The Induction of Saccharomyces cerevisiae Hsp104 Synthesis by Heat Shock Is Controlled by Mitochondria (original) (raw)

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

E. G. Rikhvanov, E. I. Rachenko, N. N. Varakina, T. M. Rusaleva, G. B. Borovskiĭ, and V. K. Voinikov
Siberian Institute of Plant Physiology and Biochemistry, Siberian Division, Russian Academy of Sciences, Irkutsk, 664033 Russia; fax: (3952)51-07-54; e-mail: eugene@sifibr.irk.ru

Received March 14, 2003

Abstract

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.

INTRODUCTION

Changing the incubation temperature of Saccharomyces cerevisiae from 25 to 38∘C38^{\circ} \mathrm{C} suppresses synthesis of ordinary proteins and induces synthesis of 50 or more heat shock proteins (HSPs) [1]. There is evidence for the role of HSP with molecular mass of 104 kDa (Hsp104) in induced thermotolerance of S. cerevisiae [2]. According to these results, the dynamics of cell death in the wild type and mutant hsp104 was similar under drastic action of lethal temperature. However, an increase in the rate of Hsp104 synthesis after exposure to mild heat shock ensured a markedly higher level of the thermotolerance in wild-type cells than in cells of mutant hsp104 [2]. In a number of S. cerevisiae strains, the constitutive synthesis of Hsp104 is rather high; thus, differences in the thermotolerance of wild-type cells and mutant cells hsp104 may be observed without preliminary heat-shock-inducing treatments [3-5]. This agrees with the role of Hsp104 in the cell at physiological temperature. As shown in the example of cytoplasmic inheritance of the exrachromosomal genetic element [PSI∗]\left[P S I^{*}\right], the conformal state of yeast prions is modulated by Hsp104 [6, 7].

Yeast Saccharomyces cerevisiae can grow due either to respiration and fermentation depending on carbon source. Glucose is mainly utilized to ethanol by fermentation. This causes a repression of respiration, but no complete inhibition of respiratory activity was observed [8]. Incubation of S. cerevisiae on a medium with nonfermentable carbon sources leads to an increase in the constitutive level of Hsp104 and in the thermotolerance, compared to the medium with glucose [5, 9]. This fact suggests a connection between Hsp104 content in the cell and respiratory activity. In
this work, we compared the induction of Hsp104 synthesis and its functions essential to the maintenance of yeast thermotolerance in respiratory incompetent cells for establishing the dependence of gene HSP104 expression on yeast energy metabolism. For this, the mitochondrial inhibitor sodium azide and respirationdeficient mutants (petites) of the S. cerevisiae parent type and mutant hsp104 were used.

MATERIALS AND METHODS

The S. cerevisiae parent strain Ψ−74−D694\Psi-74-D 694 (MATa, ade1-14(UGA), trp1-289(UAG), his3 Δ−200\Delta-200, ura3-52, leu2-3, 112 [psi −]\left.{ }^{-}\right]) and the isogenic mutant hsp104 of 74-D694 hspΔ−1Lh s p \Delta-1 L were used. Yeast were grown on the YEPD medium of the following composition ( g/l\mathrm{g} / \mathrm{l} ): yeast extract, 5 ; peptone, 10 ; glucose, 20 . For solid media, agar (15 g/l)(15 \mathrm{~g} / \mathrm{l}) was added. In the course of this experiment, yeast were kept on the YEPD medium at 30∘C30^{\circ} \mathrm{C}.

Strain S. cerevisiae Ψ−74\Psi-74-D694 contains the UGA mutation ade1-14 that leads to auxotrophy for adenine and to the accumulation of red pigment on YEPD, whereas petite mutants form small white colonies because of respiration deficiency. For isolating petite mutants, a fresh yeast culture was inoculated to the YEPD medium with 20μ g/l20 \mu \mathrm{~g} / \mathrm{l} of ethidium bromide, an inducer of petites in S. cerevisiae [10]. After a 16-h incubation at 30∘C30^{\circ} \mathrm{C}, the cell suspension was plated on the solid YEPD medium. The majority of S. cerevisiae colonies of both the parent type and mutant hsp104 became white and did not grow on the medium with ethanol.

To examine thermotolerance, yeasts were grown in a thermostatic shaker in a liquid medium for 14−16 h14-16 \mathrm{~h} at

30∘C30^{\circ} \mathrm{C}. A definite volume of the overnight yeast culture was inoculated into fresh medium and incubated for 3 to 4 h until the logarithmic growth phase. Next, tubes containing 1 ml of the cell suspension each were incubated at 45 or 50∘C50^{\circ} \mathrm{C}. For analyzing the effect of sodium azide on basal thermotolerance, a sample of 0.15 mM NaN3\mathrm{NaN}_{3} was added to the cell suspension immediately before heat shock at 45∘C45^{\circ} \mathrm{C}. The effect of sodium azide on induced thermotolerance was examined in yeast cells that had previously been kept for 30 min at 37∘C37^{\circ} \mathrm{C} in the presence or absence of 0.15mMNaN30.15 \mathrm{mM} \mathrm{NaN}_{3}. Cells were then pelleted, washed three times with a fresh medium, resuspended in the initial volume of the nutrient medium, and incubated at 45∘C45^{\circ} \mathrm{C}. At the completion of exposure to heat shock, the cell suspension was cooled, appropriately diluted, and plated on the solid YEPD medium. We recorded the number of the generated colonies after incubation for 24−72 h24-72 \mathrm{~h} at 30∘C30^{\circ} \mathrm{C}. Yeast survival was calculated as the percentage ratio of colonies appearing after the definite period of exposure to heat shock to the number of colonies before heat shock.

To study the induction of Hsp104 synthesis, yeast were grown on the YEPD medium at 26 or 30∘C30^{\circ} \mathrm{C} and incubated for 30 min at 37 or 39∘C39^{\circ} \mathrm{C} in the presence or absence of 0.15mMNaN30.15 \mathrm{mM} \mathrm{NaN}_{3}. Cells were then pelleted, washed with K-Na-phosphate buffer ( pH 7.0 ), and kept for one day at −20∘C-20^{\circ} \mathrm{C} until the protein was extracted. To disrupt cell wall, yeast biomass was resuspended in the buffer for isolating the protein ( pH7.4−7.6\mathrm{pH} 7.4-7.6 ), frozen in liquid nitrogen, and ground with quartz powder. The protein fraction was purified from crude cell components by centrifugation ( 15000 g,10 min15000 \mathrm{~g}, 10 \mathrm{~min} ), and the protein was precipitated with 10%10 \% trichloroacetic acid. The pellet was washed with acetone and dissolved in the buffer ( pH 6.8 ). The concentration of protein was measured according to Lowry [11]. Proteins were separated by SDS electrophoresis in 10%10 \% PAAG and probed with anti-Hsp104 antibodies [12].

RESULTS

A comparative study of the thermotolerance of parent strains and petite mutants has previously been conducted repeatedly. However, these studies produced conflicting results. Some authors showed that respira-tion-deficient mutants are more sensitive to heat shock than respiration-competent strains [13], whereas the other authors obtained opposite results [14]. In our experiments, a comparison of thermotolerance in yeast strains demonstrated that after exposure to heat shock at 45∘C45^{\circ} \mathrm{C}, the survival of petite mutants was markedly greater than that of original strains (Fig. 1). Nevertheless, we observed a different situation at 50∘C50^{\circ} \mathrm{C} (Fig. 2). Cells of petite mutants became more resistant to heat shock than cells of respiratory competent parents only after the initial two minutes of exposure to heat shock. The difference between strain Ψ−74\Psi-74-D694 and its derivative respiration-deficient mutant disappeared 8 min
after the beginning of heat shock, and the survival of respiration-competent mutant hsp104h s p 104 was even slightly greater than that of the petite mutant originated from the former mutant (Fig. 2). Previously, we obtained identical results when comparing the thermotolerance of strain S. cerevisiae W303-1B and the isogenic petite mutant [15]. Apparently, results of comparative thermotolerance studies in petite mutants and original cultures may vary, depending on properties of the employed strains and on the intensity and duration of heat shock.

The survival of respiration-competent cells of strain Ψ−74\Psi-74-D694 (PT) after incubation at 45 and 50∘C50^{\circ} \mathrm{C} was markedly greater than that of mutant hsp104h s p 104 with functional mitochondria (Figs. 1 and 2). These differences in the basal thermotolerance between the parent strain and a strain defective for Hsp104 synthesis were also observed by other authors [3-5]. Hence, Hsp104 is present in the SS. cerevisiae strain employed by us at the temperature optimal for incubation and executes its functions essential to providing cell thermotolerance without additional induction of its synthesis via mild heat shock.

Despite respiratory deficiency, petite mutant with an intact gene HSP104 was far more tolerant to heat shock at 45 and 50∘C50^{\circ} \mathrm{C} than the petite mutant obtained on the basis of the strain with a deletion of HSP104 (Figs. 1b and 2). Obviously, a disturbance in the respiratory activity does not prevent Hsp104 from accomplishing its functions in the cell.

A comparative study of the constitutive synthesis of Hsp104 showed that the contents of this protein in a petite mutant and in the isogenic strain Ψ−74\Psi-74-D694 do not differ significantly at 26∘C26^{\circ} \mathrm{C} (Fig. 3a, 1 and 3). Identical results were obtained by other authors in measuring the amount of mRNA HSP104 in the parent strain and a petite mutant [14]. Increasing the temperature to 39∘C39^{\circ} \mathrm{C} caused substantial induction of Hsp104 synthesis in strain Ψ\Psi-74-D694 (Fig. 3, 4). An increase in Hsp104 content induced by heat shock was observed in the petite mutant as well (Fig. 3, 2), but the level of induction was lower than in a respiration-competent analog. Weitzel et al. [16] obtained similar results in comparative studies of induction levels of some HSP in petite mutant and the parent strain. These results suggested that the induction of Hsp104 synthesis during heat shock is partially controlled by mitochondria, although this protein is synthesized and functions in a respira-tion-deficient cell.

We conducted a new series of experiments on a disturbance in mitochondria functioning using sodium azide, an inhibitor of the cytochrome oxidase complex [17] that completely inhibited the respiration of SS. cerevisiae at a concentration of 0.15mM[18,19]0.15 \mathrm{mM}[18,19]. At this concentration, the effect of sodium azide depends on yeast energy metabolism, which suggests the association between the effect of this inhibitor on the thermotolerance and its ability to suppress respiratory activity.

The addition of sodium azide directly before heat shock (at 45∘C45^{\circ} \mathrm{C} ) significantly enhanced the thermotolerance of S. cerevisiae strain Ψ−74\Psi-74-D694 upon its growth on a medium with glucose, but exactly the opposite effect of the inhibitor on the thermotolerance was observed on a medium with galactose [19].

It is known that exposure to azide and heat shock induce the appearance of exactly the same puffs in Drosophila polytene chromosomes [20]. Nevertheless, the protective effect of NaN3\mathrm{NaN}_{3} on S. cerevisiae thermotolerance upon incubation on a medium with glucose can hardly be connected with the functioning of Hsp104, because addition of this agent increased cell survival of both the parent type and mutant hsp104 with approximately the same efficiency (Fig. 4). In this respect, the effect of sodium azide on the thermotolerance resembles the phenotypic expression of petite mutation, which, as NaN3\mathrm{NaN}_{3}, leads to markedly increased cell survival after heat shock at 45∘C45^{\circ} \mathrm{C} (Fig. 1).

Exposure to 37∘C37^{\circ} \mathrm{C} for 30 min induced significant increase in the thermotolerance of strain Ψ−74\Psi-74-D694 to the subsequent lethal effect of heat shock at 45∘C45^{\circ} \mathrm{C} (Fig. 4a). The induction of thermotolerance was observed in cells defective for Hsp104 biosynthesis, although to a lesser extent (Fig. 4b). Apparently, other proteins, such as Hsp70 [21], catalase [22], and enzymes of glutathione biosynthesis [23], whose synthesis is also induced during mild heat shock, protected cells of hsp104 mutant against heat-induced cell death. Despite the fact that addition of sodium azide increased basal thermotolerance of SS. cerevisiae, the presence of NaN3\mathrm{NaN}_{3} in a yeast suspension during the time of pretreatment at 37∘C37^{\circ} \mathrm{C}, led to noticeable inhibition of induced thermotolerance formation in both yeast strains (Fig. 4). Likewise, sodium azide and another inhibitor of the cytochrome oxidase complex (sodium cyanide) inhibited induced thermotolerance of S. cerevisiae strain W3031B (data not shown).

Since the formation of induced thermotolerance is largely determined by the induction of Hsp104 synthesis, the effect of sodium azide on the basal and inducible level of this protein was examined in strain S. cerevisiae Ψ−74\Psi-74-D694. Incubation for 30 min at 30∘C30^{\circ} \mathrm{C} in the presence of NaN3\mathrm{NaN}_{3} did not substantially change the amount of Hsp104 in yeast cells (Fig. 3b, 5 and 6). However, the addition of sodium azide partially inhibited an increase in Hsp104 amount after heat treatment at 37∘C37^{\circ} \mathrm{C} (Fig. 3b, 7 and 8). Note that we registered a similar decrease in heat induction of Hsp104 synthesis in a petite mutant (Fig. 3a, 2 and 4).

Sodium azide is a multifactor inhibitor, which may inhibit, apart from cytochrome oxidase, the activity of F1 F0\mathrm{F}_{1} \mathrm{~F}_{0}-ATP synthase in the direction of ATP hydrolysis [24]. Many chaperones, Hsp104 among them, possess ATPase activity, and a disturbance in this activity leads to the inactivation of their functions. Since NaN3\mathrm{NaN}_{3} is also an inhibitor of antioxidant enzymes, it cannot be ruled out that the observed effect of the inhibitor on induced
img-0.jpeg

Fig. 1. Thermotolerance of the parent type (PT) S. cerevisiae Ψ−74\Psi-74-D694, mutant hsp104, and isogenic petite mutants at 45∘C45^{\circ} \mathrm{C}. Yeasts were grown on the YEPD medium at 30∘C30^{\circ} \mathrm{C} and incubated at 45∘C45^{\circ} \mathrm{C} for 0−60 min(a)0-60 \mathrm{~min}(\mathrm{a}) or 0−120 min( b)0-120 \mathrm{~min}(\mathrm{~b}), and cells were then plated to a solid YEPD medium by a replicator and incubated at 30∘C30^{\circ} \mathrm{C} for 3 days. © Diagrammatic representation of data obtained: (1) PT; (2) hsp104 mutant; (3) PT petite; (4) petite hsp104 mutant. Typical data of one from three experiments, which each produce similar results, are presented.
thermotolerance is connected with the inhibition of defense mechanisms activated by a preliminary exposure to heat. To verify this assumption, we kept yeasts at 37∘C37^{\circ} \mathrm{C} for 30 min without inhibitor to allow induction of cell defense systems, and added sodium azide to the yeast suspension just before heat treatment at 45∘C45^{\circ} \mathrm{C}.

img-1.jpeg

Fig. 2. Thermotolerance of the parent type (PT) S. cerevisiae Ψ\Psi-74-D694, mutant hsp104, and isogenic petite mutants at 50∘C50^{\circ} \mathrm{C}. Yeasts were grown on the YEPD medium at 30∘C30^{\circ} \mathrm{C} and incubated at 50∘C50^{\circ} \mathrm{C} for 0−8 min0-8 \mathrm{~min} (a), and cells were then plated to a solid YEPD medium by a replicator and incubated at 30∘C30^{\circ} \mathrm{C} for 3 days. (b) Diagrammatic representation of data obtained: (1) PT; (2) hsp104 mutant; (3) PT petite; (4) petite hsp104 mutant. Typical data of one from three experiments, which each produce similar results, are presented.

Under these experimental conditions (Fig. 5), no negative effect of NaN3\mathrm{NaN}_{3} on induced thermotolerance was observed. The above data do not allow us to conclude with confidence that the inhibition of chaperone hydro-
img-2.jpeg

Fig. 3. Repression of heat induction of Hsp104 synthesis in S. cerevisiae with impaired mitochondrial functions. (a) Cells of strain Ψ\Psi-74-D694 (3, 4) or isogenic petite mutant (1,2)(1,2) were grown on the YEPD medium at 30∘C30^{\circ} \mathrm{C} and incubated either at 26∘C(1,3)26^{\circ} \mathrm{C}(1,3) or at 39∘C(2,4)39^{\circ} \mathrm{C}(2,4). (b) Cells of strain Ψ\Psi-74-D694 were grown on the YEPD medium at 30∘C30^{\circ} \mathrm{C} and incubated for 30 min at 30∘C(5,6)30^{\circ} \mathrm{C}(5,6) or 37∘C(7,8)37^{\circ} \mathrm{C}(7,8) in the presence (6,8)(6,8) or absence (5,7)(5,7) of 0.15mMNaN30.15 \mathrm{mM} \mathrm{NaN}_{3}. Proteins were separated in 10%10 \% PAAG in the presence of DDC-Na, transferred onto nitrocellulose membrane, and incubated with anti-Hsp104 antibodies.
lytic activity by sodium azide is principally impossible. The protective role of Hsp104 is attributed to the restoration of heat-aggregated protein molecules, but the repair process occurred at the completion of heat shock [25]. In this experiment, a yeast suspension with the inhibitor was diluted at least 100 times immediately after heat shock and just prior to plating on the solid nutrient medium. Therefore, the process of restoration determined by Hsp104 and other chaperones proceeded virtually in the absence of sodium azide. However, it is obvious that the inhibition of SS. cerevisiae induced thermotolerance by sodium azide is determined by the repression of synthesis of stress proteins, Hsp104 for instance, rather than by the inhibition of defense systems.

The repression of Hsp104 induction may be not the only reason for the inhibition of SS. cerevisiae induced thermotolerance by sodium azide, since a similar effect was observed in cells defective for Hsp104 synthesis (Fig. 4b). Apparently, the induction of synthesis of many heat shock proteins, which, like Hsp104, may affect the survival of SS. cerevisiae cells after lethal heat shock, are also controlled by mitochondria. Supporting this view was the observation that the formation of induced thermotolerance in S. cerevisiae also depends on the induction of synthesis of γ\gamma-glutamylcysteine

img-3.jpeg

Fig. 4. Effect of sodium azide on basal and induced thermotolerance of S. cerevisiae. Cells of strain Ψ\Psi-74-D694 (a) or isogenic hsp104 mutant (b) were grown on the YEPD medium at 30∘C30^{\circ} \mathrm{C} and were either immediately subjected to heat treatment at 45∘C45^{\circ} \mathrm{C} without (1) or with (2) 0.15 mM NaN3\mathrm{NaN}_{3} or had previously been kept for 30 min at 37∘C37^{\circ} \mathrm{C} in the absence (3) or in the presence (4) of 0.15mMNaN30.15 \mathrm{mM} \mathrm{NaN}_{3}. In the latter case, yeasts were precipitated and washed with fresh medium for removing the inhibitor prior to heat treatment at 45∘C45^{\circ} \mathrm{C}. Arithmetic mean values obtained in six experiments and their standard errors are represented. In some cases, erroneous bands were smaller than those of the marker.
synthetase and glutathione synthetase encoded by genes GSH1 and GSH2, respectively [26]. The induction of expression of these genes by heat shock was not observed, when heat treatment was conducted under anaerobic conditions or upon inhibition of respiratory activity in cells using another mitochondrial inhibitor, potassium cyanide [23].

Thus, a disturbance in mitochondria functioning caused by petite mutation or by respiration inhibition by sodium azide gave virtually the same effect-inhibition of the induction of Hsp104 synthesis by heat
img-4.jpeg

Fig. 5. Effect of sodium azide on S. cerevisiae defense systems induced by heat shock. Cells of strain Ψ\Psi-74-D694 (1,2)(1,2) and isogenic hsp104 mutant (3,4)(3,4) were grown on the YEPD medium at 30∘C30^{\circ} \mathrm{C}. These cells had previously been incubated for 30 min at 37∘C37^{\circ} \mathrm{C} in the absence of the inhibitor and subjected to heat shock at 45∘C45^{\circ} \mathrm{C} in the absence (1,3)(1,3) or in the presence (2,4)(2,4) of 0.15mMNaN30.15 \mathrm{mM} \mathrm{NaN}_{3}. Arithmetic mean values from four experiments and their standard errors are presented. In some cases, erroneous bands were smaller than those of the marker.
shock. These results suggest the relationship between S. cerevisiae HSP104 gene expression after exposure to heat shock and the functional state of yeast mitochondria.

DISCUSSION

The expression of stress genes after heat shock is induced by binding the transcription factors Hsf and Msn2p/Msn4p to sequences HSE and STRE, respectively [27]. The promoter region of gene HSP104 contains three copies of the STRE element that ensure the induction of its expression in heat shock response even in yeast cells with mutation hsf1-m3 blocking Hsf activation [28]. The activity of Msn2p and Msn4p is under the negative control of cAMP-dependent protein kinase A (PKA) [27]. Exogenous addition of cAMP during heat shock activates PKA, which inhibits Msn2p and Msn4p, resulting in the inhibition of expression of genes with STRE elements [1].

There is good reason to believe that SS. cerevisiae PKA activity depends on the functional state of mitochondria. First, mutants with hyperactive PKA, as a rule, do not grow on a medium containing nonfermentable carbon sources. Second, PKA activity in cells growing due to glucose utilization is relatively high, whereas respiratively grown cells have a decreased activity [20]. Third, a mutation in gene IRA2 causing an increase in the activity of the signal pathway of PKA,

simultaneously enhances a respiration intensity [30], and, finally, addition of mitochondrial inhibitors, such as azide and dinitrophenol rapidly increases the level of S. cerevisiae cAMP [31]. From these data we infer that PKA activity may depend on mitochondrial functions.

Thus, the effect of repression of heat-induced synthesis of Hsp104 and probably other SS. cerevisiae stress proteins described in this work can have the following explanation. Exposure to mild heat shock brings about an increase in SS. cerevisiae respiration intensity [23]. This results in activity decrease of cAMP-dependent PKA, which ensures the activation of Msn2p and Msn4p factors [27], which are responsible for the expression of stress-induced genes. Petite mutation or blockage of respiration by azide enhance PKA activity. As a result, Msn2p and Msn4p became inactivated, and no heat induction of expression of genes with STRE elements occurs.

Since complete repression of Hsp104 heat-induced synthesis does not occur, one can assume that after mitochondrial functions are impaired, the induction of HSP gene expression caused by binding the transcription factor Hsf to HSE elements is either not changed or is even increased. We cannot state that sodium azide induced S. cerevisiae Hsp104 synthesis at 30∘C30^{\circ} \mathrm{C} (Fig. 3b, 5 and 6). However, a treatment with another mitochondrial inhibitor, sodium arsenite, was shown to bring about an increase in Hsp104 synthesis [9]. In addition, it is known that mitochondrial inhibitors induce the appearance of new puffs in polytene chromosomes of Drosophila salivary gland [20]. We can probably assume that in both cases, the expression of stress genes was accomplished via Hsf activation. Note that constitutive expression of HSF1 gene was 1.6 times higher in cells of petite mutant than in wild-type SS. cerevisiae [14]. However, the possible transcription activation resulting from a disturbance in the process of mitochondrial biogenesis in SS. cerevisiae strains used in this work, did not compensate for an increase in the expression of stress genes in response to heat shock (Fig. 3).

Undoubtedly, the above assumption about the regulatory mechanism of stress genes that operates in dependence on the functional state of mitochondria requires further verification. The results of Traven et al. [14], who showed that the constitutive expression of some genes with STRE elements is higher than in the wild type, somewhat contradict the proposed hypothesis. However, these authors did not detect an increase in the level of mRNA for such STRE-regulated genes as HSP104, SSA3, HSP26, CTT1, and DDR2 [14]. We hope to confirm our assumption using SS. cerevisiae mutants with hyperactive and attenuated PKA activity.

ACKNOWLEDGMENTS

The authors are grateful to S. Lindquist (Whitehead Institute for Biomedical Research, United State) for providing SS. cerevisiae strains and antibodies for

Hsp104, to Yu.O. Chernoff (Georgia Institute of Technology, United States) for critical reading of the manuscript and helpful comments.

REFERENCES

  1. Boy-Marcotte, E., Lagniel, G., Perrot, M., et al., The Heat Shock Response in Yeast: Differential Regulations and Contributions of the Msn2p/Msn4p and Hsf1p Regulons, Mol. Microbiol., 1999, vol. 33, no. 2, pp. 274283.
  2. Sanchez, Y. and Lindquist, S., HSP104 Required for Induced Thermotolerance, Science, 1990, vol. 248, no. 4959, pp. 1112-1115.
  3. Ferreira, P.C., Ness, F., Edwards, S.R., et al., The Elimination of the Yeast [PSI*] Prion by Guanidine Hydrochloride Is the Result of Hsp104 Inactivation, Mol. Microbiol., 2001, vol. 40, no. 6, pp. 1357-1369.
  4. Jung, G. and Masison, D.C., Guanidine Hydrochloride Inhibits Hsp104 Activity in Vivo: A Possible Explanation for Its Effect in Curing Yeast Prions, Curr. Microbiol., 2001, vol. 43, no. 1, pp. 7-10.
  5. Newnam, G.P., Wegrzyn, R.D., Lindquist, S.L., and Chernoff, Y.O., Antagonistic Interactions between Yeast Chaperones Hsp104 and Hsp70 in Prion Curing, Mol. Cell. Biol., 1999, vol. 19, no. 2, pp. 1325-1333.
  6. Chernoff, Y.O., Lindquist, S.L., Ono, B., et al., Role of the Chaperone Protein Hsp104 in Propagation of the Yeast Prion-like Factor [psi∗]\left[p s i^{*}\right], Science, 1995, vol. 268, no. 5212, pp. 880-884.
  7. Wegrzyn, R.D., Bapat, K., Newnam, G.P., et al., Mechanism of Prion Loss after Hsp104 Inactivation in Yeast, Mol. Cell. Biol., 2001, vol. 21, no. 14, pp. 4656-4669.
  8. De Deken, R.H., The Crabtree Effect: A Regulatory System in Yeast, J. Gen. Microbiol., 1966, vol. 44, no. 2, pp. 149-156.
  9. Sanchez, Y., Taulien, J., Borkovich, K.A., and Lindquist, S., Hsp104 Is Required for Tolerance to Many Forms of Stress, EMBO J., 1992, vol. 11, no. 6, pp. 2357-2364.
  10. Slonimski, P.P., Perrodin, G., and Croft, J.H., Ethidium Bromide-Induced Mutation of Yeast Mitochondria: Complete Transformation of Cells into Respiratory Deficient Non-Chromosomal “Petites,” Biochem. Biophys. Res. Commun., 1968, vol. 30, no. 3, pp. 232-239.
  11. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem., 1951, vol. 193, no. 1, pp. 265275.
  12. Timmons, T.M. and Dunbar, B.S., Protein Blotting and Immunodetection, Methods Enzymol., 1990, vol. 182, pp. 679-688.
  13. Gauze, G.F. and Kuzovkova, L.I., Themperature Sensitivity of Respiration-Deficient Mutants of Yeast, Izv. Akad. Nauk SSSR, Ser. Biol., 1970, vol. 26, no. 2, pp. 305-308.
  14. Traven, A., Wong, J.M., Xu, D., et al., Interorganellar Communication: Altered Nuclear Gene Expression Profiles in a Yeast Mitochondrial DNA Mutant, J. Biol. Chem., 2001, vol. 276, no. 6, pp. 4020-4027.
  15. Rikhvanov, E.G., Varakina, N.N., Rusaleva, T.M., et al., Respiratory Changes in Yeast Saccharomyces cerevisiae

Exposed to Heat Shock, Mikrobiologiya, 2001, vol. 70, no. 4, pp. 531-535.
16. Weitzel, G., Pilatus, U., and Rensing, L., The Cytoplasmic pH, ATP Content and Total Protein Synthesis Rate during Heat Shock Protein-Inducing Treatments in Yeast, Exp. Cell Res., 1987, vol. 170, no. 1, pp. 64-79.
17. Wilson, D.F. and Chance, B., Azide Inhibition of Mitochondrial Electron Transport: I. The Aerobic Steady State of Succinate Oxidation, Biochim. Biophys. Acta, 1967, vol. 131, no. 3, pp. 421-430.
18. Rikhvanov, E.G., Varakina, N.N., Rusaleva, T.M., et al., The Effect of Sodium Azide on Heat Shock Resistance of Yeasts Saccharomyces cerevisiae and Debaryomyces vanriji, Mikrobiologiya, 2001, vol. 70, no. 3, pp. 300304.
19. Rikhvanov, E.G., Varakina, N.N., Rusaleva, T.M., et al., Sodium Azide Reduces the Thermotolerance of Respiratively Grown Yeasts, Curr. Microbiol., 2002, vol. 45, no. 6, pp. 394-399.
20. Ashburner, M. and Bonner, J.J., The Induction of Gene Activity in Drosophila by Heat Shock, Cell (Cambridge, Mass.), 1979, vol. 17, no. 2, pp. 241-254.
21. Sanchez, Y., Parsell, D.A., Taulien, J., et al., Genetic Evidence for a Functional Relationship between Hsp104 and Hsp70, J. Bacteriol., 1993, vol. 175, no. 20, pp. 6484-6491.
22. Wieser, R., Adam, G., Wagner, A., et al., Heat Shock Factor-Independent Heat Control of Transcription of the CTTI Gene Encoding the Cytosolic Catalase T of Saccharomyces cerevisiae, J. Biol. Chem., 1991, vol. 266, no. 19, pp. 12 406-12 411.
23. Sugiyama, K., Izawa, S., and Inoue, Y., The Yap1pDependent Induction of Glutathione Synthesis in Heat Shock Response of Saccharomyces cerevisiae, J. Biol. Chem., 2000, vol. 275, no. 20, pp. 15 535-15 540.
24. Weber, J. and Senior, A.E., Effects of the Inhibitors Azide, Dicyclohexylcarbodiimide, and Aurovertin on Nucleotide Binding to the Three F1\mathrm{F}_{1}-ATPase Catalytic Sites Measured Using Specific Tryptophan Probes, J. Biol. Chem., 1998, vol. 273, no. 50, pp. 33 210-33 215.
25. Parsell, D.A., Kowal, A.S., Singer, M.A., and Lindquist, S., Protein Disaggregation Mediated by Heat-Shock Protein Hsp104, Nature, 1994, vol. 372, no. 6505, pp. 475-478.
26. Sugiyama, K., Kawamura, A., Izawa, S., and Inoue, Y., Role of Glutathione in Heat Shock-Induced Cell Death of Saccharomyces cerevisiae, Biochem. J., 2000, vol. 352, part 1, pp. 71-78.
27. Estruch, F., Stress-Controlled Transcription Factors, Stress-Induced Genes and Stress Tolerance in Budding Yeast, FEMS Microbiol. Rev., 2000, vol. 24, no. 4, pp. 469-486.
28. Lindquist, S. and Kim, G., Heat-Shock Protein 104 Expression Is Sufficient for Thermotolerance in Yeast, Proc. Natl. Acad. Sci. USA, 1996, vol. 93, no. 11, pp. 5301-5306.
29. Thevelein, J.M. and de Winde, J.H., Novel Sensing Mechanisms and Targets for the cAMP-Protein Kinase A Pathway in the Yeast Saccharomyces cerevisiae, Mol. Microbiol., 1999, vol. 33, no. 5, pp. 904-918.
30. Bussereau, F., Dupont, C.H., Boy-Marcotte, E., et al., The CCS1 Gene from Saccharomyces cerevisiae Which Is Involved in Mitochondrial Functions Is Identified as IRA2, an Attenuator of RAS1 and RAS2 Gene Products, Curr. Genet., 1992, vol. 21, nos. 4-5, pp. 325-329.
31. Thevelein, J.M., Beullens, M., Honshoven, F., et al., Regulation of the cAMP Level in the Yeast Saccharomyces cerevisiae: Intracellular pH and the Effect of Mem-brane-Depolarizing Compounds, J. Gen. Microbiol., 1987, vol. 133, part 8, pp. 2191-2196.