The role of trehalose synthesis for the acquisition of thermotolerance in yeast. II. Physiological concentrations of trehalose increase the thermal stability of proteins in vitro.

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.

[1]  Gabriele H. Marchler,et al.  Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae. , 1991, The Journal of biological chemistry.

[2]  F. Hartl,et al.  Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria , 1989, Nature.

[3]  K. Watson,et al.  Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast , 1984, FEBS letters.

[4]  S. Lindquist,et al.  Hsp26 is not required for growth at high temperatures, nor for thermotolerance, spore development, or germination , 1986, Cell.

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  M. B. Cole,et al.  Induction of increased thermotolerance in Saccharomyces cerevisiae may be triggered by a mechanism involving intracellular pH. , 1991, Journal of general microbiology.

[7]  J. François,et al.  Effects of heat shock on the level of trehalose and glycogen, and on the induction of thermotolerance in Neurospora crassa , 1991, FEBS letters.

[8]  E. Martegani,et al.  Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae , 1993, Molecular microbiology.

[9]  A. Laszlo.  The thermoresistant state: protection from initial damage or better repair? , 1992, Experimental cell research.

[10]  S. Lindquist,et al.  Hsp104 is required for tolerance to many forms of stress. , 1992, The EMBO journal.

[11]  J. Delaney.  A cya deletion mutant of Escherichia coli develops thermotolerance but does not exhibit a heat-shock response. , 1990, Genetical research.

[12]  H. Bergmeyer.  Methoden der enzymatischen Analyse , 1962 .

[13]  T. Boller,et al.  A method to study the rapid phosphorylation‐related modulation of neutral trehalase activity by temperature shifts in yeast , 1991, FEBS letters.

[14]  R. Rudolph,et al.  Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of alpha-glucosidase from yeast. , 1991, Biochemistry.

[15]  P. Piper,et al.  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. , 1992, European journal of biochemistry.

[16]  J. Horwitz.  Alpha-crystallin can function as a molecular chaperone. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  P. Piper,et al.  Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp104 and in the absence of protein synthesis , 1991, FEBS letters.

[18]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[19]  D. Finkelstein,et al.  Identification and expression of a cloned yeast heat shock gene. , 1983, The Journal of biological chemistry.

[20]  N. Kalkkinen,et al.  Cloning of two related genes encoding the 56-kDa and 123-kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae. , 1993, European journal of biochemistry.

[21]  T. Boller,et al.  The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae. , 1992, European journal of biochemistry.

[22]  Y. Hiromi,et al.  Actin gene mutations in Drosophila; heat shock activation in the indirect flight muscles , 1985, The EMBO journal.

[23]  P. J. Stephens,et al.  Reduced ribosomal thermal denaturation in Listeria monocytogenes following osmotic and heat shocks. , 1993, FEMS microbiology letters.

[24]  C. Georgopoulos,et al.  Biological role and regulation of the universally conserved heat shock proteins. , 1991, The Journal of biological chemistry.

[25]  M. Yaffe,et al.  Uncoupling thermotolerance from the induction of heat shock proteins. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M J Schlesinger,et al.  Heat shock proteins. , 1990, The Journal of biological chemistry.

[27]  S M Kane,et al.  Carbohydrate Metabolism During Ascospore Development in Yeast , 1974, Journal of bacteriology.

[28]  S. H. Lillie,et al.  Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation , 1980, Journal of bacteriology.

[29]  J. Thevelein,et al.  The RAS-adenylate cyclase pathway and cell cycle control in , 1992 .

[30]  A. Papavassiliou,et al.  Renaturation of denatured λ repressor requires heat shock proteins , 1990, Cell.

[31]  J. Knabe.  Methoden der enzymatischen Analyse, 3. Auflage. Herausgeb. v. H. U. Bergmeyer, LXXXI, 2353 Seiten in 2 Bänden. Preis DM 460,–, Verlag Chemie, Weinheim/Bergstr., 1974 , 1974 .

[32]  C. Gross,et al.  Is hsp70 the cellular thermometer? , 1991, Trends in biochemical sciences.

[33]  L. Nover.  Heat shock response of eukaryotic cells , 1984 .

[34]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[35]  R. Rothstein.  One-step gene disruption in yeast. , 1983, Methods in enzymology.

[36]  T. Boller,et al.  Correlation of trenalose content and heat resistance in yeast mutants altered in the RAS/adenylate cyclase pathway: is trehalose a thermoprotectant? , 1989, FEBS letters.

[37]  P. Piper,et al.  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 , 1990 .

[38]  S. Lindquist,et al.  HSP104 required for induced thermotolerance. , 1990, Science.

[39]  J. Lepock,et al.  Thermal analysis of CHL V79 cells using differential scanning calorimetry: Implications for hyperthermic cell killing and the heat shock response , 1988, Journal of cellular physiology.

[40]  T. Boller,et al.  Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts , 1987, FEBS letters.

[41]  C. De Virgilio,et al.  Heat shock induces enzymes of trehalose metabolism, trehalose accumulation, and thermotolerance in Schizosaccharomyces pombe, even in the presence of cycloheximide , 1990, FEBS letters.

[42]  E. Gerner,et al.  Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts , 1986, Molecular and cellular biology.

[43]  G. C. Johnston,et al.  Thermotolerance is independent of induction of the full spectrum of heat shock proteins and of cell cycle blockage in the yeast Saccharomyces cerevisiae , 1990, Journal of bacteriology.

[44]  S. Lindquist,et al.  hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function , 1989, Molecular and cellular biology.

[45]  A. Goldberg,et al.  Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. , 1986, Science.

[46]  A. Panek,et al.  Role of the trehalose carrier in dehydration resistance of Saccharomyces cerevisiae. , 1993, Biochimica et biophysica acta.

[47]  H. Holzer,et al.  Molecular analysis of the neutral trehalase gene from Saccharomyces cerevisiae. , 1993, The Journal of biological chemistry.

[48]  J. Ellis.  Proteins as molecular chaperones , 1987, Nature.

[49]  P. van der Zee,et al.  Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation. , 1992, European journal of biochemistry.

[50]  M. Blázquez,et al.  The fdp1 and cif1 mutations are caused by different single nucleotide changes in the yeast CIF1 gene. , 1993, FEMS microbiology letters.

[51]  B. Wright,et al.  Trehalose synthesis during differentiation in Dictyostelium discoideum. 3. In vitro unmasking of trehalose 6-phosphate synthetase. , 1972, The Journal of biological chemistry.

[52]  J. Sambrook,et al.  Protein folding in the cell , 1992, Nature.

[53]  W. Boos,et al.  Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli , 1991, Journal of bacteriology.

[54]  F. Neidhardt,et al.  Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli. , 1987, Genes & development.

[55]  B. Hall.  Yeast thermotolerance does not require protein synthesis , 1983, Journal of bacteriology.

[56]  W. Dewey,et al.  Thermotolerance induced by heat, sodium arsenite, or puromycin: Its inhibition and differences between 43°C and 45°C , 1988, Journal of cellular physiology.

[57]  Raymond S. Norton,et al.  Organic Osmoregulatory Solutes in Cyanobacteria , 1984 .

[58]  A. Goldberg,et al.  Production of abnormal proteins in E. coli stimulates transcription of ion and other heat shock genes , 1985, Cell.

[59]  B. Wright,et al.  Trehalose synthesis during differentiation in Dictyostelium discoideum. Preparation, stabilization and assay of trehalose-6-phosphate synthetase. , 1975, Archives of biochemistry and biophysics.

[60]  P. Attfield.  Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response , 1987, FEBS letters.

[61]  R. W. Davis,et al.  Efficient isolation of genes by using antibody probes. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[62]  H. Taguchi,et al.  Chaperonin from Thermus thermophilus can protect several enzymes from irreversible heat denaturation by capturing denaturation intermediate. , 1993, The Journal of biological chemistry.

[63]  Elizabeth A. Craig,et al.  A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides , 1988, Nature.

[64]  W. Welch,et al.  Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. , 1990, Science.

[65]  P. Niederberger,et al.  Permeabilization of microorganisms by Triton X-100. , 1978, Analytical biochemistry.

[66]  T. Boller,et al.  The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant. , 1994, European journal of biochemistry.

[67]  E. Nudler,et al.  Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[68]  BARNETT ROSENBERG,et al.  Quantitative Evidence for Protein Denaturation as the Cause of Thermal Death , 1971, Nature.

[69]  T. Boller,et al.  Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity. , 1993, European journal of biochemistry.

[70]  S. Lindquist.  The heat-shock response. , 1986, Annual review of biochemistry.

[71]  A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle. , 1991, Molecular and cellular biology.

[72]  S. Lindquist,et al.  The heat-shock proteins. , 1988, Annual review of genetics.

[73]  Elizabeth A. Craig,et al.  Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins , 1990, Nature.

[74]  A. Strøm,et al.  A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant. , 1993, FEMS microbiology letters.

[75]  A. Wiemken,et al.  Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae , 1987, Journal of bacteriology.