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Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae

Elena Garre, Roberto Pérez-Torrado, José V. Gimeno-Alcañiz, Emilia Matallana
DOI: http://dx.doi.org/10.1111/j.1567-1364.2008.00453.x 52-62 First published online: 1 February 2009


The role of the acid trehalase encoded by the ATH1 gene in the yeast Saccharomyces cerevisiae is still unclear. In this work, we investigated the regulation of ATH1 transcription and found a clear involvement of the protein kinase Hog1p in the induction of this gene under severe stress conditions, such as high salt. We also detected changes in the acid trehalase activity and trehalose levels, indicating a role of the acid trehalase in intracellular trehalose mobilization. Finally, the growth analysis for different mutants in neutral and acid trehalases after high salt stress implicates acid trehalase activity in saline stress resistance.

  • Saccharomyces cerevisiae
  • stress resistance
  • acid trehalase


The disaccharide trehalose [α-d-glucopyranosyl (1-1)-α-d-glucopyranoside] is present in numerous living creatures, from simple organisms such as bacteria to resurrection plants or insects, where different roles have been proposed. It may serve as an energy and carbon reserve, as part of the protection mechanism against several stress conditions (cold, heat or oxidative damage), such as a sensing compound and/or a growth regulator, and also as a structural component (reviewed in Elbein, 2003). In the budding yeast, Saccharomyces cerevisiae, the main function of this metabolite is to act as a protective molecule in stress response. This effect can be achieved by two different ways: i.e. by protecting membrane integrity through the union with phospholipids (reviewed in Crowe, 1992) and by preserving the native conformation of proteins and preventing aggregation of partially denatured proteins (Singer & Lindquist, 1998a). When yeast cells undergo determined stresses, such as heat shock or NaCl addition, they accumulate large amounts of trehalose (Blomberg, 2000). This accumulation can be explained easily by the described mechanisms of gene expression induction and activation of the trehalose synthase complex, but, surprisingly, the trehalose degradation metabolism is also induced under these conditions (Parrou, 1997; Blomberg, 2000). The key role of trehalose degradation in metabolic control (Thevelein & Hohmann, 1995) and in appropriate stress response and recovery has been revealed by several studies (Nwaka, 1995b; Pedreño, 2002).

Two kinds of trehalase activities have been described in S. cerevisiae (reviewed in Nwaka & Holzer, 1998). The best characterized is the neutral trehalase activity encoded by the NTH1 gene. Nth1p is localized in the cytosol and has maximal activity at neutral pH. This enzyme is transcriptionally regulated by glucose repression and by the general stress response pathway through the interaction of Msn2p/Msn4p transcriptional factors with STRE sequences present in its promoter (Zahringer, 2000). Posttranslational regulatory mechanisms have also been described, such as cAMP-dependent phosphorylation (Zahringer, 1998), followed by full activation mediated by 14-3-3-binding proteins (Bmh1p, Bmh2p) (Panni, 2007), and also inhibition by interaction with the protein Dcs1p (De Mesquita, 2003). The function of Nth1p is to hydrolyze intracellular trehalose both in normal growth and under different stress conditions (San Miguel & Argüelles, 1994). NTH1 gene expression is induced in the presence of toxic chemical agents (CuSO4 or NaAsO2), hydrogen peroxide or heat shock, but transcriptional induction is accompanied by a higher trehalose accumulation in yeast cells only under heat shock (Zahringer, 1997, 2000). The NTH2 gene shares 77% identity at the protein level with the NTH1 gene, but its function is still unknown. Changes in transcript levels have been detected, but no effects in trehalose levels or neutral trehalase activity have been found in nth2 mutants (Nwaka, 1995a, b). Recent work by Jules and coworkers suggests its involvement in trehalose mobilization in yeasts cells lacking both the TPS1 and the NTH1 genes and grown on trehalose (Jules, 2008). The ATH1 gene codes for acid trehalase, a trehalase activity with a low optimal pH. Ath1p has been classically considered vacuolar (Harris & Cotter, 1988; Alizadeh & Klionsky, 1996), but recently, new data suggest the possibility of a dual localization both in the periplasmic space and in the vacuole (Jules, 2004; Parrou, 2005; Huang, 2007). This enzyme has been related to extracellular trehalose degradation for its use as a carbon source for growth (Nwaka, 1996; Jules, 2004; Parrou, 2005), although its participation in degradation of intracellular trehalose has not been ruled out (Nwaka, 1995b). The enzymatic regulation of Ath1p is not clear; apparently, the protein needs to be synthesized de novo to be active and acid trehalase activity is detected only in glucose-grown resting cells and on respiratory substrates (San Miguel & Argüelles, 1994). To date, this activity has been considered not to be regulated by stress because of the lack of STRE sequences at the ATH1 gene promoter. Recently, an Ath1p-dependent trehalose mobilization has been described, when mutant tps1 yeast cells are forced to accumulate trehalose by growing them on a medium with the disaccharide and lacking glucose (Jules, 2008).

When the budding yeast S. cerevisiae is subjected to saline conditions, it undergoes rapid dehydration and growth arrest (Hohmann & Mager, 2003). This activates a cellular response that consists of (1) Na+ exclusion toward the extracellular space through the Pmr2p/Ena1p sodium transporter and the Nha1p Na+/H+ antiporter or compartmentalization in the vacuole mediated by the Nhx1p Na+/H+ antiporter, (2) intracellular accumulation of glycerol as a compatible solute and (3) accumulation of trehalose (Sharma, 1997) just like in response to other stresses such as heat shock (De Virgilio, 1994) or acetic acid stress (Lewis, 1997).

In this work, we have attempted to shed light on the implication of Ath1p in the saline stress response and yeast adaptation. For this purpose, we have analyzed the transcriptional profile of the ATH1 gene and also acid trehalase activity in the presence of a high salt concentration in both wild-type and hog1 mutant strains. By construction of several mutants in neutral and acid trehalase activities, we have been able to detect effects of the lack of one of them on the activity of the other, analyze the participation of Ath1p in intracellular trehalose mobilization and also investigate its implication in saline stress resistance.

Materials and methods

Yeast strains and growth conditions

The strains used in this study were MCY1264 (S288c genetic background) and mutants MCY1264-hog1, MCY1264-nth1, MCY1264-ath1 and MCY1264-nth1ath1. Different stress conditions were tested. For osmotic stress and glucose depletion, cells were grown in YPD medium (2% glucose, 2% peptone and 1% yeast extract) up to OD600 nm 1.0 with vigorous shaking at 30 °C; cells were then harvested by centrifugation, suspended in fresh YPD medium containing 0.9 M sorbitol or in fresh YP medium (2% peptone and 1% yeast extract) and incubated for 1, 3 and 6 h or 8 and 24 h, respectively. For saline stress experiments, cells were grown up to the exponential OD600 nm 1.0 and postdiauxic OD600 nm 6.0 phases, suspended in fresh YPD containing 1.2 M NaCl and incubated for 1, 3, 6 and 24 h. In general, all the stresses were developed on fresh YPD medium, except glucose deprivation, in order to detect specific ATH1 inductions, not the previously described induction driven by glucose exhaustion. Alternatively, saline stress was produced by directly adding salt up to 1.2 M concentration to postdiauxic cultures. Then, different samples were taken for total RNA isolation, trehalase activity measurement and trehalose content determination. Viability after stress was assayed by plating serial dilutions from previous OD600 nm 1.0 dilution (1 × 10−2, 5 × 10−3, 2.5 × 10−3, 1.25 × 10−3, 6.25 × 10−4 and 3.125 × 10−4) of cells in YPD and checking growth after 25 h of incubation at 30 °C.

Construction of disrupted mutant strains

Disruptions of the NTH1, ATH1 and HOG1 genes were performed by homologous recombination with a deletion cassette as described elsewhere (Güldener, 1996). The primers used for synthesis of deletion cassettes are shown in Table 1. These primers had between 40 and 60 homologous bases to the ends of the gene-coding sequence (underlined letters). PCR reactions using plasmid pUG6 as a template were as follows: 2 min at 94 °C; 30 cycles of 15 s at 94 °C, 30 s at 50 °C, a variable time (1 min by kb) at 72 °C and 5 min at 72 °C. Cells were transformed with the PCR product using the LiAc protocol (Gietz, 1995). After heat shock, cells were incubated for 3 h in YPD liquid medium at 30 °C. Finally, transformed cells were selected on YPD plates with geneticin (200 mg L−1).

View this table:

Primers used for deletion cassettes and probes synthesis

PrimersSequence (5′→3′)Use
  • aUnderlined sequence is the 5′ end (S1) or 3′ end (C2) of the corresponding gene. The end of primer sequence (no underlined sequence) is homologous to the pUG6 plasmid (Güldener, 1996).

RNA purification and RNA blot hybridization analysis

Total RNA from 50 mg yeast cells was extracted using an automated device Mini-Bead Beater (Biospec Products) in 0.5 mL LETS buffer [LiCl 0.1 M, EDTA 0.01 M, pH 8.0, Tris-HCl 0.01 M, pH 7.4, and SDS 0.2% (p/v)], 0.5 mL phenol, pH 4.3, (Amresco), and 0.5 mL glass beads. Supernatants were extracted with phenol : chloroform (5 : 1) (v : v) and phenol : chloroform : isoamylic alcohol (25 : 24 : 1) (v : v : v). A RNA precipitate was obtained after incubation with one volume of LiCl 5 M at −20 °C for at least 3 h. RNA (30–50 μg) was separated by electrophoresis in denaturing 1% (w/v) agarose gel [2.2 M formaldehyde, 1 × MOPS (20 mM MOPS, 8 mM NaAc and 1 mM EDTA, pH 7.0)] and blotted on Hybond N+ membranes (Amersham) (Sambrook & Rusell, 2001). NTH1 and ATH1 probes were obtained by PCR using the primers shown in Table 1. The NTH1 probe was obtained by EcoRI digestion of the PCR product. PCR reactions were carried out with genomic DNA as a template. Probes were labelled with commercial kits (Rediprime, Amersham; High Prime, Roche) and [α-32P]dCTP (Amersham). High-stringency conditions were used both for hybridizations and for washes. Quantification of Northern blots was performed with a phosphoimager FLA3000 and the image gauge software (Fujifilm), and the 28S rRNA gene was used for normalization. mRNA levels are relative to the 1-h sample.

Assay of neutral and acid trehalase activities

Cell-free extracts from 70 mg yeast cells and trehalase determinations were carried out as reported previously (San Miguel & Argüelles, 1994; Pedreño, 2002). The glucose concentration was determined in the supernatants using the glucose oxidase/peroxidase assay. Specific activity is expressed as nmol of glucose liberated per min per mg total protein. The total protein content was determined using commercial kits (Bio Rad Protein Assay, Bio Rad Laboratories GmbH).

Determination of intracellular trehalose

Permeabilized cell mixtures from 10 mg of cultured cells were obtained as described elsewhere (Parrou & François, 1997). Trehalose was measured after enzymatic degradation with commercial trehalase (Sigma). The glucose released was determined by the glucose oxidase/peroxidase assay. The amount of trehalose is expressed as μg of trehalose per mg of cells dry weight.


Expression of ATH1 is induced by severe saline stress

In order to investigate the putative involvement of acid trehalase Ath1p in the yeast stress response, we carried out different treatments in exponential and postdiauxic growing cultures of the MCY1264 strain as described in Materials and methods, and cell samples were taken at different time points for several analyses. We first analyzed the ATH1 mRNA profiles along the stress treatments and also the expression of the NTH1 gene for comparison. The ATH1 transcriptional level increased at different time points during a salt stress challenge. Figure 1 shows the growth profiles and the results of the ATH1 and NTH1 gene expression analysis under saline treatment on postdiauxic cultures, the condition we selected for further studies where ATH1 induction was clearly observed. As expected, the addition of salt caused a growth delay as shown in Fig. 1c. As can be seen in Fig. 1a (left), ATH1 mRNA levels increased after 3 h in the presence of 1.2 M NaCl (white bars). Maximal transcript accumulation was observed at the 6-h time point, when a three- to fourfold increase in the mRNA level was observed as compared with the YPD control condition at the same time point. A high ATH1 mRNA level was still observed after 24 h, where other environmental factors, such as the nutritional status, could have been driving ATH1 induction. Under control conditions on YPD (black bars), a slight decay was observed during the first hours in fresh YPD medium that then recovered after 24 h. The response to high salt was different for the neutral trehalase gene NTH1, as can be seen in Fig. 1a (right), and no significant long-term induction was detected at the 24-h time point. However, clearer changes were produced in the NTH1 gene expression during growth on YPD after inoculation in fresh medium, where a 50% decrease in the NTH1 mRNA level during the first 6 h was observed. The presence of 2% glucose in fresh YPD can explain this repression.


Expression of ATH1 and NTH1 genes (a), quantification of their corresponding enzymatic activities (b), and growth profiles (c) during saline stress in the MCY1264 strain. Growth, mRNA levels and trehalase activities were determined after treatment with 1.2 M NaCl in YPD medium (white bars and symbols) or under control conditions (black bars and symbols) in YPD during 1, 3, 6 and 24 h. mRNA levels were normalized to rRNA. Data are shown as the means±SD from at least two independent experiments. Statistical analysis was performed by means of the Student t-test with P-value <0.05 (*).

Trehalase activities were also measured in fresh extracts from cell samples obtained at the same time points. In contrast to mRNA levels, the increase in acid trehalase activity was not clearly significant until 24 h with a high salt concentration (Fig. 1b, left, white bars), negating that posttranslational mechanisms may regulate the new Ath1p synthesized after stress. However, in the case of the neutral trehalase (Fig. 1b, right), its activity was reduced in parallel to the mRNA level during the growth on YPD (black bars), but under saline stress (white bars) the activity increased during the first 6 h, when the mRNA level was similar to that of the 1-h sample.

The same analysis of gene expression and trehalase activities was performed under other stress conditions as described in Materials and methods. ATH1 induction was also detected under severe osmotic stress and long-term glucose deprivation (data not shown).

Although these experiments showed that ATH1 induction and acid trehalase activity could be related to the saline stress, changes in glucose availability also affect the expression of trehalase genes and activities; hence an alternative salt stress condition was created by directly adding salt to a final 1.2 M concentration to postdiauxic MCY1264 cultures, where the ATH1 gene is already induced and a high Ath1p activity is detectable. The growth profiles and trehalase activities from this experiment are shown in Fig. 2. The effect of salt on the growth profiles (Fig. 2b) was still clear although the growth rate was lower due to the lack of glucose. Even under these conditions, a very high acid trehalase activity (Fig. 2a, left) was detected as a long-term response under saline treatment, reaching very high levels after 6 and 24 h in salt. Maximal acid activity under this salt stress under glucose-deprivation conditions was up to fourfold higher than the activity in fresh YPD plus salt (compare with Fig. 1b, left). In contrast, neutral activity was extremely low during the time course of the experiment (Fig. 2a, right), although a slight induction was also detected during long incubations in salt.


Acid and neutral trehalase activities (a) and growth profiles (b) in the MCY1264 strain during saline stress under glucose-starvation conditions. Growth and trehalase activities were determined after adding 1.2 M NaCl to three different postdiauxic cultures (white bars and symbols) or under control conditions without added salt (black bars and symbols) during 1, 3, 6 and 24 h. Data and statistical treatment as described in Fig. 1.

The expression of ATH1 and NTH1 genes was also analyzed in the MCY1264-nth1 and ath1 mutant strains, respectively. Identical saline stress experiments in fresh YPD were carried out, and both transcript levels and acid trehalase activity were determined. The growth profiles and the results of these experiments are shown in Fig. 3. Deletion of the NTH1 gene does not affect the pattern of ATH1 gene response along saline stress (Fig. 3a, upper panel, white bars). Transcript levels were up to twofold higher in the nth1 mutant strain than in the control strain in the experiments described in Fig. 1 (see Fig. 1a, left, white bars), whereas differences in the short-term ATH1 induction were minimal in others (see Fig. 4a, lower panel). A slightly higher level of ATH1 mRNA in the nth1 mutant was visible for the long-term induction observed at 24 h during growth under control conditions in YPD (black bars, Fig. 3a, upper panel), indicating a clear induction of the ATH1 gene in the postdiauxic phase of normal growth on glucose medium. In parallel to the gene expression experiment, acid trehalase activity was also determined (Fig. 3a, lower panel). Maximal activity was detected after 24 h, as observed for the control strain. The difference in acid trehalase activity under saline stress between both strains was not as high as the difference in the mRNA level. However, a slight effect of the nth1 mutation in the Ath1p activity could be observed after 24 h of growth on YPD (black bars). We wanted to see whether this influence of the absence of neutral trehalase on acid trehalase expression was reciprocal; hence, we investigated NTH1 expression and neutral trehalase activity in the ath1 mutant strain (Fig. 3b). ATH1 deletion seems to affect the level of NTH1 gene induction both during growth on YPD (Fig. 3b, upper panel, black bars) and, especially, during long incubations under saline stress (Fig. 3b, upper panel, white bars), although the magnitude of this influence was dependent on the experiment. However, neutral trehalase activity was lower in the ath1 mutant (Fig. 3b, lower panel) than in the control strain (see Fig. 1b, right). As in the first set of experiments (Fig. 1), a general pattern of decrease (except for the 3-h time point) was observed in YPD control experiments (Fig. 3b, black bars). The NTH1 gene expression and the neutral trehalase activity were repressed after exposure to YPD where glucose repression mechanisms were found, but the presence of NaCl diminished this effect.


Expression of ATH1 (a) or NTH1 genes (b), quantification of their corresponding enzymatic activities, and growth profiles (c) during saline stress in mutant strains, nth1 (triangles) and ath1 (squares). Growth, mRNA levels and trehalase activities were determined after treatment with 1.2 M NaCl (white bars and symbols) or under control conditions (black bars and symbols) during 1, 3, 6 and 24 h. mRNA levels were normalized to rRNA. Data and statistical treatment as described in Fig. 1.


Effect of HOG1 gene deletion on the expression of ATH1 (a) and NTH1 (b) genes during saline treatment. Northern blots (upper panels) and their quantifications (lower panels) for wild-type (black bars) and hog1 (gray bars) strains after treatment with 1.2 M NaCl (striped bars) or under control conditions (closed bars) during 1, 3, 6 and 24 h. mRNA levels were normalized to rRNA. Data and statistical treatment as described in Fig. 1. Columns labeled with the same letter are statistically different (P-value<0.05).

Saline stress induction of ATH1 depends on the presence of Hog1p

The long-term transcriptional induction of ATH1 in response to both saline and osmotic stress prompted us to check its dependence on a functional HOG pathway. An isogenic hog1 mutant was constructed by deletion of the HOG1 gene in the MCY1264 background, and the saline stress experiments described above were performed. Figure 4 displays the results of the gene expression analysis for both the NTH1 and the ATH1 genes in the hog1 mutant. Although the expression of both genes was dependent on the presence of HOG1, different behavior was observed in the hog1 background. As can be seen in Fig. 4a, both the growth-dependent and the salt-dependent inductions of ATH1 were affected in the hog1 mutant strain. The low-level induction still observed in the hog1 mutant during growth in salt does not have statistical significance (P>0.05) and the induction at 6 and 24 h on YPD was completely abolished. Short-time NTH1 expression was minimally affected by the absence of Hog1p; however, the induction observed after 24 h under saline treatment (Fig. 4a) was not present in the hog1 mutant strain.

Ath1p is involved in intracellular trehalose mobilization

Our data from the gene expression and enzymatic activity experiments implicate acid trehalase in the mobilization of intracellular trehalose during saline stress. In order to investigate this possibility, we used the same time point samples to determine the intracellular trehalose levels and compared the trehalose accumulation in the wild-type strain and the different trehalase mutants including the double nth1ath1 mutant strain. Data from these experiments are shown in Fig. 5. Both the nth1 and the ath1 trehalase single mutants (Fig. 5b and c, respectively) showed higher trehalose accumulation than the wild-type strain (Fig. 5a) during the treatment with 1.2 M NaCl, indicating that neutral and acid trehalase activities were involved in trehalose metabolism during this stress. The double nth1ath1 mutant (Fig. 5d) accumulated the largest amounts of trehalose but only at determinate times of incubation. It is interesting to observe the behaviour of the different strains during the growth on YPD (black bars). During 24 h of growth on glucose, very low trehalose levels were detected in the control strain (Fig. 5a) and a higher increase was observed for the single nth1 and the double nth1ath1 mutants (Fig. 5b and d, respectively). However, a significant increase in trehalose levels was observed for the single ath1 mutant (Fig. 5c), according to the above-mentioned enzymatic activity data, reinforcing the role of acid trehalase in intracellular trehalose mobilization during saline stress and also during normal growth on glucose.


Effect of mutations in NTH1 and ATH1 genes on the trehalose content upon saline treatment. Wild-type (a), nth1 (b), ath1 (c) and nth1ath1 (d) strains were subjected to 1.2 M NaCl (white bars) or control conditions (black bars). Results in this figure are representative of at least two independent experiments.

A role for Ath1p in saline stress resistance

Our gene expression and biochemical data indicate the involvement of acid trehalase activity in intracellular trehalose metabolism and suggest a role in the response to saline stress. To assess the physiological meaning of the ATH1 induction and the consequent increase in acid trehalase activity, viability experiments were designed to analyze the phenotypic growth effects of the different trehalase mutations. Viability after stress was assayed by growing cells on YPD plates after treatment with 1.2 M NaCl for the same time periods used in the experiments described above. The results of these experiments are shown in Fig. 6. We found that the wild-type strain and the nth1 mutant had delayed growth after saline treatment during 1, 3 and 6 h. This delay in growth was not present in the ath1 and the double nth1ath1 mutants. The adaptation to longer incubation in the presence of 1.2 M NaCl minimized the differences among strains, and then all the strains showed approximately the same growth on YPD plates when the period of incubation in a high salt concentration was 24 h.


Effect of mutations in NTH1 and ATH1 genes on the viability after saline stress. Dilutions of wild-type (WT), nth1, ath1 and nth1ath1 strains were plated on YPD after incubation with 1.2 M NaCl or without sal (control) during 1, 3, 6 and 24 h and then incubated for 25 h at 30°C. Results in this figure are representative of at least two independent experiments and correspond to the 10−2-dilution sample. Growth profiles for the double nth1ath1 mutant during the salt stress experiment are shown in (b).


Our results first demonstrate that both the expression of the ATH1 gene and the acid trehalase activity are induced by severe saline stress in postdiauxic cells, suggesting a new function for acid trehalase other than its previously described role in external trehalose utilization (Nwaka, 1996) or its implication in trehalose mobilization by tps1 mutant yeast cells grown with trehalose and without glucose, which has been described recently (Jules, 2008). The dependence of this induction on a functional HOG pathway reinforces its relationship with adaptation to stress, as this is the main pathway for osmotic and saline stress responses (Hohmann & Mager, 2003). The different effects of the HOG1 deletion on the expression of NTH1 and ATH1 genes indicate that although both are affected by the presence of the Hog1p kinase pathway, the two trehalase genes are differentially regulated. The rapid induction of NTH1 expression in response to osmotic stress (Zahringer, 2000) and also after short incubations under glucose-deprivation conditions, or during normal growth on glucose when the carbon source becomes limiting has been described (Parrou, 1999; François & Parrou, 2001). In all these cases, induction of NTH1 seems to depend on the general stress response mediated by the Msn2p/Msn4p transcriptional factors through the STRE elements present in its promoter (Zahringer, 2000). It is interesting to note that in these situations, NTH1 induction does not parallel the increase in neutral trehalase activity due to the phosphorylation regulatory mechanism acting posttranslationaly (Jorge, 1997; Nwaka & Holzer, 1998). However, our results indicate that under the high salt stress, the induction of NTH1 gene results in an increase in neutral trehalase activity, as has been reported previously under other stress conditions such as high temperature, hydrogen peroxide addition and the presence of metals (Nwaka, 1995a; Zahringer, 1997). In our experiments, this neutral trehalase activity dependent on de novo protein synthesis was not affected by a functional HOG pathway because deletion of the HOG1 gene does not affect the induction of NTH1 during the first 6 h. In the case of the acid trehalase gene, two overlapped inductions were detected in our experiments and both were affected significantly by the hog1 deletion. However, additional experiments must be carried out to clarify the molecular relation between Hog1p and the ATH1 gene. The correlation between acid trehalase activity and ATH1 induction in the presence of 1.2 M NaCl and also during normal growth on glucose is better than that of neutral trehalase activity and NTH1 expression, suggesting that Ath1p activity is mainly dependent on de novo protein synthesis; however, posttranslational regulation cannot be discarded because timing differences between mRNA synthesis and enzymatic activity were observed.

Although both NTH1 and ATH1 genes are regulated in response to stress, they display different expression patterns, as could be expected from the absence of the consensus sequence of STRE elements in the ATH1 promoter. The induction of ATH1 resembles the HOG1-dependent long-term responses described previously under severe saline stress (Van Wuytswinkel, 2000), whereas the rapid induction of NTH1 is a typical general stress response. As our experiments indicate, a long-term NTH1 response could also occur and this is clearly affected by the hog1 deletion.

In addition to the saline stress response, we have also documented the transcriptional induction of the ATH1 gene during normal growth on glucose and the involvement of the acid trehalase activity in the maintenance of the trehalose intracellular levels. Data on the trehalose content in the different strains demonstrate that both Nth1p and Ath1p participate in intracellular trehalose mobilization, as the deletion of any of them leads to higher trehalose accumulation than the wild-type strain under both control and stress conditions, although the stress-controlled biosynthetic activities also have to be taken into account (Winderickx, 1996). The deletion of ATH1 had even greater impact on trehalose levels than NTH1 under saline stress. This possibility had never been completely ruled out (Nwaka, 1995b) but only indirect evidences were available (Nwaka & Holzer, 1998). Here, we show a correlation between the absence of Ath1p activity and a higher intracellular trehalose content. This new role of Ath1p is proposed not only under severe saline stress but also in the postdiauxic phase during normal growth in YPD, where trehalose accumulated at higher levels in the ath1 mutant than in the wild-type strain growing in YPD for 24 h. This result is consistent with early descriptions of high ATH1 expression (Destruelle, 1995) and acid trehalase activity (San Miguel & Argüelles, 1994) during the stationary phase of growth. It was also interesting to note the dramatic effect of the salt treatment on trehalose content when ath1 stationary cells were transferred to fresh YPD, which suggested that the salt stress is somehow blocking the phosphorylation-dependent activation of the neutral trehalase activity in glucose. The involvement of acid trehalase in intracellular trehalose mobilization is consistent with the classically accepted subcellular localization of this enzyme (Harris & Cotter, 1988; Alizadeh & Klionsky, 1996), proved recently (Huang, 2007), and argues against the finding of acid trehalase activity in the cell wall (Parrou, 2005), although a dual localization could also be possible. It is worth noting that the double-mutant nth1ath1 strain did not show an additive effect on the content of trehalose caused by the loss of two trehalase activities. These data could support the idea that excessive accumulation of trehalose is harmful for yeast cells (Singer & Lindquist, 1998b).

Few previous data are available about the effect of ath1 mutations on stress responses (Nwaka, 1995b; Kim, 1996) and some of them, consistent with our results, were difficult to interpret because they were affected by the differences in auxotrophies between mutant and reference strains (Chopra, 1999). It is puzzling that the ath1 and the double nth1ath1 mutant display better growth both during and after saline stress than the wild type and the nth1 mutant, but due to the strategy for construction of our mutant strains, no other differences exist with respect to the parental strain. As can be seen in Figs 5 and 6, the enhanced growth after saline stress for these two mutants was more evident for the short salt treatments, when they display a higher difference in trehalose levels with respect to the parental strain; hence, their behavior might be due to the high trehalose content. This explanation drives us to hypothesize that some other unidentified trehalase activity is responsible for trehalose degradation required for cell growth recovery after stress. As mentioned in the Results, analyses of the trehalase enzymatic activities and trehalose accumulation in the mutant strains suggest a mutual influence between both trehalase activities. This interdependence between trehalases as well as the relationship between them and the activity of the trehalose synthase complex have been reported previously (Pedreño, 2002). The nature of these crossed effects is not known but might also be related to the behaviour of the double nth1ath1 mutant. Further experiments are underway in order to determine the role of the different trehalases during yeast recovery from stress.


This work was supported by grants ALI99-1224-CO2-02, AGL2002-01109 and AGL 2005-00508. R.P.-T. and J.V.G.-A. were supported by fellowships from Generalitat Valenciana and E.G. was a fellow of the FPI program of the Ministerio de Educación y Ciencia (Spain).


  • Editor: Ian Dawes


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