The heat inducibility of the yeast heat-shock response (HSR) pathway has been shown to be critically dependent on the level of unsaturated fatty acids present in the cell. Here the inducibility by heat or salt of the independently regulated general stress response (GSR) pathway is shown to be affected in the same way. An increase in the percentage of unsaturated fatty acids in heat-or salt-acclimated cells correlated with a decrease in the induction of a general stress-response-promoter-element (STRE)-driven reporter gene by either stress. Despite inducing reporter gene expression, sorbic acid treatment did not confer salt cross-tolerance on the cells. This failure correlated with a failure to increase the percentage of unsaturated fatty acids in the cells, suggesting that GSR pathway induction, in the absence of lipid changes, is insufficient for the induction of cross-tolerance. Cells grown with fatty acid supplements under anaerobic conditions provided further evidence for a potential role for lipids in the acquisition of stress resistance. These cells contained different fatty acid profiles depending on the fatty acid supplement supplied, exhibited differential sensitivity to both heat and salt stress, but had not undergone STRE induction. These results suggest that heat-and salt-stress induction of the GSR are sensitive to the level of unsaturated fatty acids present in the cell and that stress cross-tolerance may be a lipid-mediated phenomenon. Given that an increased level of unsaturated fatty acids also down-regulates heat induction of the HSR pathway, these observations lead to the provocative hypothesis that lipid modifications, rather than HSR or GSR pathway induction, are a major contributor to the induced heat and salt tolerance of yeast cells.
The stress-sensing systems leading to the cellular heat shock response (HSR) and the mechanism responsible for the desensitizing of this response in stressacclimated cells are largely unknown. Here it is demonstrated that there is a close correlation between a 3 O C increase in the temperature required for maximal activation of a heat-shock (HS)-inducible gene in Saccharomyces cerevisiae and an increase in the percentage of cellular unsaturated fatty acids when cells are subjected to extended periods of growth at 37 O C . The latter occurs with the same kinetics as HS gene down-regulation during a prolonged HS and is reversed by reacclimation to growth at 25 OC. The transient nature of the HS may therefore be due to a lipid-mediated decrease in cellular heat sensitivity. Further evidence that unsaturated fatty acids desensitize cells to heat, with a resultant down-regulation of the HSR, is provided by demonstrating a 9 O C increase in the temperature required for maximal induction of this HS-inducible gene in cells containing high levels of unsaturated fatty acids assimilated during anaerobic growth at 25 OC.
Moderate levels of reactive oxygen species (ROS) have been implicated as second messengers in a number of biochemical pathways, and in animal cells have been associated with the activation of the heat shock response (HSR). Here, using an intracellular probe, we demonstrate that differential accumulation of ROS in the yeast Saccharomyces cerevisiae is strongly associated with differential induction of an HS reporter gene over a range of heat shock temperatures. There was a good correlation between cellular ROS levels and the levels of HS-induced reporter gene expression between 37• C and 44 • C, both reaching maximal values at 41 • C. Furthermore, the addition of 150 µM H 2 O 2 to the yeast cells during heat treatment resulted in a 3• C decrease in the temperature required for maximal induction of the HS expression vector -an increased HS sensitivity that corresponded to a concomitant increase in ROS levels at these lower HS temperatures. Conversely, cells treated with 10 mM of the antioxidant ascorbic acid required a temperature that was 2• C above that required in untreated controls for maximal induction of the HS expression vector. This decreased HS sensitivity corresponded to a decrease in ROS levels at these higher HS temperatures. Finally, cell viability assays reveal that intrinsic thermotolerance remains high in control cells despite concomitant decreases in HS-reporter gene expression and ROS accumulation between 41• C and 44 • C. We conclude that the sensitivity of the yeast HSR is strongly associated with ROS accumulation, and suggest that ROS-mediated signalling ensures cooperation between the HS and the antioxidant responses.
The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock, When S . cerevisiae growing exponentially on glucose media was shifted from 25 "C to 38 "C transient increases of 6 -7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rnal .I mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress.Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 "C to 38 "C) or severe (25 "C to 42 "C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heatshock and translated efficiently at supra-optimal temperatures.Upon exposure to a heat shock the cells of virtually all species display alterations in their physiology and protein synthesis that together constitute the 'heat-shock response' (reviewed in [l -31). While this response involves primarily an altered control over gene transcription, it also operates in eukaryotes partly through effects on the translocation of mRNAs from the nucleus or the stabilisation and selective translation of mRNAs in the cytoplasm [2, 31. The transcriptional changes involve stimulation of a small number of heat-shock protein (HSP) genes and the simultaneous repression of many, if not most, of the genes that were previously active. Of the 17 proteins whose synthesis is enhanced when Escherichia coli cells are heat-shocked, a few have been assigned either an enzymatic function (e.g. the 1ysU and lon gene products) or a role in the control of enzyme activities (e. g. rpoD which encodes the regulatory sigma subunit of E. coli RNA polymerase) [I]. All 17 genes are activated during heat-shock by the product of the htpR locus, a protein which displays both a striking structural similarity to sigma and the apparent ability to antagonize the activity of sigma [l].Correspondence to P. W. Piper, Department of Biochemistry, University College London, Gower Street, London, England WC1 E 6BTAbbreviations and nomenclature. The nomenclature used for yeast heat-shock ...
The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was inserted on a high copy plasmid into cells having a TRP1 gene disruption of the solitary chromosomal PGK locus. This enabled PGK transcripts of plasmid and chromosomal origin to be distinguished by virtue of their different sizes. Certain deletions lacking UASPGK displayed activities that were very low in unstressed cells, but which increased fifty to one-hundred fold after heat shock. With UASPGK present heat shock had only a relatively small or negligible effect on PGK mRNA levels. Heat shock activation was abolished when the -256 to -377 region with homology to the heat shock element consensus of eukaryotes was deleted in addition to UASPGK, but was unaffected by the deletion of regions further downstream containing TATA- and CAAT- sequence motifs. This is the first demonstration of a heat shock element, an activator site normally found upstream of eukaryotic heat shock protein genes, as a natural constituent of a high efficiency glycolytic promoter. It is proposed that PGK may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock.
The formation of cross-links between bovine serum albumin and DNA in the presence of chromium(III) chloride was found to be highly pH dependent. In vitro, such lesions were only formed at acidic values of pH, but were not detected at neutral pH. Complexes of chromium(III) and GSH/GSSG similarly failed to induce DNA-protein cross-links at physiological values of pH. Our findings indicate that the cross-links generated in vitro at acidic pH may not be directly relevant to the observed formation of such lesions in cultured cells and that a physiologically relevant in vitro model for the efficient cross-linking of proteins to DNA has yet to be devised.
When Mat a cells are treated with alpha-factor prior to being protoplasted and fused, the frequency of karyogamy is higher than in unarrested controls.
Heat shock enhances the very high level of transcription of the phosphoglycerate kinase (PGK) gene in fermentative cultures of Saccharomyces cerevisiae. This response of PGK mRNA levels was not found on gluconeogenic carbon sources, and could be switched on or off subject to availability of fermentable carbon source. The addition of glucose to yeast growing on glycerol resulted in acquisition, within 30-60 min, of the ability to elevate PGK mRNA levels after heat shock. In addition, in aerobic cultures growing on glucose the exhaustion of the medium glucose coincided with a loss of the heat-shock effect on PGK mRNA and a switch-over to slower growth by aerobic respiration. Levels of hsp26 mRNA were analysed during these experiments. Contrasting with this requirement for fermentable catabolite for manifestation of a heat-shock response of PGK mRNA levels, the PGK enzyme was not synthesized at a greater level in heat-shocked fermentative than in gluconeogenic cultures. PGK is one of only a few proteins made efficiently after mild heat shock of yeast. Thus, heat-stress-induced elevation of PGK mRNA levels does not appreciably increase PGK synthesis during exposure to high temperatures and so its role may be to assist cells repressed in mitochondrial function during recovery following a heat shock.
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