The Induction of Saccharomyces cerevisiae Hsp104 Synthesis by Heat Shock Is Controlled by Mitochondria

Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104,the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria.     

The Effect of Sodium Azide on Basic and Induced Thermotolerance in Saccharomyces cerevisiae

The action mechanism of the mitochondrial inhibitor sodium azide on thermotolerance in Saccharomyces cerevisiae was studied. At ambient growth temperature, pretreatment with sodium azide was shown to improve the thermotolerance of parent cells and the hsp104 mutant. Treating with the inhibitor during a mild heat shock suppressed the development of induced thermotolerance due to the inhibition of heat shock protein (Hsp104) synthesis. Treating with the inhibitor immediately before lethal heat shock produced a variety of effects on thermotolerance depending on whether the yeast metabolism was oxidative or fermentative. The conclusions are: (1) the protective effect of sodium azide on the thermotolerance of S. cerevisiae cells grown on glucose-containing medium is not related to Hsp104 functioning, and (2) the mechanisms of basic and induced thermotolerance differ considerably.     

Induction of synthesis of Hsp104 of Saccharomyces cerevisiae in heat shock is controlled by mitochondria

Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104, the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria.     

Effect of amiodarone on thermotolerance and Hsp104p synthesis in the yeast Saccharomyces cerevisiae

Amiodarone (AMD) is known to induce a transient increase in cytosolic Ca²⁺ level in cells of the yeast Saccharomyces cerevisiae. In the present study the effect of AMD on the thermotolerance and Hsp104p synthesis of the yeast was studied. AMD induced Hsp104p synthesis and increased survival of the yeast after a severe heat shock (50°C). The development of thermotolerance to a considerable extent depended on the presence of Hsp104p. The same effect was achieved by treatment with the classical uncoupler CCCP, which is also known to increase the cytosolic Ca²⁺ level. It is supposed that the change in intracellular Ca²⁺ concentration plays an important role in activation of the HSP104 gene expression and in increasing the thermotolerance of the yeast. The possible link between mitochondrial activity and calcium homeostasis is discussed.     

The molecular chaperone Hsp78 confers compartment-specific thermotolerance to mitochondria

Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

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.     

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.     

Plant Hsp100 family with special reference to rice

Heat shock proteins (Hsps) represent a group of specific proteins which are synthesized primarily in response to heat shock in almost all biological systems. Members of Hsp100 family have been directly implicated in induction of thermotolerance in microbial and animal cells. Yeast cells harbouring defectivehsp104 gene do not show thermotolerance under conditions in which the normal cells do. Several plant species have been shown to synthesize Hsps in the range of 100 kDa. Rice Hsp104 (OsHsp104) is rapidly and predominantly accumulated in heat-shocked cells. Western blotting analysis show that anti rice Hsp104 antibodies (generated against purified Hsp104 protein from cultivated riceOryza sativa L.) cross-react with the same-sized high temperature inducible protein in 15 different wild rices. It was further found that anti rice Hsp104 antibodies also cross-react with a major high temperature regulated protein ofEscherichia coli. We have previously shown that a 110 kDa stress regulated protein in rice (OsHsp110) is immunologically related to yeast Hsp104 protein. In this paper, we present a comparative account of characteristics of the OsHsp104 and OsHsp110 proteins.     

Mechanism of Saccharomyces cerevisiae yeast cell death induced by heat shock. Effect of cycloheximide on thermotolerance

The mechanism of yeast cell death induced by heat shock was found to be dependent on the intensity of heat exposure. Moderate (45°C) heat shock strongly increased the generation of reactive oxygen species (ROS) and cell death. Pretreatment with cycloheximide (at 30°C) suppressed cell death, but produced no effect on ROS production. The protective effect was absent if cycloheximide was added immediately before heat exposure and the cells were incubated with the drug during the heat treatment and recovery period. The rate of ROS production and protective effect of cycloheximide on viability were significantly decreased in the case of severe (50°C) heat shock. Treatment with cycloheximide at 39°C inhibited the induction of Hsp104 synthesis and suppressed the development of induced thermotolerance to severe shock (50°C), but it had no effect on induced thermotolerance to moderate (45°C) heat shock. At the same time, Hsp104 effectively protected cells from death independently of the intensity of heat exposure. These data indicate that moderate heat shock induced programmed cell death in the yeast cells, and cycloheximide suppressed this process by inhibiting general synthesis of proteins.     

An Arabidopsis heat shock protein complements a thermotolerance defect in yeast.

The heat shock protein Hsp104 of the yeast Saccharomyces cerevisiae plays a key role in promoting survival at extreme temperatures. We found that when diverse higher plant species are exposed to high temperatures they accumulate proteins that are antigenically related to Hsp104. We isolated a cDNA corresponding to one of these proteins from Arabidopsis. The protein, AtHSP101, is 43% identical to yeast Hsp104. DNA gel blot analysis indicated that AtHSP101 is encoded by a single- or low-copy number gene. AtHsp101 mRNA was undetectable in the absence of stress but accumulated to high levels during exposure to high temperatures. When AtHSP101 was expressed in yeast, it complemented the thermotolerance defect caused by a deletion of the HSP104 gene. The ability of AtHSP101 to protect yeast from severe heat stress strongly suggests that this HSP plays an important role in thermotolerance in higher plants.     

Acquisition of heat shock tolerance by regulation of intracellular redox states

In the yeast Saccharomyces cerevisiae, a mild heat treatment strongly induces Hsp104p which provides acquisition of thermotolerance. The mechanism by which Hsp104p protects cells from the severe heat shock has not yet been completely elucidated. In this study, a pivotal role of Hsp104p as an efficient scavenger of the reactive oxygen species (ROS) is investigated. In our previous study, we reported that Hsp104p acted as a regulator in the mitochondrial respiration pathway. In this report, the recombinant wild-type and hypersensitive ras mutants (ira2Delta) with the extrachromosomal plasmids wild-type and mutant hsp104 genes were studied. The resulting strains successfully expressed both wild-type and mutant Hsp104p and showed the thermotolerance phenotype in the strain with the functional wild-type Hsp104p expressed. Upon treatment with H2O2 and menadione, the strains with the functional Hsp104p expressed showed higher survival rates than the other mutants, indicating the protective role of Hsp104p from the oxidative stress. Fluorescence measurement of the oxidation-dependent probe, 2',7'-dichlorofluoroscein diacetate (H2DCFDA), also indicated that Hsp104p significantly reduced the amount of ROS. Resistance to the oxidative stress was independent of the amount of the glutathione in the hyperactivated ras mutants. Taken all together, this study confirms that Hsp104p plays a crucial role in keeping cells from being damaged by the oxidative stress, thus acting as a modulator of the intracellular redox state.     

Changes in heat-shock protein synthesis and thermotolerance of Arabodopsis thaliana seedlings resulting from Hsp90 inhibition by geldanamycin

The influence of geldanamycin (GA), a specific inhibitor of heat-shock protein Hsp90, on the synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA under normal conditions induced synthesis of these stress proteins. Treatment of seeds with the Hsp90 inhibitor resulted in elevated constitutive levels of Hsp70 and Hsp90 in seedlings, as well as increased induction of their synthesis under heat shock. The GA effect increased with its concentration. Hsp up-regulation promoted thermotolerance of seedlings. The findings suggest autoregulation of heatshock protein synthesis and regulation of plant tolerance by Hsp90.     

Thermotolerance is independent of induction of the full spectrum of heat shock proteins and of cell cycle blockage in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock. We show here through the use of cell cycle inhibitors that blockage of yeast cells in the G1, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells. These G1- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37 degrees C. One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins. Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37 degrees C. Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells. These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.     

Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent substrate recognition by disrupting oligomerization and cause high temperature inactivation

Hsp104 is an important determinant of thermotolerance in yeast and is an unusual molecular chaperone that specializes in the remodeling of aggregated proteins. The structural requirements for Hsp104-substrate interactions remain unclear. Upon mild heat shock Hsp104 formed cytosolic foci in live cells that indicated co-localization of the chaperone with aggregates of thermally denatured proteins. We generated random amino acid substitutions in the C-terminal 199 amino acid residues of a GFP-Hsp104 fusion protein, and we used a visual screen to identify mutants that remained diffusely distributed immediately after heat shock. Multiple amino acid substitutions were required for loss of heat-inducible redistribution, and this correlated with complete loss of nucleotide-dependent oligomerization. Based on the multiply substituted proteins, several single amino acid substitutions were generated by site-directed mutagenesis. The singly substituted proteins retained the ability to oligomerize and detect substrates. Intriguingly, some derivatives of Hsp104 functioned well in prion propagation and multiple stress tolerance but failed to protect yeast from extreme thermal stress. We demonstrate that these proteins co-aggregate in the presence of other thermolabile proteins during heat treatment both in vitro and in vivo suggesting a novel mechanism for uncoupling the function of Hsp104 in acute severe heat shock from its functions at moderate temperatures.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Ssd1 is required for thermotolerance and Hsp104-mediated protein disaggregation in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Hsp104-mediated disaggregation of protein aggregates is essential for thermotolerance and to facilitate the maintenance of prions. In humans, protein aggregation is associated with neuronal death and dysfunction in many neurodegenerative diseases. Mechanisms of aggregation surveillance that regulate protein disaggregation are likely to play a major role in cell survival after acute stress. However, such mechanisms have not been studied. In a screen using the yeast gene deletion library for mutants unable to survive an aggregation-inducing heat stress, we find that SSD1 is required for Hsp104-mediated protein disaggregation. SSD1 is a polymorphic gene that plays a role in cellular integrity, longevity, and pathogenicity in yeast. Allelic variants of SSD1 regulate the level of thermotolerance and cell wall remodeling. We have shown that Ssd1 influences the ability of Hsp104 to hexamerize, to interact with the cochaperone Sti1, and to bind protein aggregates. These results provide a paradigm for linking Ssd1-mediated cellular integrity and Hsp104-mediated disaggregation to ensure the survival of cells with fewer aggregates.