School of Biological Sciences, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK1
Author for correspondence: Brendan P. G. Curran. Tel: +44 207 775 3013. Fax: +44 208 983 0973. e-mail: B.Curran{at}qmw.ac.uk
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ABSTRACT |
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Keywords: Dioctyl phthalate, lipids, yeast, heat shock response
Abbreviations: HSE, heat shock element; HSR, heat shock response
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INTRODUCTION |
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Intriguingly, the plasma membrane has been implicated in mechanisms of cellular stress (reviewed by Vigh et al., 1998 ), and membrane fluidity varies with changes in environmental temperature in a wide variety of different organisms, including Saccharomyces cerevisiae (Hunter & Rose, 1972
; Cronan, 1978
; Okuyama et al., 1979
; Suutari et al., 1990
). Coote et al. (1991)
noted that the yeast plasma membrane becomes leaky as a consequence of heat stress, resulting in an increased permeability to extracellular protons. They suggested that this intracellular acidification acted as a trigger for inducing tolerance to an otherwise lethal temperature challenge. Furthermore, Coote et al. (1994)
used an ATPase mutant of Saccharomyces cerevisiae to demonstrate that this membrane-bound proton pump plays a key role in cellular thermotolerance. The activity of this enzyme has also been shown to affect both the level of expression of heat shock genes and the duration of time over which they are induced (Panaretou & Piper, 1990
) whereas in a separate study, minor intracellular acidification arising from ionophore treatment has been correlated with the expression of HSP70 in yeast (Weitzel et al., 1987
). Furthermore, Kamada et al. (1995)
provided evidence that thermal stress triggers the expression of the yeast PKC1 gene, by stretching the plasma membrane. Thus a number of disparate observations provide support for the view that cellular stress sensitivity is intimately associated with membrane structure and function. The direct manipulation of cellular fatty acids provides further evidence for this (Carratu et al., 1996
; Chatterjee et al., 1997
).
Carratu et al. (1996) found a decrease in the cellular heat shock sensitivity and an increase in unsaturated fatty acids in yeast cells overexpressing the ole1gene product. Previously published work from this laboratory (Chatterjee et al., 1997
) demonstrated an equally strong correlation between the heat shock sensitivity of yeast and the type of fatty acid present in the cells. Expression of ß-galactosidase from a heat shock reporter gene revealed that the temperature of maximum induction was 45 °C, 47 °C or 49 °C, depending on the type and percentage of unsaturated fatty acids present in cells after they had been grown on different lipid sources under anaerobic conditions. We also demonstrated that cells grown at 37 °C have 29% more unsaturated fatty acids and a 3 °C higher maximal HSR than cells grown at 25 °C; an alteration in lipid profile and HSR sensitivity that we propose provides a possible mechanism to explain the transient nature of the HSR.
Despite multiple reproducible repeats of experiments that revealed these differences, suddenly the profiles of ß-galactosidase induction from cells grown at 25 °C and 37 °C became indistinguishable from one another. Even more intriguingly, for no apparent reason the lipid profiles from the two sets of cells also became the same. Here, after many months of exhaustive analysis we provide an explanation for this abrupt change; an explanation that adds even further weight to the hypothesis that the HSR is intimately associated with membrane structure.
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METHODS |
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Overnight growth.
DBY747-HSE1 was grown to exponential phase (2x106 cells ml-1) in 8x60 ml aliquots in 250 ml conical flasks in a selection of yeast nitrogen base (YNB) media supplemented with histidine (20 mg l-1), leucine (30 mg l-1) and tryptophan (20 mg l-1). Cells were grown at 25 °C or acclimatized in a 37 °C shaking water bath for a period of 16 h (during which time cells were diluted with YNB selective medium to maintain exponential growth). Samples were analysed for temperature profiles of HSElacZ induction and/or lipid profiles at the specified time points.
Commercial YNB medium.
YNB (0·67%, w/v) without amino acids (Difco), in old or new plastic containers, containing 2·0% (w/v) glucose was used.
Home-made YNB.
Stock solutions of the compounds detailed below were made up and autoclaved, or where specified, filter-sterilized. Stocks were stored at room temperature except where stated otherwise. Concentrations stated represent the final concentration in 1 l medium. Monobasic potassium phosphate (850 mg l-1) and dibasic potassium phosphate (150 mg l-1) were made up to a stock concentration of 100xstated specification, and calcium chloride (100 mg l-1) was made up to a stock concentration of 1000xstated specification. Ammonium sulphate (5 g l-1), sodium chloride (100 mg l-1) and magnesium sulphate (500 mg l-1) were each made up to a stock concentration of 50xstated specification. L-Methionine (20 mg l-1) was filter-sterilized. Dioctyl phthalate (serially diluted in ethanol) was added to the final concentration indicated, where appropriate.
The following vitamins were made up in a stock solution of 1000xstated specification, filter-sterilized and stored in aliquots of 1 ml at -20 °C: biotin (20 µg l-1), calcium pantothenate (2 mg l-1), inositol (10 mg l-1), pyridoxine hydrochloride (400 µg l-1); thiamin hydrochloride (400 µg l-1), folic acid (2 µg l-1), p-aminobenzoic acid (200 µg l-1), niacin (400 µg l-1) and riboflavin (200 µg l-1).
The following trace elements were made up in a stock solution of 1000xstated specification, filter-sterilized and stored in aliquots of 1 ml at -20 °C: boric acid (500 µg l-1), copper sulphate (40 µg l-1), zinc sulphate (400 µg l-1), potassium iodide (100 µg l-1), manganese sulphate (400 µg l-1) and sodium molybdate (200 µg l-1). Ferric chloride (200 µg l-1) was made up as a stock solution of 500xstated specification, filter-sterilized and stored in aliquots of 1 ml at -20 °C
Determination of temperature profiles of HSElacZ.
Aliquots (10 ml) of exponentially growing cells were added to flasks containing 20 ml liquid YNB medium or home-made YNB as specified, pre-heated to the stated temperature (in 250 ml conical flasks) in shaking water baths and subjected to a 10 min heat shock. The flasks were then placed in a 25 °C shaking water bath for a further 50 min to allow ß-galactosidase expression from the induced transcripts.
Measurement of ß-galactosidase activity from total cell extracts.
ß-Galactosidase activity was measured from a total cell extract as previously described (Chatterjee et al., 1997 ). One representative set of experimental results (from at least three replicates) is presented for each temperature profile of HSElacZ expression. The absolute level of ß-galactosidase varied between experiments but these profiles were reproduced in repeated separate experiments.
Determination of the cellular lipid profile.
Lipids were extracted and analysed using a modification of the method described by Hossack & Rose (1976) as previously described (Chatterjee et al.,1997
). The mean value and range from at least two separate experiments are presented in each figure.
HPLC.
Comparative HPLC was carried out on an LKB system using a 2150 hplc pump and a LKB 2151 variable-wavelength monitor. The system was de-gassed with helium before a 10 µl sample was loaded on a Li-Chromosorb column RP-18 (7 µm particle size) and run on 35% acetonitryl/65% water (v/v) at a flow rate of 1 ml min-1. Preparative HPLC was carried out under the same conditions except that repeated 10 µl aliquots of a 10-fold concentrated YNB solution were added to the column until 0·5 ml of the 22 min peak had been collected.
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RESULTS |
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Dioctyl phthalate increases the percentage of unsaturated fatty acids and decreases the cellular HSR sensitivity in yeast cells grown at 25 °C
Within limits, the percentage of unsaturated fatty acids present in the cells was a reflection of the concentration of dioctyl phthalate present in the medium (data not shown). The percentage of unsaturated fatty acids found in cells that had been exposed to concentrations of dioctyl phthalate up to and including 18 µM was indistinguishable from the 41% found in cells grown in the complete absence of this compound. However, there was a 21% increase in the percentage of unsaturated fatty acids in cells that had been exposed to 36 µM dioctyl phthalate and an additional 15% increase, to 77%, in cells exposed to 72 µM dioctyl phthalate. A further doubling of dioctyl phthalate to 144 µM failed to increase the percentage of unsaturated fatty acids above 77%. The growth rates of these cultures were indistinguishable from one another but there was a significant decrease in the heat shock sensitivity of cells grown in the presence of dioctyl phthalate. The HSR profile of cells grown in the presence of 36 µM dioctyl phthalate, the minimum level found to induce an increase in unsaturated fatty acids, is presented in (Fig. 4).
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DISCUSSION |
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The first indication that the aberrant lipid and HSR profiles were associated with commercially available YNB medium was when cells grown in home-made YNB produced the same lipid and HSR profiles as cells grown in our earlier experiments. This can be seen in Fig. 2 where cells growing in new YNB had 71% unsaturated fatty acids whereas cells growing in home-made YNB only contained 44%. Some idea of the potency of the lipid modifying compound can be gleaned from the fact that cells grown in a dilution series (up to 1:600) of new YNB in the home-made variety still produced more unsaturated fatty acids than that found in the control cells (Fig. 2
). This suggested that the new medium contained rather than lacked a lipid-metabolism-modifying constituent and indeed, a comparative HPLC analysis revealed a novel elution peak at 22 min that was unique to the new YNB.
The results presented in Fig. 3 indicate that this fraction does indeed contain the mystery contaminant. Cells produced 73% unsaturated fatty acids when grown in 60 ml home-made YNB in the presence of 100 µl of the 22 min fraction from the new YNB, but only 42% unsaturated fatty acids when 100 µl from the equivalent peak of the old YNB eluate was added (Fig. 3
). Identified as dioctyl phthalate by mass spectroscopy, the results in Fig. 4
confirm the correlation between an increase in the level of unsaturated fatty acids in cells that have been grown in the presence of dioctyl phthalate and a decrease in their heat shock sensitivity. Cells grown at 25 °C in the absence of dioctyl phthalate contained 41% unsaturated fatty acids and responded maximally to heat shock at 40 °C, whereas cells grown in the presence of 36 µM dioctyl phthalate had 62% unsaturated fatty acids and maximal induction of the reporter gene at 43 °C (Fig. 4
).
The mechanism by which phthalate has this effect on yeast cell lipid metabolism is unknown. In mammalian cells however, phthalate is known to induce peroxisome proliferation (reviewed by Latruffe & Vamecq, 1997 ). Peroxisomes serve a major function in fatty acid oxidation (reviewed by Small et al., 1997
) and indeed Rottensteiner et al. (1996)
have shown that they are induced in Saccharomyces cerevisiae when oleic acid is supplied as the sole carbon source. It is unlikely however, that peroxisomes are induced under the growth conditions used here because peroxisomal structures and proteins are hardly detectable when glucose is present in the growth medium (Veenhuis et al., 1987
; Filipits et al., 1993
).
Phthalate is also known to increase the activity of protein kinase C (Bojes & Thurman, 1996 ) in mammalian cells. It is possible therefore, that phthalate could affect the equivalent protein in yeast affecting MAP kinase pathways such as the cascade found to be involved in cell-wall metabolism in Saccharomyces cerevisiae (Hunter & Plowman, 1997
) or indeed by exerting an effect on yeast cell-cycle control (reviewed by Livneh & Fishman, 1997
). It is not difficult to imagine subsequent knock-on effects occurring on lipid metabolism under such circumstances.
The details of how this molecule brings about these changes remains to be elucidated but given that a 1 h exposure to 36 µM dioctyl phthalate failed to induce ß-galactosidase expression from this heat shock reporter gene (data not shown), dioctyl phthalate treatment appears to confer a decrease in yeast HSR sensitivity without inducing the response. Moreover, the growth rate of the cells used in these experiments was the same regardless of whether they were grown in 0, 18, 36, 72 or 144 µM dioctyl phthalate, indicating that dioctyl phthalate does not significantly compromise overall cellular metabolism.
The results presented in this paper agree with earlier work from this laboratory that demonstrated that the percentage and type of fatty acid present in the cells determine the heat shock sensitivity of yeast cells (Chatterjee et al., 1997 ). It also agrees with the work of Carratu et al. (1996
), who decreased the HSR sensitivity of yeast cells grown at 25 °C by overexpressing the Ole1 gene product. Heat stress (Chatterjee et al., 1997
), overexpression of the Ole1 gene product (Carratu et al. 1996
), fatty acid supplements (Chatterjee et al., 1997
) and dioctyl phthalate (Fig. 4
) all increase the percentage of cellular unsaturated fatty acids and decrease the sensitivity of the cellular HSR system, yet only the first of these is associated with induction of the HSR pathway. This indicates that even in the absence of stress-induced proteins, an increase in the percentage of unsaturated fatty acids is sufficient to decrease the HSR sensitivity of yeast cells.
Phthalate contamination of YNB prevented us from reproducing our earlier results for many months. It is indeed a strange irony that rather than undermining the original observations, it eventually provided even further support for the counterintuitive hypothesis that the down-regulation of the yeast heat-sensing mechanism is intimately associated with an increase in the percentage of unsaturated fatty acids present in the cell.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Carratu, L., Franceschelli, S., Pardini, C. L., Kobayashi, G. S., Horvath, I., Vigh, L. & Maresca, B. (1996). Membrane lipid perturbation modifies the set point of the temperature of heat shock response in yeast. Proc Natl Acad Sci U S A 93, 3870-3875.
Chatterjee, M. T., Khalawan, S. A. & Curran, B. P. G. (1997). Alterations in cellular lipids may be responsible for the transient nature of the yeast heat shock response. Microbiology 143, 3063-3068.[Abstract]
Coote, P. J., Cole, M. C. & Jones, M. V. (1991). Induction of increased thermotolerance in Saccharomyces cerevisiae may be triggered by a mechanism involving intracellular pH. J Gen Microbiol 137, 1701-1708.[Medline]
Coote, P. J., Jones, M. V., Seymour, I. J., Rowe, D. L., Ferdinando, D. P., McArthur, A. J. & Cole, M. B. (1994). Activity of plasma membrane H+-ATPase is a key physiological determinant of thermotolerance in Saccharomyces cerevisiae. Microbiology 140, 1881-1890.[Abstract]
Cronan, J. E. (1978). Molecular biology of bacterial membrane lipids. Annu Rev Biochem 47, 163-189.[Medline]
Filipits, M., Simon, M. M., Rapatz, W., Hamilton, B. & Ruis, H. (1993). A Saccharomyces cerevisiae upstream activating sequence mediates induction of peroxisome proliferation by fatty acids. Gene 132, 49-55.[Medline]
Hossack, J. A. & Rose, A. H. (1976). Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols. J Bacteriol 127, 67-75.[Medline]
Hunter, T. & Plowman, G. D. (1997). The protein kinases of budding yeast: six score and more. Trends Biochem Sci 22, 18-21.[Medline]
Hunter, K. & Rose, A. H. (1972). Lipid composition of Saccharomyces cerevisiae as influenced by growth temperature. Biochim Biophys Acta 260, 639-653.[Medline]
Kamada, Y., Jung, U. S., Piotrowski, J. & Levin, D. E. (1995). The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev 9, 1559-1571.[Abstract]
Kondo, K. & Inouye, M. (1991). TIP1, a cold shock-inducible gene of Saccharomyces cerevisiae. J Biol Chem 266, 17537-17544.
Latruffe, N. & Vamecq, J. (1997). Peroxisome proliferators and peroxisome proliferator activated receptors (PPARs) as regulators of lipid metabolism. Biochimie 79, 81-94.[Medline]
Livneh, E. & Fishman, D. D. (1997). Linking protein kinase C to cell-cycle control. Eur J Biochem 248, 1-9.[Abstract]
Mager, W. H. & De Kruijff, A. J. J. (1995). Stress-induced transcriptional activation. Microbiol Rev 59, 506-531.[Abstract]
Miller, M. J., Xuong, N. & Geiduschek, E. P. (1979). A response of protein synthesis to temperature shift in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 76, 5222-5225.[Abstract]
Okuyama, H., Saito, M., Joshi, V. C., Gunsberg, S. & Wakil, S. L. (1979). Regulation by temperature of the chain length of fatty acids in yeast. J Biol Chem 254, 12281-12284.[Medline]
Panaretou, B. & Piper, P. W. (1990). Plasma-membrane ATPase action affects several stress tolerances of Saccharomyces cerevisiae and Schizosaccharomyces pombe as well as the extent and duration of the heat shock response. J Gen Microbiol 136, 1763-1770.
Panaretou, B. & Piper, P. W. (1992). The plasma membrane of yeast acquires a novel heat shock protein (Hsp30) and displays a decline in proton-pumping ATPase levels in response to both heat shock and the entry to stationary phase. Eur J Biochem 206, 635-640.[Abstract]
Parsell, D. A. & Lindquist, S. (1993). The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27, 437-496.[Medline]
Rottensteiner, H., Kal, A. J., Filipits, M., Binder, M., Hamilton, B., Tabak, H. F. & Ruis, H. (1996). Pip2p: a transcriptional regulator of peroxisome proliferation in the yeast Saccharomyces cerevisiae. EMBO J 15, 2924-2934.[Abstract]
Slater, M. R. & Craig, E. A. (1987). Transcriptional regulation of an Hsp70 heat shock gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7, 1906-1916.[Medline]
Small, G. M., Karpichev, I. V. & Luo, Y. (1997). Regulation of peroxisomal fatty acyl-CoA oxidase in the yeast, Saccharomyces cerevisiae. Adv Exp Med Biol 422, 157-166.[Medline]
Sorger, P. K. (1990). Yeast heat shock factor contains separable transient and sustained response transcriptional activators. Cell 62, 793-805.[Medline]
Sorger, P. K. (1991). Heat shock factor and the heat shock response. Cell 65, 363-366.[Medline]
Sorger, P. K. & Pelham, H. R. B. (1987). Purification and characterization of a heat shock element binding protein from yeast. EMBO J 6, 3035-3041.[Abstract]
Sorger, P. K., Lewis, M. J. & Pelham, H. R. B. (1987). Heat shock factor is regulated differently in yeast and HeLa cells. Nature 329, 81-84.[Medline]
Suutari, M., Liukkonen, K. & Laakso, S. (1990). Temperature adaptation in yeasts: the role of fatty acids. J Gen Microbiol 136, 1469-1474.[Medline]
Veenhuis, M., Mateblowski, M., Kunau, W. H. & Harder, W. (1987). Proliferation of microbodies in Saccharomyces cerevisiae. Yeast 3, 77-84.[Medline]
Vigh, L., Maresca, B. & Harwood, J. L. (1998). Does the membranes physical state control the expression of heat shock and other genes? Trends Biochem Sci 10, 369-374.
Weitzel, G., Pilatus, U. & Rensing, L. (1987). The cytoplasmic pH, ATP content and total protein synthesis rate during heat shock inducing treatments in yeast. Exp Cell Res 170, 64-79.[Medline]
Received 11 April 2000;
revised 25 May 2000;
accepted 9 June 2000.
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