In the yeast Saccharomyces cerevisiae, accumulation of the non-reducing disaccharide trehalose is triggered by various stimuli that activate the heat-schock response. Several studies have shown a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. In this study, we have examined mutants defective in genes coding for key enzymes involved in trehalose metabolism with respect to the heat-induced and stationary-phase-induced accumulation of trehalose and the acquisition of thermotolerance. Inactivation of either TPSl or TPS2, encoding subunits of the trehalose-6-phosphate synthase/phosphatase complex, caused an inability to accumulate trehalose upon a mild heat-shock or upon initiation of the stationary phase and significantly reduced the levels of heat-induced and stationary-phase-induced thermotolerance. Deletion of NTHZ, the gene coding for the neutral trehalase, resulted in a defect in trehalose mobilization during recovery from a heat shock which was paralleled by an abnormally slow decrease of thermotolerance. Our results provide strong genetic evidence that heat-induced synthesis of trehalose is an important factor for thermotolerance induction. In an accompanying study [Hottiger, T
Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae. In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40°C. When the temperature was readjusted to 27°C, the accumulated trehalose was rapidly degraded. In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature. Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the ceils at 27°C. In pulse-labeling experiments with [}4C]glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo. Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.The nonreducing disaccharide trehalose (a-D-glucopyranosyl-1,1-a*-D-glucopyranoside) is ubiquitously found in fungi, in which it is supposed to function as a reserve carbohydrate (24). In Saccharomyces cerevisiae, trehalose accumulates during periods of reduced growth, for example during starvation for nitrogen, phosphorus, or sulfur (13,19).Trehalose is also abundant in chemostat cultures maintained at low dilution rates (11,12) and in batch cultures during adaptation to new carbon sources or transition to the stationary phase (13,18). Under some of these conditions, trehalose can account for up to 23% of the dry weight of the cells (13).Trehalose biosynthesis in yeast proceeds in two steps. First, trehalose-6-phosphate (P)-synthase (UDP-glucose:Dglucose-6-P-1-glucosyltransferase, EC 2.4.1.15) condenses UDPG and glucose-6-P to yield trehalose-6-P. Second, a specific phosphatase (trehalose-6-P phosphohydrolase, EC 3.1.3.12) cleaves off phosphate from trehalose-6-P (3). Trehalose degradation is mediated by trehalase (trehalose 1-glucohydrolase, EC 3.2.1.28) (24). Two trehalases with different pH optima have been found in S. cerevisiae (15). One of them, neutral trehalase, has attracted much attention because its activity is regulated by cyclic AMP (cAMP)-dependent phosphorylation (14,(25)(26)(27). Current research has therefore focused on trehalose catabolism, whereas the study of anabolism has been neglected.It has been reported that yeast cells growing at 37°C continuously contain appreciable amounts of trehalose (7)
The trehalose content of exponentially growing Saccharonzyces cerevisiae cells rapidly increased in response to a temperature shift from 27 to 40°C and decreased again when the temperature was shifted back from 40 to 27°C. These changes were closely correlated with increases and decreases in the thermotolerance and desiccation tolerance of the cells. Our results support the hypothesis that trehalose functions as a protectant against heat and desiccation.
In baker's yeast (Saccharomyces cerevisiae), accumulation of the non‐reducing disaccharide, trehalose, is triggered by stimuli that activate the heat‐shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 179–186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose‐6P‐dehydrogenase, phosphoglucose‐isomerase) as well as enzymes of non‐yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat‐induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose‐6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.
Exponentially growing cells of the fission yeast, Schizosuccharontyees pombe, contained virtually no trehalose at 27°C but rapidly accumulated large quantities during heat shock at 40°C. Activities of trehalose-6-phosphate synthase and trehalase also increased upon heat shock. Thermotolerance of the cells, measured as survival at 52°C increased in parallel to trehalose accumulation and decreased in parallel to the trehalose levels when cells were shifted back to 27°C. Trehalose levels, activities of enzymes of trehalose metabolism and thermotolerance strongly increased upon heat shock even in the presence of cycloheximide, indicating that none of these effects requires protein synthesis. The data support the hypothesis that trehalose acts as a thermoprotectant in Schizosaccharomycespombe.
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