per 1.5 ml tube) were rapidly frozen and incubated for the period indicated at 30 °C. Frozen cells were thawed and plated on YPD medium plates after dilution with 10 mM potassium phosphate buffer. Percent survival was expressed relative to the initial viability prior to freezing. Results are representative of three independent experiments. Circles, wild-type cells; squares, msn2Dmsn4D cells, triangles, yap1D cells.
temperature (0 °C) for 2 days [1], indicating that Msn2p and Msn4p were activated in cells during this period. However, interestingly, these mRNAs were immediately degraded upon the shift from 0 °C to a growth temperature (30 °C) [1]. This finding may imply that Msn2p and Msn4p do not maintain the activated forms during the recovery process from 0 °C. On the other hand, in our experiments, levels of these mRNAs were increased after the shift to 28 °C from a freezing temperature (30 °C) (Fig. 2B). To see whether Msn2p and Msn4p are activated during the recovery process from 30 °C but not from 0 °C, we compared the levels of these mRNAs after the return to a growth temperature (28 °C) from 30 °C or 0 °C (Fig. 4A). In our conditions, cells showed an increase in the levels of TPS1, TPS2, and HSP104 mRNAs upon the return to a growth temperature from both temperatures, and the quick degradation of these mRNAs was not observed upon the shift to 28 °C from 0 °C (Fig. 4A). Additionally, the nuclear accumulation of Msn2p-GFP and Msn4p-GFP was observed for at least 15 min after the shift to 28 °C from 0 °C (Fig. 4B). These results clearly indicate that Msn2p and Msn4p were activated during the recovery process from a near freezing temperature. Such a discrepancy between the previous report [1] and ours is presumably caused by the difference in the period of storage of cells at 0 °C. Whereas cells were stored at 0 °C for 2 days in the study by Kandror et al. [1], cells were incubated at 0 °C for 1 h in our experiment. Under our experimental conditions, chilled cells as well as frozen-thawed cells responded actively to a return to growth temperatures through the induction of STREgenes, suggesting that Msn2p and Msn4p play a role in the recovery from a near freezing temperature as well as a freezing temperature.
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Fig. 4. Changes in the levels of TPS1, TPS2, and HSP104 mRNAs and intracellular localization of Msn2p/Msn4p upon return to a growth temperature. Cells were incubated at 30 °C or 0 °C for 1 h, and subsequently at 28 °C with shaking for the period indicated. A Northern blot analysis (A) and microscopic analysis of Msn2p-GFP and Msn4pGFP (from 0 °C to 28 °C) (B) were carried out. Total RNA was prepared and 15 lg of RNA was applied to each lane. BF, before freezing.
Discussion Importance of the activation of Msn2p/Msn4p after thawing To acquire sufficient resistance to stress, intracellular recovery systems as well as defense systems should function properly. An elevation in levels of intracellular cryoprotectants such as trehalose and glycerol before freezing and during the cooling process presumably function as defense systems in the resistance to freeze-thaw stress [1,4–8]. On the other hand, there have been almost no reports to date about the recovery systems for damage caused by freezethaw stress. We here focused on the cellular responses in the thawed cells, and demonstrated that Msn2p and Msn4p were activated and caused the increased expression of STRE-genes (Figs. 1A and 2B). These findings led us to suppose that Msn2p and Msn4p contribute to the recovery from freezing injury. To verify this idea, we examined the physiological importance of the activation of Msn2p and Msn4p in the thawed cells. As shown in Fig. 2B, increased expression of STRE-genes was not observed in cells kept at 30 °C for 1 h (compare lanes of cells before freezing and after 5 min of thawing). These findings indicate that Msn2p
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and Msn4p were hardly activated at all during the cooling process and the period of freezing under our experimental conditions. This is not surprising since substantial activation of Msn2p and Msn4p is relatively slow and inefficient at near freezing temperatures and during the cooling process [1]. Additionally, we minimized the activation of Msn2p and Msn4p in the cooling process by rapidly freezing a small amount of cells for the freeze-thaw stress tolerance test (Fig. 3). Taking into account these results, the difference in resistance to freeze-thaw stress between the wild-type and msn2Dmsn4D mutant (Fig. 3) is probably due in part to the activation of Msn2p/Msn4p after thawing. At least in our experiments, the activation of Msn2p/ Msn4p during the cooling process has presumably little effect on the resistance to freeze-thaw stress. These results support the idea that Msn2p and Msn4p play a role in the thawed cells and probably contribute to the recovery from the damage due to freezing. Recovery from a near freezing temperature After the shift back from a near freezing temperature (0 °C) to a growth temperature, quick degradation of TPS1, TPS2, and HSP104 mRNAs was not observed in the cells incubated at 0 °C for 1 h (Fig. 4A). However, in the case of cells incubated at 0 °C for 2 days, quick degradation of these mRNAs upon the return to a growth temperature was reported by Kandror et al. [1]. This difference between their results and ours may be due to the difference in the storage period at 0 °C, and probably reflects the provision for chilling injury. As they mentioned, the cellular response to near freezing temperatures is relatively slow and more than 20 h is required for maximal accumulation of trehalose at 0 °C [1]. Indeed, the expression of TPS1, TPS2, and HSP104 was not sufficiently increased in the cells after 1 h at 0 °C (compare lanes of cells before freezing and 0 min after shift from 0 to 28 °C in Fig. 4A). It is likely that cells were not able to prepare sufficiently for the adaptation to low temperatures during the incubation at 0 °C for only 1 h (short-term treatment). In contrast, cells with long-term treatment (2 days) at 0 °C presumably had enough time to get adjusted to the chilled conditions. Thus, cellular responses after the shift back to a growth temperature would be much more important for cells with short-term treatment at 0 °C than cells with long-term treatment, and further induction of the expression of STRE-genes after the return to a growth temperature might be essential for the short-term treated cells to recover from the chilling injury. Msn2p and Msn4p seem to play important roles after chilling (not for a long-term) as well as after freezing during the process of recovery from damage caused by low temperatures. Oxidative stress in the freeze-thawing process Here, we showed that the expression of SOD1, SOD2, and CTT1 genes was induced in the thawed cells
(Fig. 2B). It has been reported that superoxide anions (O2) are generated in yeast cells as a result of freezing and thawing, and that superoxide dismutase (SOD) is required for the resistance to freeze-thaw stress [17]. Taking into account these findings, the induction of SOD in the thawed cells seems quite reasonable. SOD converts superoxide anions to hydrogen peroxide (H2O2), and catalase disproportionates H2O2 to water and O2. Therefore, increased expression of the catalase gene (CTT1) in the thawed cells also seems to be a physiologically reasonable way to detoxify superoxide anions and H2O2. In contrast, the activity of Yap1p and the expression of its target genes such as GPX2 and TRR1 were not affected by freeze-thaw stress (Fig. 1B, 2A and C). Since peroxide stress is critical for the activation of Yap1p [16,19], these results may also suggest that the induced catalase scavenges hydrogen peroxide sufficiently and prevents the activation of Yap1p in the thawed cells. Additionally, Park et al. reported that yap1D cells show the same resistance to freeze-thaw stress as the wild-type cells [17], and our results also imply that Yap1p is not a key factor in the freeze-thaw resistance (Fig. 3). Taken together, these findings indicate that the expression of Yap1p-target genes is less important for the recovery from freezing and acquirement of resistance to freeze-thaw stress. Acknowledgments We are sincerely grateful to Drs. D. Engelberg, S. Kuge, C. Schu¨ller, and K. Weis for providing materials and the constructive discussion. This research was supported by Bio-oriented Technology Research Advancement Institution. References [1] O. Kandror, N. Bretschneider, E. Kreydin, D. Cavalieri, A.L. Goldberg, Yeast adapt to near-freezing temperatures by STRE/ Msn2, 4-dependent induction of trehalose synthesis and certain molecular chaperones, Mol. Cell. 13 (2004) 771–781. [2] M.T. Martinez-Pastor, G. Marchler, C. Schu¨ller, A. Marchler-Bauer, H. Ruis, F. Estruch, The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE), EMBO J. 15 (1996) 2227–2235. [3] W. Go¨rner, E. Durchschlag, M.T. Martinez-Pastor, F. Estruch, G. Ammerer, B. Hamilton, H. Ruis, C. Schu¨ller, Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity, Genes Dev. 12 (1998) 586–597. [4] J. Panadero, C. Pallotti, S. Rodrı´gez-Vargas, F. Randez-Gil, J. Prieto, A downshift in temperature activates the high osmolarity glycerol (HOG) pathway, which determines freeze tolerance in Saccharomyces cerevisiae, J. Biol. Chem. 281 (2006) 4638–4645. [5] C. Coutinho, E. Bernardes, D. Felix, A.D. Panek, Trehalose as cryoprotectant for preservation of yeast strains, J. Biotechnol. 7 (1988) 23–32. [6] R. Hirasawa, K. Yokoigawa, Leavening ability of baker’s yeast exposed to hyperosmotic media, FEMS Microbiol. Lett. 194 (2001) 159–162. [7] S. Izawa, M. Sato, K. Yokoigawa, Y. Inoue, Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol dehydrogenase genes and
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