Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis.

Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C. A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning. Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment. The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain. Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent. These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.     

Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe

Yeast cells show an adaptive response to a mild heat shock, resulting in thermotolerance acquisition. This is accompanied by induction of heat-shock protein (hsp) synthesis and rapid accumulation of trehalose. Genetic approaches to determine the specific role of trehalose in heat-induced thermotolerance in Saccharomyces cerevisiae have been hampered by the finding that deletion of TPS1 , the gene encoding trehalose-6-phosphate synthase, causes a variety of pleiotropic effects, including inability to grow on glucose-containing media. Here, we have studied a tps1 mutant of the yeast Schizosaccharomyces pombe that reportedly has no such growth defects. We show that tps1 mutants have a serious defect in heat shock-induced acquisition of thermotolerance if conditioned at highly elevated temperatures (40–42.5°C), which, in wild-type cells, prevent hsp but not trehalose synthesis. In contrast, hsp synthesis appears to become particularly important under conditions in which trehalose synthesis is either absent (in tps1 mutant strains) or not fully induced (conditioning at moderately elevated temperatures, i.e. 35°C). In addition, pka1 mutants deficient in cAMP-dependent protein kinase were examined. Unconditioned pka1 cells had low levels of trehalose but a high basal level of thermotolerance. It was found that pka1 mutant cells, contrary to wild-type cells, accumulated large amounts of trehalose, even during a 50°C treatment. pka1 tps1 double mutants lacked this ability and showed reduced intrinsic thermotolerance, indicating a particularly important role for trehalose synthesis, which takes place during the challenging heat shock.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Acquisition of thermotolerance during development of Blastocladiella emersonii

In the fungus Blastocladiella emersonii the synthesis of heat-shock proteins is developmentally regulated; particular subsets of heat-shock proteins are induced by heat shock during sporulation, germination and growth and some heat shock-related proteins are spontaneously expressed during sporulation (Bonato et al., 1987, Eur. J. Biochem., in press). Nevertheless, acquisition of thermotolerance can be induced at any stage of the life cycle. The development of thermotolerance is correlated with the enhanced synthesis of some heat-shock proteins: hsp 82a, hsp 82b, hsp 76, hsp 70, hsp 60, hsp 25, hsp 17b. Other hsps are not specifically involved in thermotolerance.     

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation.

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.     

Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae.

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 degrees C. When the temperature was readjusted to 27 degrees 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 cells at 27 degrees C. In pulse-labeling experiments with [14C]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.     

Induction of the heat shock response and translational thermotolerance in day 15 ovine trophectoderm

The objectives of this study were to determine the ability of trophectoderm from preimplantation ovine embryos to synthesize hsp70 in response to heat shock and to identify conditions which induce translational thermotolerance in this tissue. Day 15 embryos were collected, and proteins synthesized in 1.5-mm sections of trophectoderm were radioactively labeled with (35)S-methionine. One-dimensional SDS-PAGE gels, two-dimensional gel electrophoresis and Western blots were utilized to characterize the heat shock response and to examine the induction of translational thermotolerance. Increased synthesis of the 70 kDa heat shock proteins and a protein with an approximate molecular weight of 15 to 20 kDa was observed with heat shock (> or = 42 degrees C). Total protein synthesis decreased (P < 0.05) with increased intensity of heat shock. At 45 degrees C, protein synthesis was suppressed with little or no synthesis of all proteins including hsp70. Recovery of protein synthesis following a severe heat shock (45 degrees C for 20 min) occurred faster (P < 0.05) in trophectoderm pretreated with a mild heat shock (42 degrees C for 30 min) than trophectoderm not pretreated with mild heat. In summary, trophoblastic tissue obtained from ovine embryos exhibit the characteristic "heatshock" response similar to that described for other mammalian systems. In addition, a sublethal heat shock induced the ability of the tissue to resume protein synthesis following severe heat stress. Since maintaining protein synthesis is crucial to embryonic survival, manipulation of the heat-shock response may provide a method to enhance embryonic survival.     

Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells.

In this study we have investigated the acquisition of thermotolerance in a Xenopus laevis kidney A6 epithelial cell line at both the level of cell survival and translation. In cell survival studies, A6 cells were incubated at temperatures ranging from 22 to 35 degrees degrees C for 2 h followed by a thermal challenge at 39 degrees degrees C for 2 h and a recovery period at 22 degrees C for 24 h. Optimal acquisition of thermotolerance occurred at 33 degrees degrees C. For example, exposure of A6 cells to 39 degrees degrees C for 2 h resulted in only 3.4% survival of the cells whereas prior exposure to 33 degrees C for 2 h enhanced the survival rate to 69%. This state of thermotolerance in A6 cells was detectable after 1 h at 33 degrees C and was maintained even after 18 h of incubation. Cycloheximide inhibited the acquisition of thermotolerance at 33 degrees C suggesting the requirement for ongoing protein synthesis. The optimal temperature for the acquisition of translational thermotolerance also occurred at 33 degrees C. Treatment of A6 cells at 39 degrees C for 2 h resulted in an inhibition of labeled amino acid incorporation into protein which recovered to approximately 14% of control after 19 h at 22 degrees C whereas cells treated at 33 degrees C for 2 h prior to the thermal challenge recovered to 58% of control levels. These translationally thermotolerant cells displayed relatively high levels of the heat shock proteins hsp30, hsp70, and hsp90 compared to pretreatment at 22, 28, 30, or 35 degrees C. These studies demonstrate that Xenopus A6 cells can acquire a state of thermotolerance and that it is correlated with the synthesis of heat shock proteins.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

The heat shock gene, htpG, and thermotolerance in the cyanobacterium, Synechocystis sp. PCC 6803

The htpG null mutant was obtained by inserting a chloramphenicol resistance cassette (Cm(r)) in the htpG coding sequence. The htpG null mutant (delta htpG), delta hsp16.6, and the double mutant, delta htpG::hsp16.6 cells showed little growth disadvantage at 30 degrees C and 37 degrees C, but not at 40 degrees C. This suggests that HtpG and HSP16.6 proteins do not have an essential role during growth at normal and mildly elevated temperatures. Cell growth, cell survival rate, and oxygen electrode measurements demonstrated that delta htpG, delta hsp16.6, and delta htpG::hsp16.6 cells were sensitive to heat stress. Decreased basal and acquired thermotolerance was observed when mutants were heat shocked, with delta htpG::hsp16.6 being the most sensitive. A comparison of mutants showed that delta hsp16.6 was more sensitive to heat shock than delta htpG.     

Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose.

The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined. The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids. Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins. Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different. However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type. Furthermore, trehalose accumulation was also not closely related to stress tolerance. On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol. We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.     

Comparative studies between the glucose-induced phosphorylation signal and the heat shock response in mutants of Saccharomyces cerevisiae

Addition of glucose to derepressed yeast cells, as well as a heat shock treatment, trigger the phosphorylation of trehalase and of trehalose-6-phosphate synthase. In the present paper, evidence is provided for the requirement of the RAS protein in the transduction of the glucose signal. On the other hand, a heat shock at 52 degrees C for 2 min was able to produce a significant phosphorylating effect even in mutant strains deficient in the GTP binding protein. Moreover, it was shown that this treatment does not affect exclusively the cAMP-dependent protein kinase. The use of a series of mutant strains confirmed that low levels of cAMP favor thermotolerance; the role of trehalose in yeast viability is also discussed.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Changes in hsp70 alter thermotolerance and heat-shock regulation in Drosophila.

To test the role of the heat shock protein hsp70 in induced thermotolerance and in the regulation of the heat-shock response, we established cell lines with altered expression of the Hsp70 gene. Underexpressing cells were created by transformation with antisense Hsp70 genes, and overexpressing cells by transformation with extra copies of the wild-type gene. Expression at normal temperatures was achieved by placing Hsp70 coding sequences under the control of the metallothionein promoter. Cells that expressed mutant hsp70s were created by transforming cells with deletion and frameshift mutations. The results indicate that hsp70 plays a major role in both thermotolerance and regulation. Surprisingly, they also indicate that these functions can be separated. Overexpression affected thermotolerance more than regulation; underexpression affected regulation more than thermotolerance. A carboxyl-terminal deletion of Hsp70 had a severe dominant-negative effect on thermotolerance but only a minor effect on regulation; an amino-terminal deletion strongly affected regulation but not thermotolerance. A model that explains these observations is presented.     

Time course and magnitude of synthesis of heat-shock proteins in congeneric marine snails (Genus tegula) from different tidal heights

The time course and magnitude of the heat-shock response in relation to severity of thermal stress are important, yet poorly understood, aspects of thermotolerance. We examined patterns of protein synthesis in congeneric marine snails (genus Tegula) that occur at different heights along the subtidal to intertidal gradient after a thermal exposure (30 degrees C for 2.5 h, followed by 50 h recovery at 13 degrees C) that induced the heat-shock response. We monitored the kinetics and magnitudes of protein synthesis by quantifying incorporation of 35S-labeled methionine and cysteine into newly synthesized proteins and observed synthesis of putative heat-shock proteins (hsp's) of size classes 90, 77, 70, and 38 kDa. In the low- to mid-intertidal species, Tegula funebralis, whose body temperature frequently exceeds 30 degrees C during emersion, synthesis of hsp's commenced immediately after heat stress, reached maximal levels 1-3 h into recovery, and returned to prestress levels by 6 h, except for hsp90 (14 h). In contrast, in the low-intertidal to subtidal species, Tegula brunnea, for which 2.5 h at 30 degrees C represents a near lethal heat stress, synthesis of hsp's commenced 2-14 h after heat stress; reached maximal levels after 15-30 h, which exceeded magnitudes of synthesis in T. funebralis; and returned to prestress levels in the case of hsp90 (50 h) and hsp77 (30 h) but not in the case of hsp70 and hsp38. Exposures to 30 degrees C under aerial (emersion) and aquatic (immersion) conditions resulted in differences in hsp synthesis in T. brunnea but not in T. funebralis. The different time courses and magnitudes of hsp synthesis in these congeners suggest that the vertical limits of their distributions may be set in part by thermal stress.     

Role of glutathione and hsp 70 in the acquisition of thermotolerance in postimplantation rat embryos

Studies were initiated to determine the extent to which reduced glutathione (GSH) may be involved in the capacity of cultured rat embryos to develop heat-induced tolerance to the deleterious effects of exposure to high temperatures (heat shock). Investigations of the modulation of dysmorphogenic responses of embryos to heat shock (43 degrees C, 30 min) as well as to the expression of the hsp70 gene and subsequent formation of hsps indicated that the acquisition of thermotolerance by rat embryos could be significantly influenced by the inhibition of GSH synthesis. Treatment of conceptuses with L-buthionine-S,R-sulfoximine (BSO) reduced intracellular GSH concentrations and compromised the capacity of embryos to mount a thermotolerance response as assessed by alterations in indices of growth and development. Embryonic thermotolerance elicited by preexposure to 42 degrees C for 30 min was accompanied by increases in GSH to levels greater than those measured in control embryos at 37 degrees C just prior to the subsequent 43 degrees C heat exposure. Expression of hsp70 mRNA was detectable soon after elevation of the temperature to 42 degrees C and reached its highest level of accumulation 1.5 hr after the 43 degrees C heat shock. BSO treatment had little if any effect on hsp70 message levels or on the synthesis of hsp70. The fact that BSO-treatment attenuated the thermotolerance response but did not produce a decrease in hsp70 RNA or the synthesis of hsp70 suggests that hsp70 alone is not sufficient to confer thermotolerance upon cultured rat embryos.     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.     

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.