Abstract

Yeasts used in bread making are exposed to freeze–thaw stress during frozen-dough baking. To clarify the genes required for freeze–thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze–thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze–thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H+-ATPase. Next, the cross-sensitivity of the freeze–thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze–thaw stress. The results showed that defects in the functions of vacuolar H+-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze–thaw injury: oxidative stress generated during the freeze–thaw process, and defects in cell wall assembly.

Introduction

Frozen-dough baking has become a key technology, because it improves labor conditions for bakers and enables bakers to provide fresh baked goods to consumers. Because baker's yeast cells in frozen dough suffer freeze–thaw injuries during the freeze–thaw process, the fermentation activity of yeast cells after thawing dramatically decreases (Kline & Sugihara, 1968; Hsu, 1979a).

Freeze–thaw injuries of yeast cells depend on numerous factors, including the physiological condition of the yeast cells, genetic background of the yeast strains, and freezing conditions, such as freezing period and freezing rate. Changes in cell physiology caused by the onset of fermentation might decrease freeze–thaw tolerance (Lewis, 1993; Van Dijck, 1995; Versele, 2004), possibly due to activation of the cAMP pathway (Park, 1997). Decrease in freeze–thaw tolerance is reportedly strongly related to trehalose degradation, and the level of intracellular trehalose affects the freeze–thaw tolerance of baker's yeast (Van Dijck, 1995; Shima, 1999). In commercial frozen-dough processes, freezing of prefermented dough is desirable, because bread made from prefermented frozen dough has the correct texture and taste (Hsu, 1979b; Teunissen, 2002).

The freeze–thaw tolerance of baker's yeast is determined by the genetic characteristics of the yeast. Some yeast strains with higher freeze–thaw tolerance have been isolated from natural sources and constructed by gene manipulations (Oda, 1986; Hino, 1987; Hahn & Kawai, 1990; Shima, 1999; Nishida, 2004). The freezing period is a critical parameter for freeze–thaw injury; extended freezing periods seriously damage yeast cells, due to the growth of ice crystals (Mazur, 1970; Toner, 1993). Free radicals are generated during freezing and thawing (Hermes-Lima & Storey, 1993; Lewis, 1997; Du & Takagi, 2005), and in Saccharomyces cerevisiae, cytoplasmic Cu/Zn-superoxide dismutase is required for tolerance to freeze–thaw stress (Park, 1998). On the basis of DNA microarray analysis, Odani (2003) suggested that freeze–thaw stress causes damage to the structure of the cell wall and to cellular organelles. Thus, despite extensive studies on freeze–thaw injuries in baker's yeast, the molecular mechanisms behind the freeze–thaw tolerance of baker's yeast remain unclear.

A yeast deletion mutant collection is a powerful tool for determining the gene function by analyzing the phenotype of mutants lacking the gene (so-called ‘phenomics’) (Giaever, 2002; Warringer, 2003; Fernandez-Ricaud, 2005). An international consortium has carried out a systematic deletion of all of the ORFs of S. cerevisiae using a PCR-mediated gene deletion strategy (Giaever, 2002). Analysis using this deletion mutant collection relies on the number of genes whose mutation affects components of an important pathway and results in a phenotype of sensitivity or resistance (Birrell, 2002; Warringer, 2003; Fernandez-Ricaud, 2005). We previously reported a comprehensive functional analysis of genes required for tolerance to high-sugar stress; this tolerance is important for high-sugar dough baking (Ando, 2006). Giaever (2002) and Warringer (2003) reported phenotype analyses of the deletion mutant collection under high osmotic stress conditions caused by NaCl and sorbitol. As far as we know, however, phenotypic analysis under freeze–thaw stress conditions has not been done.

In this study, to clarify the pathway involved in tolerance to freeze–thaw stress, the genes required for tolerance to freeze–thaw stress in S. cerevisiae were first screened using the yeast deletion mutant collection. These deleted genes, which were considered to be required for tolerance to freeze–thaw stress, were then classified on the basis of cell function and subcellular localization of their products. Next, to gain further insights into freeze–thaw tolerance, the cross-sensitivity of the freeze–thaw-sensitive deletion mutants to oxidative stress (an environmental stress closely related to freeze–thaw stress) and to a cell wall-challenging reagent, calcofluor white (CFW), was examined. Finally, the correlation between freeze–thaw stress and oxidative stress and that between freeze–thaw stress and cell wall damage were determined.

Materials and methods

Yeast strains and media

Saccharomyces cerevisiae BY4743 (MATahis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0) and the complete collection of diploid deletion strains (MATa/α) constructed by insertion of kanMX4 cassettes (geneticin resistance) as selective markers into the genome of BY4743 (Giaever, 2002) were obtained from EUROSCARF (European Saccharomyces cerevisiae Achive for Functional Analysis).

Deletion strains were maintained on YPD medium containing 10 g of yeast extract (Difco laboratories, Detroit), 20 g of peptone (Difco), and 20 g of glucose (per liter). Deletion mutant strains were maintained on YPD agar supplemented with 200 μg mL−1 of geneticin (Sigma-Aldrich, St Louis). YP4D medium (YPD medium containing 4% glucose) was used for prefermentation in examination of freeze–thaw sensitivity.

Identification of gene deletion yielding hypersensitivity to freeze–thaw stress

Deletion mutant and wild-type strains were inoculated into 50 μL of YPD liquid medium in 96-well microtiter plates (Corning Incorporated, Corning) using a 48-pin replicator (Funakoshi, Tokyo, Japan) and then cultivated at 30°C for 2 days (preculture). To start prefermentation, 50 μL of YP4D was added to the preculture, because it is necessary to add sufficient fermentable sugar. The prefermentation was carried out by incubation for 5 h at 30°C. The prefermented cultures were exposed to freeze–thaw stress by being frozen at −25°C for 5 days and then thawed at 30°C for 15 min. Portions of the frozen-thawed cultures were inoculated into 100 μL of fresh YPD medium and incubated for 23 h at 30°C, and the cell density (OD630 nm) was measured using a microplate reader (Elx800, BioTek, Winooski). As nonfrozen controls, portions of the prefermented culture before freezing were immediately inoculated into 100 μL of YPD medium, and incubated for 16 h at 30°C; the cell density (OD630 nm) was then measured. To maximize standardization and reproducibility for each run, the quantitative growth data measured in each microtiter plate were normalized by the average response of three replicates of the wild-type strain included in each run. The runs for freeze–thaw sensitivity were carried out in duplicate.

Four parameters were used in the data analysis of the sensitivity to freeze–thaw stress in the deletion mutants: (1) AM, defined as the OD630 nm of mutant strains after freeze–thaw stress; (2) AW, defined as the OD630 nm of BY4743 (the wild-type strain) after freeze–thaw stress; (3) BM, defined as the OD630 nm of mutant strains under nonstress conditions; and (4) BW, defined as the OD630 nm of BY4743 under nonstress conditions. Mutants that showed considerable growth inhibition (i.e. BM/BW<0.1) under nonstress conditions were excluded from further data analysis. The value of (AM/AW)/(BM/BW) was used as the parameter for sensitivity to freeze–thaw stress. In this calculation, BM/BW values were used for compensation of growth defects under nonstress conditions. All the experiments for identification of freeze–thaw-sensitive mutant strains were performed in duplicate, and the average of the values was used for the evaluation of sensitivity.

Classification of function and subcellular localization of gene products

The function and subcellular localization of gene products were classified on the basis of the MIPS database (Munich Information Centre for Protein Sequence). In the MIPS database, the function and subcellular localization of yeast gene products are placed into 17 and 20 classes, respectively.

Assessment of H2O2 sensitivity of freeze–thaw-sensitive mutants

Sensitivity to oxidative stress of mutants that were identified as freeze–thaw-sensitive mutants was assessed using hydrogen peroxide (H2O2) as follows. BY4743 and freeze–thaw-sensitive mutants were grown in 100 μL of YPD medium in a 96-well microtiter plate for 2 days at 30°C without shaking (preculture). A portion of the preculture was then inoculated into 100 μL of YPD medium supplemented or not supplemented with 2 mM H2O2. After incubation at 30°C for 15 h in YPD medium or for 17.5 h in YPD-H2O2 medium, the cell density (OD630 nm) was measured.

Four parameters were used in the data analysis of the sensitivity to oxidative stress in the deletion mutants: (1) HM, defined as the OD630 nm of mutant strains under oxidative conditions; (2) HW, defined as the OD630 nm of BY4743 under oxidative conditions; (3) YM, defined as the OD630 nm of mutant strains under nonstress conditions; and (4) YW, defined as the OD630 nm of BY4743 under nonstress conditions. The value of (HM/HW)/(YM/YW) was used as the parameter for sensitivity to oxidation. Measurements were performed in triplicate, and the average of the values was used for the evaluation of sensitivity. Mutants that exhibited a sensitivity value below 0.5 were defined as sensitive to H2O2.

Assessment of CFW sensitivity of freeze–thaw-sensitive mutants

CFW is a cell wall-challenging reagent that specifically binds to chitin in the cell wall and inhibits cell wall assembly (Ram, 1994). Here, the sensitivity of freeze–thaw-sensitive mutants to CFW was assessed. Except for the challenging reagent, the method of assessment was basically the same as that described above to assess H2O2 sensitivity. Instead of H2O2, 8.2 μM CFW was used as a challenging reagent.

Four parameters were used in the data analysis of the sensitivity to CFW in the deletion mutants: (1) CM, defined as the OD630 nm of mutant strains in YPD medium containing CFW; (2) CW, defined as the OD630 nm of BY4743 in YPD medium containing CFW; (3) YM, defined as the OD630 nm of mutant strains in YPD medium; and (4) YW, defined as the OD630 nm of BY4743 in YPD medium. The value of (CM/CW)/(YM/YW) was used as the parameter for sensitivity to CFW. Measurements were performed in triplicate, and the average of the values was used for the evaluation of sensitivity. Mutants that exhibited a sensitivity value below 0.5 were defined as sensitive to CFW.

Results and discussion

Experimental design for screening of mutants sensitive to freeze–thaw stress

First, we constructed a comprehensive evaluation system for sensitivity to freeze–thaw stress. In this evaluation system, yeast cells after 5 h of fermentation were used, because in general, yeast cells whose freeze–thaw sensitivity has decreased as a result of prefermentation (Hino, 1990) are frozen and thawed in actual frozen-dough baking. To determine suitable freezing periods for evaluation of freeze–thaw-sensitivity, after various freezing times (Fig. 1a) we measured the viabilities of wild-type strain BY4743 and a pro1 deletion mutant that was known to be hypersensitive to freeze–thaw stress (strain number 33659) (Morita, 2003; Terao, 2003). The results showed that both BY4743 and the pro1 mutant suffered freeze–thaw injuries under all conditions tested here, and that there was a considerable difference in freeze–thaw tolerance between the wild type and the pro1 mutant after freezing for 5 days.

1

Viabilities (a) and growth curves (b) of the wild-type strain (BY 4743) and pro1 deletion mutant under freeze–thaw stress conditions. Values are expressed as mean±standard deviation from triplicate experiments.

1

Viabilities (a) and growth curves (b) of the wild-type strain (BY 4743) and pro1 deletion mutant under freeze–thaw stress conditions. Values are expressed as mean±standard deviation from triplicate experiments.

To conveniently assess viabilities after freeze–thaw stress, we then monitored the growth of the wild-type and pro1 strains in fresh YPD medium after exposure to freeze–thaw stress (Fig. 1b). In comparison with the growth of yeast cells under nonstress conditions, the onset of growth of the wild-type and pro1 strains was clearly delayed after exposure to freeze–thaw stress; in particular, that of the pro1 strain was strongly inhibited. These growth results suggest that monitoring the growth of yeast cells after exposure to freeze–thaw stress is effective for convenient evaluation of freeze–thaw sensitivity. On the basis of these results, we screened the freeze–thaw-sensitive mutants by monitoring growth after 5 days of freezing.

Screening of freeze–thaw-sensitive deletion mutants from the deletion mutant collection

The complete deletion mutant collection of diploid strains derived from BY4743, which consisted of c. 4700 strains, was examined here using the method described in Materials and methods. To obtain an overall view of the effects of gene deletions on freeze–thaw sensitivity, we plotted the distribution of freeze–thaw sensitivity [shown as values of (AM/AW)/(BM/BW)] of deletion mutants examined here as a frequency distribution plot (Fig. 2). The results revealed that among the c. 4700 deletion mutants tested here, c.1.2% (58 strains) showed freeze–thaw tolerance less than 50% of that of the wild-type strain. We defined these strains as hypersensitive to freeze–thaw stress.

2

Frequency distribution of freeze–thaw tolerance of gene deletion mutants. Freeze–thaw tolerance is expressed as (AM/AW)/(BM/BW) (see Materials and methods). Mutants with (AM/AW)/(BM/BW) below 0.5 were defined as freeze–thaw sensitive.

2

Frequency distribution of freeze–thaw tolerance of gene deletion mutants. Freeze–thaw tolerance is expressed as (AM/AW)/(BM/BW) (see Materials and methods). Mutants with (AM/AW)/(BM/BW) below 0.5 were defined as freeze–thaw sensitive.

Table 1 summarizes the genes whose deletions yielded yeast cells hypersensitive to freeze–thaw stress. In Table 1, the genes were tentatively sorted into functional classes, although single genes could often be placed into several different functional classes in the MIPS database. Figure 3a shows the classification results by gene function based on the MIPS database, which uses 17 classes. This classification helps to clarify the gene functions involved in tolerance to freeze–thaw stress; namely, the functional classes containing a high number of genes might represent important cellular functions for tolerance to freeze–thaw stress. The results of this classification showed that the deletions that conferred freeze–thaw sensitivity were frequently included in the ‘Interaction with the cellular environment’, ‘Protein fate’ and ‘Protein synthesis’ classes.

1

Gene deletions resulting in freeze–thaw sensitivity

ORF Gene Freeze sensitivity H2O2 sensitivity CFW sensitivity Group Description 
Interaction with the cellular environment 
YKL119C VPH2 0.01 0.02 0.48 H+-ATPase assembly protein 
YBR127C VMA2 0.01 0.02 0.22 H+-ATPase V1 domain 60-kDa subunit, vacuolar 
YEL051W VMA8 0.02 0.15 0.16 H+-ATP synthase V1 domain 32-kDa subunit, vacuolar 
YEL027W CUP5 0.02 0.04 0.28 H+-ATPase V0 domain 17-kDa subunit, vacuolar 
YGR020C VMA7 0.02 0.10 0.14 H+-ATPase V1 domain 14-kDa subunit, vacuolar 
YDL185W TFP1 0.03 0.02 0.17 Encodes three-region protein that is self-spliced into Tfp1p and PI-SceI 
YGL167C PMR1 0.05 0.31 0.17 Ca2+-transporting P-type ATPase located in Golgi 
YOR332W VMA4 0.07 0.03 0.19 H+-ATPase V1 domain 27-kDa subunit, vacuolar 
YPL234C TFP3 0.12 0.03 0.23 H+-ATPase V0 domain 17-kDa subunit, vacuolar 
YPR036W VMA13 0.22 0.10 0.35 H+-ATPase V1 domain 54-kDa subunit, vacuolar 
YOR270C VPH1 0.29 0.84 0.77 H+-ATPase V0 domain 95-kDa subunit, vacuolar 
Protein fate (folding, modification, destination) 
YGR105W VMA21 0.03 0.03 0.34 ATPase assembly integral membrane protein 
YEL036C ANP1 0.05 0.90 0.05 Required for protein glycosylation in the Golgi 
YBL058W SHP1 0.09 0.08 0.04 Potential regulatory subunit for Glc7p 
YDR245W MNN10 0.10 1.33 0.09 Subunit of mannosyltransferase complex 
YDR264C AKR1 0.27 1.23 0.03 Ankyrin repeat-containing protein 
YJL183W MNN11 0.40 0.51 0.38 Related to Mnn10p, and in a complex containing other MNN gene products 
YDR140W MTQ2 0.46 1.01 0.16 Putative S-adenosylmethionine-dependent methyltransferase localized to cytoplasm and nucleus 
YNL054W VAC7 0.46 0.08 1.39 Vacuolar protein 
Protein synthesis 
YCR003W MRPL32 0.07 0.35 0.91 Mitochondrial ribosomal protein, large subunit 
YNR037C RSM19 0.10 0.10 0.89 Mitochondrial ribosomal protein, small subunit 
YHR010W RPL27A 0.13 0.59 0.48 60S large subunit ribosomal protein L27.e 
YKR085C MRPL20 0.16 ND ND ND Mitochondrial ribosomal protein, large subunit 
YDL191W RPL35A 0.29 0.42 0.33 60S large subunit ribosomal protein 
YJL189W RPL39 0.40 0.19 1.37 60S large subunit ribosomal protein L39.e 
YDL061C RPS29B 0.42 0.56 0.37 Ribosomal protein S29.e.B 
YGR159C NSR1 0.44 0.30 1.51 Nuclear localization sequence-binding protein, required for pre-rRNA processing and ribosome biogenesis 
Transcription 
YGL070C RPB9 0.08 0.70 0.30 DNA-directed RNA polymerase II, 14.2-kDa subunit 
YJL140W RPB4 0.10 0.27 0.27 DNA-directed RNA polymerase II, 32-kDa subunit 
YOL148C SPT20 0.16 0.05 0.68 Member of the TBP class of SPT proteins that alter transcription site selection 
YFR001W LOC1 0.36 0.59 0.49 Nuclear protein involved in asymmetric localization of ASH1 mRNA 
YDL106C PHO2 0.38 0.15 0.68 Homeodomain protein 
YBR289W SNF5 0.38 0.54 0.80 Component of SWI/SNF transcription activator complex 
YDR174W HMO1 0.44 0.19 0.91 Nonhistone protein 
YMR179W SPT21 0.47 1.19 0.76 Required for normal transcription at a number of loci 
Metabolism 
YJR090C GRR1 0.01 0.05 0.48 Required for glucose repression and for glucose and cation transport 
YFR019W FAB1 0.07 0.95 1.00 Phosphatidylinositol-3-phosphate-5-kinase 
YFL001W DEG1 0.25 0.17 0.73 Pseudouridine synthase 
YDR300C PRO1 0.29 0.03 0.46 Glutamate-5-kinase 
YNL280C ERG24 0.33 0.08 0.10 C-14 sterol reductase 
YOR039W CKB2 0.42 0.53 0.88 Casein kinase II β-chain 
Others 
YCL007C CWH36 0.02 0.11 0.22 Questionable protein (ORF overlaps VMA9 gene) 
YGL095C VPS45 0.12 0.07 0.38 Vacuolar protein sorting-associated protein 
YOR080W DIA2 0.14 1.21 0.51 Protein involved in invasive and pseudohyphal growth 
YBR171W SEC66 0.15 0.56 0.08 ER protein–translocation complex subunit 
YPR135W CTF4 0.29 0.79 0.08 DNA-directed DNA polymerase α-binding protein 
YMR307W GAS1 0.33 0.33 0.96 Glycophospholipid-anchored surface glycoprotein 
YPL106C SSE1 0.34 0.33 0.21 Heat shock protein of HSP70 family 
YDR477W SNF1 0.34 0.66 0.92 Carbon catabolite derepressing Ser/Thr protein kinase 
YDL074C BRE1 0.37 1.02 0.98 E3 ubiquitin ligase for Rad6p 
Unclassified proteins 
YNL080C  0.06 0.02 0.96 Protein of unknown function 
YOR331C  0.12 0.03 0.27 Questionable protein 
YGR160W FYV13 0.24 0.67 0.53 Questionable protein 
YDR161W TCI1 0.25 1.25 0.18 Protein of unknown function localized to cytoplasm and nucleus 
YDR126W SWF1 0.29 0.00 0.00 Spore wall formation 
YBR194W SOY1 0.32 1.56 1.86 Synthetic with Old Yellow enzyme 
YJL188C BUD19 0.35 0.17 0.71 Questionable protein 
YGL218W SRF1 0.40 0.13 0.73 Questionable protein 
ORF Gene Freeze sensitivity H2O2 sensitivity CFW sensitivity Group Description 
Interaction with the cellular environment 
YKL119C VPH2 0.01 0.02 0.48 H+-ATPase assembly protein 
YBR127C VMA2 0.01 0.02 0.22 H+-ATPase V1 domain 60-kDa subunit, vacuolar 
YEL051W VMA8 0.02 0.15 0.16 H+-ATP synthase V1 domain 32-kDa subunit, vacuolar 
YEL027W CUP5 0.02 0.04 0.28 H+-ATPase V0 domain 17-kDa subunit, vacuolar 
YGR020C VMA7 0.02 0.10 0.14 H+-ATPase V1 domain 14-kDa subunit, vacuolar 
YDL185W TFP1 0.03 0.02 0.17 Encodes three-region protein that is self-spliced into Tfp1p and PI-SceI 
YGL167C PMR1 0.05 0.31 0.17 Ca2+-transporting P-type ATPase located in Golgi 
YOR332W VMA4 0.07 0.03 0.19 H+-ATPase V1 domain 27-kDa subunit, vacuolar 
YPL234C TFP3 0.12 0.03 0.23 H+-ATPase V0 domain 17-kDa subunit, vacuolar 
YPR036W VMA13 0.22 0.10 0.35 H+-ATPase V1 domain 54-kDa subunit, vacuolar 
YOR270C VPH1 0.29 0.84 0.77 H+-ATPase V0 domain 95-kDa subunit, vacuolar 
Protein fate (folding, modification, destination) 
YGR105W VMA21 0.03 0.03 0.34 ATPase assembly integral membrane protein 
YEL036C ANP1 0.05 0.90 0.05 Required for protein glycosylation in the Golgi 
YBL058W SHP1 0.09 0.08 0.04 Potential regulatory subunit for Glc7p 
YDR245W MNN10 0.10 1.33 0.09 Subunit of mannosyltransferase complex 
YDR264C AKR1 0.27 1.23 0.03 Ankyrin repeat-containing protein 
YJL183W MNN11 0.40 0.51 0.38 Related to Mnn10p, and in a complex containing other MNN gene products 
YDR140W MTQ2 0.46 1.01 0.16 Putative S-adenosylmethionine-dependent methyltransferase localized to cytoplasm and nucleus 
YNL054W VAC7 0.46 0.08 1.39 Vacuolar protein 
Protein synthesis 
YCR003W MRPL32 0.07 0.35 0.91 Mitochondrial ribosomal protein, large subunit 
YNR037C RSM19 0.10 0.10 0.89 Mitochondrial ribosomal protein, small subunit 
YHR010W RPL27A 0.13 0.59 0.48 60S large subunit ribosomal protein L27.e 
YKR085C MRPL20 0.16 ND ND ND Mitochondrial ribosomal protein, large subunit 
YDL191W RPL35A 0.29 0.42 0.33 60S large subunit ribosomal protein 
YJL189W RPL39 0.40 0.19 1.37 60S large subunit ribosomal protein L39.e 
YDL061C RPS29B 0.42 0.56 0.37 Ribosomal protein S29.e.B 
YGR159C NSR1 0.44 0.30 1.51 Nuclear localization sequence-binding protein, required for pre-rRNA processing and ribosome biogenesis 
Transcription 
YGL070C RPB9 0.08 0.70 0.30 DNA-directed RNA polymerase II, 14.2-kDa subunit 
YJL140W RPB4 0.10 0.27 0.27 DNA-directed RNA polymerase II, 32-kDa subunit 
YOL148C SPT20 0.16 0.05 0.68 Member of the TBP class of SPT proteins that alter transcription site selection 
YFR001W LOC1 0.36 0.59 0.49 Nuclear protein involved in asymmetric localization of ASH1 mRNA 
YDL106C PHO2 0.38 0.15 0.68 Homeodomain protein 
YBR289W SNF5 0.38 0.54 0.80 Component of SWI/SNF transcription activator complex 
YDR174W HMO1 0.44 0.19 0.91 Nonhistone protein 
YMR179W SPT21 0.47 1.19 0.76 Required for normal transcription at a number of loci 
Metabolism 
YJR090C GRR1 0.01 0.05 0.48 Required for glucose repression and for glucose and cation transport 
YFR019W FAB1 0.07 0.95 1.00 Phosphatidylinositol-3-phosphate-5-kinase 
YFL001W DEG1 0.25 0.17 0.73 Pseudouridine synthase 
YDR300C PRO1 0.29 0.03 0.46 Glutamate-5-kinase 
YNL280C ERG24 0.33 0.08 0.10 C-14 sterol reductase 
YOR039W CKB2 0.42 0.53 0.88 Casein kinase II β-chain 
Others 
YCL007C CWH36 0.02 0.11 0.22 Questionable protein (ORF overlaps VMA9 gene) 
YGL095C VPS45 0.12 0.07 0.38 Vacuolar protein sorting-associated protein 
YOR080W DIA2 0.14 1.21 0.51 Protein involved in invasive and pseudohyphal growth 
YBR171W SEC66 0.15 0.56 0.08 ER protein–translocation complex subunit 
YPR135W CTF4 0.29 0.79 0.08 DNA-directed DNA polymerase α-binding protein 
YMR307W GAS1 0.33 0.33 0.96 Glycophospholipid-anchored surface glycoprotein 
YPL106C SSE1 0.34 0.33 0.21 Heat shock protein of HSP70 family 
YDR477W SNF1 0.34 0.66 0.92 Carbon catabolite derepressing Ser/Thr protein kinase 
YDL074C BRE1 0.37 1.02 0.98 E3 ubiquitin ligase for Rad6p 
Unclassified proteins 
YNL080C  0.06 0.02 0.96 Protein of unknown function 
YOR331C  0.12 0.03 0.27 Questionable protein 
YGR160W FYV13 0.24 0.67 0.53 Questionable protein 
YDR161W TCI1 0.25 1.25 0.18 Protein of unknown function localized to cytoplasm and nucleus 
YDR126W SWF1 0.29 0.00 0.00 Spore wall formation 
YBR194W SOY1 0.32 1.56 1.86 Synthetic with Old Yellow enzyme 
YJL188C BUD19 0.35 0.17 0.71 Questionable protein 
YGL218W SRF1 0.40 0.13 0.73 Questionable protein 
*

Sensitivity was defined in Materials and methods.

Groups were defined as follows: A, sensitivity to all stress; B, sensitivity to freeze–thaw and H2O2 stress; C, sensitivity to freeze–thaw and CFW stress; D, sensitivity to freeze–thaw stress.

ND, not determined; ER, endoplasmic reticulum; CFW, calcofluor white.

3

Function (a) and subcellular product localization (b) of genes required for tolerance (58 genes) to freeze–thaw stress and of all genes deleted in the complete set of strains. Classifications were determined on the basis of the MIPS database (see Materials and methods).

3

Function (a) and subcellular product localization (b) of genes required for tolerance (58 genes) to freeze–thaw stress and of all genes deleted in the complete set of strains. Classifications were determined on the basis of the MIPS database (see Materials and methods).

The ‘Interaction with the cellular environment’ class contained a high number of genes for the structural components of vacuolar H+-ATPase, such as VMA2 and VMA8. Vacuolar H+-ATPase, consisting of at least 14 components, is involved in the regulation of pH homeostasis by vacuolar acidification (Forgac, 1999; Sambade & Kane, 2004). Table 1 shows that deletions of 10 genes out of the 14 known component genes of vacuolar H+-ATPase conferred freeze–thaw sensitivity. VMA genes reportedly play a protective role against environmental stresses, including pH stress and ethanol stress (Perrone, 2005; Fujita, 2006). Our results strongly suggest that the functions of vacuolar H+-ATPase are required for freeze–thaw tolerance in yeast cells.

The ‘Protein fate’ class contained genes involved in protein glycosylation, such as ANP1, MNN10, and MNN11 (Jungmann & Munro, 1998; Jungmann, 1999). These genes are required for maintaining cell wall integrity (Klis, 1994; Schmidt, 2005). Therefore, cell wall integrity is probably important for freeze–thaw tolerance in yeast cells.

The ‘Protein synthesis’ functional class contained several genes for ribosomal and mitochondrial ribosomal proteins, such as RPL27A and MRPL32 (Graack & Wittmann-Liebold, 1998; Planta & Mager, 1998). This indicates that unimpaired functions of the ribosome are required for tolerance to freeze–thaw stress.

Figure 3b shows the classification results based on subcellular localization of gene products using the MIPS database. In the MIPS database, the localization of yeast proteins is assigned to 20 categories of cellular components. On the basis of Fig. 3, the high numbers of gene products from genes required for freeze–thaw tolerance were located in the vacuole, endoplasmic reticulum (ER), and Golgi apparatus. In agreement with the functional classification, which assigned a high number of genes involved to functions of vacuolar H+-ATPase, the highest numbers of gene products were localized in the vacuole. The functions of ER and Golgi apparatus are required for vacuolar biogenesis (Klionsky, 1990; Bryant & Stevens, 1998). The importance of vacuolar function for freeze–thaw tolerance in yeast cells is highlighted here by the high frequency of subcellular localizations to the ER and Golgi apparatus (Fig. 3b). The functions of the ER and Golgi apparatus are also required for correct cell wall biogenesis. An impaired cell wall phenotype was previously observed in mutants of genes, such as PMR1, SEC66, and MNN10, whose gene products are localized in these organelles (Okorokov & Lehle, 1998; Page, 2003; Schmidt, 2005). These functional and subcellular classifications suggest that the function of the vacuole as well as that of the cell wall is important for freeze–thaw tolerance in yeast cells.

Cross-sensitivity of freeze–thaw-sensitive mutants to oxidative stress and cell wall-challenging reagent

Because free radicals are generated during freezing and thawing (Hermes-Lima & Storey, 1993; Du & Takagi, 2005), the overlap of freeze–thaw sensitivity and oxidative stress sensitivity was assessed here by examining the cross-sensitivities of the mutants identified as freeze–thaw-sensitive mutants. To determine suitable measurement conditions, we cultivated BY4743 cells in YPD media containing various concentrations of H2O2. In YPD medium containing 2 mM H2O2, growth was inhibited by c. 50% (Fig. 4a). Table 1 lists all the freeze–thaw-sensitive mutants examined under this oxidative condition. Cross-sensitivity to oxidative stress was detected in c. 60% of the freeze–thaw-sensitive mutants, suggesting that the mechanisms of freeze–thaw tolerance overlap those of oxidative reagent tolerance.

4

Growth of the wild-type strain in YPD medium containing (a) 0–5 mM H2O2, and (b) YPD medium containing 0–21.8 μM calcofluor white. OD630 nm values are expressed as mean±standard deviation from triplicate experiments.

4

Growth of the wild-type strain in YPD medium containing (a) 0–5 mM H2O2, and (b) YPD medium containing 0–21.8 μM calcofluor white. OD630 nm values are expressed as mean±standard deviation from triplicate experiments.

To determine the correlation between freeze–thaw sensitivity and cell wall damage, which might cause deterioration of freeze–thaw tolerance, we evaluated the sensitivity of freeze–thaw-sensitive mutants to a cell wall-challenging reagent, CFW. To determine suitable measurement conditions, we cultivated BY4743 cells in YPD media containing various concentrations of CFW. In YPD medium containing 8.2 μM CFW, growth was inhibited by c. 50% (Fig. 4b). Sensitivity to CFW under this culture condition was examined for all freeze–thaw-sensitive mutants (Table 1). The results revealed that c. 60% of the freeze–thaw-sensitive mutants showed cross-sensitivity to CFW, suggesting that defects in biogenesis or in cell wall assembly are responsible for freeze–thaw sensitivity in many freeze–thaw-sensitive strains.

Figure 5 shows a profile of the overlap in sensitivities to freeze–thaw stress, H2O2 stress, and cell wall stress. Of the 58 freeze–thaw-sensitive mutants tested, 22 exhibited sensitivity to all stress conditions (group A), 13 to both freeze–thaw stress and H2O2 stress (group B), 12 to both freeze–thaw stress and CFW (group C), and 10 to only the freeze–thaw stress (group D). Higgins (2002) and Thorpe (2004) identified the genes required for tolerance to oxidative stress using H2O2 and other oxidative reagents. However, almost all genes identified in these previous studies, such as MOG1 and YJL055w, were not contained in any group, suggesting that only a fraction of the genes required for tolerance to oxidative stress are involved in freeze–thaw tolerance.

5

Venn diagram of number of freeze–thaw-sensitive deletion mutants that showed cross-sensitivity to oxidative stress caused by H2O2 and/or to cell wall stress caused by calcofluor white. Deleted genes in the mutants included in groups A–D are shown.

5

Venn diagram of number of freeze–thaw-sensitive deletion mutants that showed cross-sensitivity to oxidative stress caused by H2O2 and/or to cell wall stress caused by calcofluor white. Deleted genes in the mutants included in groups A–D are shown.

Figure 6 summarizes the subcellular localization of gene products included in each group. In group A, the highest numbers of gene products were localized in the vacuole, and most of the products were components of vacuolar H+-ATPase (Figs 5 and 6). This result implies that vacuolar functions contribute to freeze–thaw tolerance, on the basis of their protective roles against both oxidative stress and cell wall stress. In group B, higher numbers of gene products were located in the cytoplasm and nucleus, such as Rpl39 and Nsr1 (Figs 5 and 6). These gene products might contribute to freeze–thaw tolerance by the promotion or repression of aspects of cellular metabolism, such as protein synthesis. In group C, higher numbers of gene products were located in the Golgi apparatus and ER, such as Mnn10 and Mnn11 (Figs 5 and 6). Mnn proteins are involved in cell wall biogenesis via protein glycosylation (Klis, 1994; Schmidt, 2005). Our results therefore suggest that biogenesis and assembly of cell walls are important for freeze–thaw tolerance in yeast cells. In group D, the distribution pattern of gene products was similar to that in group B. At present, the functions of the gene products included in groups B and D required for freeze–thaw tolerance cannot be determined in detail.

6

Distribution of subcellular product localization of the genes deleted in mutants included in each group defined in Fig. 5.

6

Distribution of subcellular product localization of the genes deleted in mutants included in each group defined in Fig. 5.

Although MPR1 and MPR2, encoding N-acetyltransferase, are reportedly required for freeze–thaw tolerance (Takagi, 2000; Du & Takagi, 2005), in this study the genes were not contained in any group. MPR1 and MPR2 were present in S. cerevisiaeΣ1278b, but were absent in S. cerevisiae S288C (Takagi, 2000). Because we used BY4743, which is a derivative strain of S288C, MPR1 and MPR2 could not be detected in our study. Park (1998) reported that Cu/Zn-superoxide dismutase (Sod1) was required for freeze–thaw tolerance. In our study, we did not identify the SOD1 deletion strain as a freeze–thaw-sensitive strain. This discrepancy might be due to differences in the cultivation conditions of the yeast cells. Park et al. cultivated yeast cells under aerobic condition, whereas we cultivated them under relatively anaerobic conditions, because we used a microtiter plate for cultivation.

Our results suggest at least two causes of freeze–thaw injury: oxidative stress generated during the freeze–thaw process, and defects in cell wall assembly that lead to disruption of the biomembrane, macromolecule and organelle by the growth of ice crystals. In this study, although almost all genes required for tolerance to oxidative stress were not involved in freeze–thaw tolerance, the fraction of the genes involved in oxidative stress tolerance was necessary for freeze–thaw tolerance. The functions of vacuolar H+-ATPase might protect yeast (via vacuolar acidification) from both types of injury, and also might contribute to normalization of cell wall assembly. The functions of the ER and Golgi apparatus might be required for robustness of the cell wall and for protection from macromolecular damage.

The information obtained in this study helps to clarify the mechanisms of freeze–thaw injury and freeze–thaw tolerance, and will help in the further development of molecular breeding of freeze–thaw-tolerant yeast. Cross-investigation with comprehensive gene expression analysis using a DNA microarray helps to clarify the cellular response to freeze–thaw stress. Overexpression or upregulation of these genes might increase tolerance to freeze–thaw stress. This increase in freeze–thaw tolerance might accelerate the development of manufacturing processes for frozen-dough baking.

Acknowledgements

This work was supported by a grant to H. Takagi. and J. Shima from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). We thank Setsuko Ohya (National Food Research Institute) for skillful technical assistance.

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Author notes

Editor: Hyun Kang