Neutral trehalase Nth1p of Saccharomyces cerevisiae encoded by the NTH1 gene is a multiple stress responsive protein.

We have shown previously that expression of the NTH1 gene is increased at heat stress (40 degrees C) both at the mRNA and enzymatic activity levels. This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50 degrees C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region [S. Nwaka et al., FEBS Lett. 360 (1995) 286-290; S. Nwaka et al., J. Biol. Chem. 270 (1995) 10193-10198]. We show here that expression of the NTH1 gene and its product, neutral trehalase (Nthlp), are also induced by other stressors such as H2O2, CuSO4, NaAsO2, and cycloheximide (CHX). Heat-induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose. In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions. Our data suggest that: (1) heat- and chemical stress-induced expression of neutral trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat- and chemical stress-induced expression of NTH1 at the transcriptional level. Participation of neutral trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.

Metabolic regulation of the trehalose content of vegetative yeast.

We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast. When cells of S. cerevisiae M1 growing exponentially at 30 degrees C were shifted to 45 degrees C for 20 min, or to 39 degrees C for 40 min, the concentration of trehalose increased by about 25-fold; an effect reversed upon lowering the temperature to 30 degrees C. This was compared to the more than 50-fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase. Whereas the latter was paralleled by a 12-fold increase in the activity of trehalose-6-phosphate synthase, no significant change in the activities of trehalose-synthesizing and -degrading enzymes was measured under heat shock conditions. Accordingly, cycloheximide did not prevent the heat-induced accumulation of trehalose. However, the concentrations of the substrates for trehalose-6-phosphate synthase, i.e. glucose-6-phosphate and UDP-glucose, were found to rise during heat shock by about 5-10-fold. Since the elevated levels of both sugars are still well below the Km-values determined for trehalose-6-phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions. A similar increase in the steady-state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP. This suggests that temperature-dependent changes in the kinetic parameters of glycolytic enzymes vary in steady-state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis. Trehalose, glucose-6-phosphate, UDP-glucose, and ATP, were all found to increase during the 40 min heat treatment at 39 degrees C. Since this also occurs in a mutant lacking the heat shock-induced protein HSP104 (delta hsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions. However, mutant delta hsp104, in contrast to the parental wild-type, was sensitive towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not towards a 20 min incubation at 50 degrees C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S. cerevisiae from the damage caused by a 50 degrees C treatment.

Is thermotolerance of yeast dependent on trehalose accumulation?

During heat stress, trehalose concentration increases in yeast cells in parallel to thermotolerance. This parallelism suggested that trehalose mediated thermotolerance. We show in this work that, under certain conditions, trehalose accumulation and increase in thermotolerance do not go in parallel. A mutant deficient in the trehalose-degrading neutral trehalase shows, after shift from 40 degrees C to 30 degrees C, low thermotolerance in spite of a high trehalose concentration. When glucose is added to stationary yeast cells with high trehalose concentration and high thermotolerance, trehalose concentration decreases while thermotolerance remains high. A mutant deficient in ubiquitin-conjugating genes, ubc4ubc5, shows during exponential growth a low trehalose concentration, but a high thermotolerance, in contrast to wild-type cells. Because the ubc4ubc5 mutant synthesizes heat-shock proteins constitutively, it is proposed that, under these conditions, accumulation of heat-shock proteins, and not trehalose [corrected], mediates thermotolerance.

Assay of trehalose with acid trehalase purified from Saccharomyces cerevisiae.

An enzymatic end-point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre-existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre-existing glucose can be washed out with ice-cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95 degrees C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988, J. Biol. Chem. 263, 8537-8543; Mittenbühler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose-6-phosphate, glucose-1-phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.