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Keywords:

  • Ergosterol;
  • Heat shock protein;
  • Stress tolerance;
  • Trehalose;
  • Yeast

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The role of ergosterol in yeast stress tolerance, together with heat shock proteins (hsps) and trehalose, was examined in a sterol auxotrophic mutant of Saccharomyces cerevisiae. Ergosterol levels paralleled viability data, with cells containing higher levels of the sterol exhibiting greater tolerances to heat and ethanol. Although the mutant synthesised hsps and accumulated trehalose upon heat shock to the same levels as the wild-type cells, these parameters did not relate to stress tolerance. These results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The stress response is a highly conserved physiological response elicited by virtually all organisms in response to an environmental stress. The induction of thermotolerance is a fundamental aspect of the stress response in which a prior non-lethal heat shock endows cells with the capacity to endure more severe conditions [1]. Two aspects of the yeast stress response that have received extensive analyses are the induction of heat shock proteins (hsps) [2] and the accumulation of trehalose [3, 4]. Both of these biomolecules are heat shock inducible and so have been implicated in the acquisition of thermotolerance in cells.

The cellular signals leading to the synthesis of hsps and trehalose under stressful conditions are not known. However, biological membranes have been implicated as a primary sensor of environmental stress. In this respect, sterols as modulators of membrane fluidity have been proposed as factors determining heat sensitivity of cells. Earlier studies on mammalian cells report a positive correlation between heat sensitivity and cholesterol levels [5, 6] while several studies on yeast implicate ergosterol as a factor conferring ethanol tolerance [7–9].

Although membrane sterols appear important in stress tolerance, no detailed study has emerged which links this component with associated factors of the stress response. We were particularly interested in the effect of ergosterol on yeast stress tolerance as this sterol is the most abundant in the yeast plasma membrane [10] and is related to sterols found in higher eukaryotes in terms of both structure and function. The availability of yeast sterol auxotrophs allowed quantified alterations in ergosterol content to be made under aerobic conditions and thereby provided an attractive model system in which to examine the direct effects of ergosterol levels on the yeast stress response. In this communication, we demonstrate that ergosterol does not directly affect hsps and trehalose accumulation but rather stabilises membranes against heat and ethanol stresses.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Yeast strains and culture conditions

Saccharomyces cerevisiae wild-type strain X2180-1B (MATαSUC2 mal mel gal2 cup1R) and an erg1-disruptant strain FY14 (MATαhem1 erg1::LEU2 ade2 leu2) were kind gifts from Drs L. Parks and J. Crowley (North Carolina State University, Raleigh, NC, USA). The erg1-disruptant strain is a sterol auxotroph defective in haem biosynthesis and in the early stages of ergosterol biosynthesis. The erg1-disruptant strain was maintained by weekly subculturing into YEP medium (0.5% yeast extract, 0.5% bacteriological peptone, 0.3% KH2PO4, 0.3% (NH4)2SO4, and 2% glucose, all w/v), containing ergosterol (5 μg ml−1, Sigma), Tween 80 (0.05% v/v, Sigma), ampicillin (25 μg ml−1) and streptomycin (20 μg ml−1). YEP agar plates were incubated at 28°C for 72 h and regularly checked for revertants. Experimental cultures were shaken at 25°C on a rotary shaker (200 rpm) in YEP medium. The erg1-disruptant strain was grown in medium supplemented with ergosterol (1, 10 or 20 μg ml−1). Cell growth was followed by reading optical density at 600 nm (OD600). Cells were harvested during mid-exponential phase when the OD600 was between 0.35 and 0.40. This corresponded to a cell density of between 3×106 and 9×106 cfu ml−1 for ergosterol supplemented cultures of the erg1-disruptant and approximately 1×107 cfu ml−1 for the wild-type strain.

2.2Stress conditions

Cultures for stress tolerance studies were washed twice with sterile water and resuspended in YEP without glucose. Intrinsic thermotolerance (heat stress) of cells grown at 25°C was determined by exposing cells to 48°C. Samples were taken from heat stressed cultures at various times over a 60-min time course. Induced thermotolerance was measured by preincubating cells suspended in YEP at 37°C for 30 min (heat shock) prior to heat stress exposure as described above. Following thermal stress, 1-ml samples were transferred to microfuge tubes and cooled in an ice bath to 25°C before viability determination. In addition to thermotolerance, intrinsic tolerance to ethanol (17% v/v) was also examined. Preliminary experiments determined that this concentration of ethanol and the 48°C heat stress resulted in consistent survival kinetics over the given time course.

Cell viability was determined by colony counts on YEP plates. Colonies were counted after 2 days (X2180-1B) or 4 days (FY14) incubation at 25°C. Stress tolerance was expressed as percentage survivors by comparing the colony count of stressed cells with that of an unstressed control maintained at 25°C over the time course.

2.3Biochemical analyses

Cells (400 ml of culture) for fatty acid analysis of the total phospholipid fraction were washed twice with distilled water and stored at −70°C until required. Lipid extraction and analysis were as previously described [11].

Total ergosterol (free and esterified) was determined by the spectrophotometric method of Breivik and Owades [12]. Cells from 400 ml of culture were washed twice with distilled water and stored at −70°C until required. Thawed samples were digested in 25% w/v alcoholic KOH at 85–90°C for 3 h. Saponified samples were extracted into n-heptane. Ergosterol was determined spectrophotometrically at 281.5 nm. The content of ergosterol was expressed as a percentage of the dry weight of cells. This method gave results comparable to that determined by thin-layer chromatography and gas chromatography [13].

For trehalose determination cells were harvested from 80 ml of culture and washed twice with chilled distilled water. Trehalose was extracted with cold 0.5 M trichloroacetic acid and estimated according to a modified anthrone procedure as previously described [14]. The content of trehalose was expressed as a percentage of the dry weight of cells.

2.4Analysis of protein synthesis

Cells from 20 ml culture medium were pelleted and washed in methionine-free media YNBG (0.67% yeast nitrogen base without amino acids (Difco), 0.3% KH2PO4 and 2% glucose, all w/v) and resuspended in 2 ml of YNBG. Cells in YNBG were labelled for 30 min with [35S]methionine (150 μCi; specific activity 1175 Ci mmol−1; ICN Pharmaceuticals) at 25°C (control) or 37°C (heat shock). Methionine labelling was terminated by addition of 150 μl of 100 mg ml−1 unlabelled methionine. Cells were pelleted and proteins extracted as previously described [15]. Protein concentration of extracts was quantified using a Coomassie blue protein microassay procedure (Pierce, Rockford, IL, USA) based on the method of Bradford [16]. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [17]. Stacking and resolving gels were 4% and 10% polyacrylamide respectively.

Protein samples (10 μg) were run against prestained broad range molecular mass standards (Bio-Rad, catalogue number 161-0318). Gels were dried and exposed to BioMax MR film (Integrated Sciences) at −70°C for 18 h before developing.

Autoradiograms were scanned using a gel documentation system (Ultra-Violet Products, UK) and a Phoretix densitometer software package, V.3.0 (Phoretix International, UK). Quantitation of hsps was achieved by calculating the ratio of respective band intensities relative to that of actin (Act1, 43 kDa) which is not increased upon a heat shock [18, 19]. Relative increase in protein synthesis, before and after heat shock, was expressed as the ratio of normalised pixel intensity with respect to actin.

The level of hsp104 in control and heat shocked cells was determined using Western blot analysis and was performed as previously described [20] using antibody to yeast hsp104 (Stressgen, Victoria, Canada).

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Ergosterol content and membrane fatty acid composition

Sterol enrichment of the erg1-disruptant cells produced consistent alterations in the level of ergosterol (Table 1). While supplementation with 1 μg ml−1 of ergosterol gave a relatively low value for total ergosterol, addition of 10 μg ml−1 of ergosterol resulted in an approximately three-fold increase in cellular ergosterol levels. Increasing the concentration of ergosterol supplement to 20 μg ml−1 had little further effect on the total ergosterol content. Supplementing with 10 μg ml−1 of ergosterol produced cells containing the same levels as the wild-type cells. Previous studies using a similar sterol auxotrophic mutant have shown that sterol saturation is achieved by addition of approximately 10 μg ml−1 of ergosterol to the medium [21].

Table 1.  Ergosterol content of the erg1-disruptant (FY14) and the wild-type (X2180-1B) cells
StrainErgosterol supplementErgosterol
 (μg ml−1)(% dry weight of cells)
  1. The erg1-disruptant cells were supplemented with 1, 10 or 20 μg ml−1 of ergosterol. Data presented are the mean±S.D. of five independent experiments.

FY14 (Δerg1)10.115±0.008
FY14 (Δerg1)100.332±0.023
FY14 (Δerg1)200.365±0.046
X2180-1B (WT)0.388±0.086

The fatty acid composition from the total phospholipid fraction was analysed to determine if ergosterol enrichment resulted in any compensatory changes to the fatty acid profile. The erg1-disruptant cells grown on media containing 1 μg ml−1 of ergosterol had a high content of monounsaturated fatty acid (11±0.5% palmitoleic acid and 52±1.5% oleic acid, mean±S.D. of five independent experiments) with the remainder saturated fatty acid, predominantly palmitic acid (22±1%). There was very little variation in the fatty acid profile in the erg1-disruptant cells supplied with increasing concentrations of ergosterol. The wild-type cells had a similar level of palmitic acid (19±0.5%) whereas the proportions of palmitoleic acid (16±1%) and oleic acid (54±1%) differed.

3.2Stress tolerance

Cells supplemented with only 1 μg ml−1 of ergosterol were heat sensitive with less than 20% survivors following 5 min exposure to 48°C (Fig. 1A). Intrinsic thermotolerance paralleled ergosterol content, with thermotolerance increasing with increasing ergosterol concentration. Moreover, thermotolerance was inducible in all variants of the erg1-disruptant as well as in the wild-type strain because higher levels of survivors were observed with previous exposure of the cells at 37°C for 30 min (Fig. 1B) than those without pre-exposure to 37°C (Fig. 1A). Induction and maintenance of thermotolerance over the 60-min time course was greatest in cells enriched with 1 μg ml−1 of ergosterol (Fig. 1B).

image

Figure 1. Stress tolerance of the erg1-disruptant cells (FY14) in YEP medium supplemented with 1 μg ml−1 (diagonally hatched bars), 10 μg ml−1 (white bars) or 20 μg ml−1 (horizontally hatched bars) of ergosterol and of the wild-type strain (X2180-1B) without ergosterol (black bars). Intrinsic thermotolerance (A) was measured at 48°C, while induced thermotolerance (B) was measured at 48°C following exposure of cells to 37°C for 30 min. Ethanol stress (C) was 17% v/v. Samples were taken at 5, 15, 30 and 60 min following exposure of cells to stress conditions. Cell viability was determined as described in Section 2. Results are presented as percentage survivors with respect to a non-heat shocked sample. Data presented are the mean±S.D. of three independent experiments.

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Ethanol tolerance (Fig. 1C) was also observed to increase with increasing ergosterol concentration. Over the time course, the wild-type cells displayed the greatest resistance to ethanol (Fig. 1C), while thermoresistance (Fig. 1A) was lower in comparison to the erg1-disruptant cells.

3.3Trehalose

Basal levels of trehalose were similar in the wild-type strain (0.29% dry weight) and the erg1-disruptant strain grown on medium containing 1 μg ml−1 of ergosterol (0.28% dry weight). Following heat shock, there was an approximately 10-fold increase in trehalose content in the erg1-disruptant (2.50% dry weight) and the wild-type (2.62% dry weight) strains. Similar levels of trehalose were measured in non-heat shocked and heat shocked cells of the erg1-disruptant grown on media containing 10 and 20 μg ml−1 of ergosterol.

3.4Protein synthesis

Cells were labelled with [35S]methionine in YNBG medium with or without addition of ergosterol in order to examine the relationship between ergosterol content and protein synthesis in non-heat shocked and heat shocked cells in comparison to the wild-type strain. Fig. 2 shows a typical SDS-PAGE autoradiogram of [35S]methionine-labelled protein extracted from the erg1-disruptant cells (lanes 1–6) and wild-type cells (lanes 7 and 8). Both strains exhibited increased synthesis of typical heat shock inducible proteins designated as hsp104, hsp90 and hsp70. Other heat shock inducible proteins were also observed (Fig. 2, indicated by arrows) corresponding to approximately 60, 50, 48, 44 and 38 kDa. There were no obvious differences in protein profiles of the mutant attributable to sterol enrichment (Fig. 2, lanes 1–6) and that between the erg1-disruptant and wild-type cells (lanes 7 and 8).

image

Figure 2. SDS-polyacrylamide gel autoradiogram of [35S]methionine-labelled protein extracts from FY14 and X2180-1B. Lanes 1, 3 and 5 represent 25°C non-heat shocked samples while lanes 2, 4 and 6 represent 37°C heat shocked samples. The erg1-disruptant cells (FY14) were supplemented with 1 (lanes 1 and 2), 10 (lanes 3 and 4) and 20 (lanes 5 and 6) μg ml−1 of ergosterol. Lanes 7 and 8 represent 25°C non-heat shocked samples and 37°C heat shocked samples of the wild-type strain (X2180-1B), respectively. Arrows indicate new or increased protein bands in heat shocked samples compared with non-heat shocked samples. Molecular mass standards (kDa) are as indicated.

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Densitometric analyses of protein gels (Fig. 2), using actin as an internal standard for non-heat shock inducible protein [18, 19], confirmed that heat shock proteins were substantially induced upon a heat shock. The proportional increases for hsp104 were 2.2-, 3.1- and 3.9-fold for cells grown in media supplemented with 1, 10 and 20 μg ml−1 of ergosterol respectively, and 2.8-fold for the wild-type. Similarly, hsp90 synthesis was induced 2.9-, 5.1- and 6.0-fold for cells grown in media supplemented with 1, 10 and 20 μg ml−1 of ergosterol respectively, and 4.5-fold for the wild-type.

Western blot analysis was carried out to confirm the presence of hsp104, as a number of key roles have been proposed for this hsp in stress tolerance [22]. Heat shock inducible expression (Fig. 3, lanes 2, 4, 6, 8) of hsp104 was higher than constitutive expression (Fig. 3, lanes 1, 3, 5, 7) in both the erg1-disruptant and wild-type cells. Similarly, antibodies to hsp70 and hsp90 were used to confirm the presence of these heat shock proteins in control (non-heat shocked) and heat shocked cells (results not shown). These observations confirmed the results of the SDS-PAGE protein analysis, which clearly showed heat shock induced synthesis of hsp104, hsp90 and hsp70.

image

Figure 3. Western blot analysis of hsp104 from FY14 and X2180-1B. Lanes 1, 3 and 5 represent 25°C non-heat shocked samples while lanes 2, 4 and 6 represent 37°C heat shocked samples. The erg1-disruptant cells (FY14) were supplemented with 1 (lanes 1 and 2), 10 (lanes 3 and 4) and 20 (lanes 5 and 6) μg ml−1 of ergosterol. Lanes 7 and 8 represent 25°C non-heat shocked samples and 37°C heat shocked samples of the wild-type strain (X2180-1B), respectively.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Both heat and ethanol stresses have been reported to exert considerable adverse effects on membrane associated processes (reviewed in [23]). Observations from the present study indicating a relationship between increased ergosterol content and enhanced resistance to these stressors are presumably related to the membrane stabilising effects of ergosterol. In this respect, ergosterol has been shown to be involved in the stability of the yeast plasma membrane as determined by the increased osmotic stability of sterol-enriched sphaeroplasts [24]. Early studies by Thomas et al. [7] reported that cells enriched with ergosterol were significantly more resistant to ethanol than cells enriched with cholesterol. These authors attributed ethanol resistance to a more effective membrane barrier formed against the entry of ethanol in cells enriched with a sterol containing an unsaturated side chain (ergosterol) rather than one with a saturated side chain (cholesterol). Evidence from previous studies therefore implies that ergosterol may be important for stabilising both membrane lipids and proteins against the detrimental effects of ethanol. Moreover, the fact that alterations in ergosterol levels did not modulate the fatty acid composition in the erg1-disruptant demonstrates the specific effect of ergosterol on yeast stress tolerance in the present study.

It is well established in yeast that ethanol and heat induce the synthesis of hsps [25] and the accumulation of trehalose [3]. The present study was aimed at establishing a role for trehalose and hsps in the stress response of a yeast sterol auxotroph. The induction of thermotolerance and the associated increase in percentage survivors (Fig. 1B), together with the induction of trehalose and synthesis of hsps (Fig. 2) upon heat shock, indicated that the stress response was not compromised in the erg1-disruptant strain. However, while our data clearly demonstrated that hsps were synthesised and trehalose accumulated upon heat shock, the contribution made by these biomolecules to thermotolerance was less certain. Accumulation of trehalose mirrored the ergosterol content (Table 1) in the erg1-disruptant, but failed to correspond with the viability data (Fig. 1A,B). Intrinsic and heat shock induced levels of trehalose were similar in the wild-type and erg1-disruptant cells containing different concentrations of ergosterol. However, intrinsic ethanol tolerance and thermotolerance were different in terms of maintenance of tolerance over the 60-min time course (Fig. 1). These results suggest that trehalose levels are not correlated with stress tolerance. Previous results from this [26, 27] and other [28] laboratories are in accord with the present results.

The present study also reports inconsistencies concerning the role of hsps in thermotolerance. The erg1-disruptant cells grown in media supplemented with low levels (1 μg ml−1) of ergosterol showed a typical heat shock response with substantial increase in synthesis of hsp104, hsp90 and hsp70 following a heat shock at 37°C. However, tolerance to heat (Fig. 1A,B) and ethanol (Fig. 1C) was consistently lower in the erg1-disruptant cells supplemented with low as compared to high levels of ergosterol.

In summary, the current study demonstrated that while the stress response was not compromised in the erg1-disruptant cells as judged by the ability of cells to synthesise trehalose and hsps under heat shock conditions, these parameters did not closely relate to stress tolerance. On the other hand, our data indicated a more prominent role for ergosterol, presumably as a stabiliser of membrane components, in heat and ethanol tolerance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This research was supported in part by internal research grants from the University of New England.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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