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Dehydration of yeast: Changes in the intracellular content of Hsp70 family proteins
Process Biochemistry 43 (2008) 1138–1141
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Dehydration of yeast: Changes in the intracellular content of Hsp70 family proteins Irina Guzhova a, Irina Krallish b, Galina Khroustalyova b, Boris Margulis a, Alexander Rapoport b,* a b
Laboratory of Cell Protective Mechanisms, Institute of Cytology, Russian Academy of Science, Saint Petersburg, Russia Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia, LV-1586 Riga, Latvia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 December 2007 Received in revised form 9 April 2008 Accepted 22 May 2008
Yeast is known to experience in natural and industrial conditions cycles of dehydration–rehydration. Several molecular mechanisms can be triggered in response to this and other environmental stressors and to rescue yeast cells of the cytotoxic effect. Since heat shock proteins constitute one of the most important systems of the response to stress we studied whether the pre-induced major stress protein, Hsp70, can cope with yeast cell drying. To induce Hsp70 expression the cells of two yeast species, Saccharomyces cerevisiae and Debaryomyces hansenii, were subjected to non-lethal heat shock. It was found that during yeast culture growth Hsp70 accumulation occurred at the exponential growth phase, and there was no marked change in the protein level at the stationary phase both in aerobic and anaerobic conditions. Interestingly, dehydration of sensitive to this kind of stress S. cerevisiae grown in anaerobic conditions led to the increase of Hsp70 expression; to our knowledge this finding was presented for the first time. Dehydration of yeast taken from the stationary growth phase did not cause the induction of Hsp70 expression. Irrespective of the inducer, Hsp70 did not rescue yeast cells from dehydration stress damages. This result well coincides with data of other groups found that Hsp70 in yeast possesses chaperonic activity, and the latter does not impact to an increase in protective power of the protein demonstrated in many other organisms. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Hsp70 Anhydrobiosis Dehydration–rehydration Protective reactions Yeast Saccharomyces cerevisiae Debaryomyces hansenii
1. Introduction Yeasts as many other types of microorganisms can be subjected to significant changes of humidity in nature. As the result they can undergo multiple cycles of dehydration and subsequent rehydration in their life. During evolution they worked out a variety of mechanisms which help to maintain their viability at their transfer into ‘‘non-active’’ state of anhydrobiosis. This state is characterized by a transient and reversible reduction of metabolism and also by a variety of changes at biochemical and functional levels [1]. The latter include condensation of chromatin, separation by membranes of rather big parts of nucleus and damaged areas of cytoplasm [2–4], synthesis of trehalose and polyols [5–7], stabilization of molecular organization of intracellular membranes [8], maintenance of redox homeostasis [9] and other. Heat shock proteins belonging to Hsp70 family are established to be the ubiquitous stress-responsive system in all living organisms. The accumulation of Hsp70 signals that a cell or tissue
* Corresponding author. Tel.: +371 67034891; fax: +371 67227925. E-mail address: [email protected] (A. Rapoport). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.05.012
respond to an environmental or xenobiotic harmful factor, and in most cases the increase of Hsp70 expression renders cells more resistant to repetitive stressors. Intracellular functions of Hsp70 are based on its chaperonic activity that implies assembly, folding, intracellular localization, secretion, and degradation of cellular polypeptides [10–12]. Protective power of Hsp70 thought to be linked to its chaperonic activity is proved by studies on hundreds cell and animal models. The genome of Saccharomyces cerevisiae yeast contains 14 genes comprising multigene Hsp70 family proteins [13]. This protein family includes mitochondrial proteins Ssc1 and Ssc1p [14–17], cytosolic proteins Ssa1, Ssa1p, Ssa2 and Ssa4p which accumulate in cell nucleus during yeast starvation [18]. As in other organism in yeast Hsp70 chaperones facilitate endoplasmic reticulum-associated degradation of ‘‘defective’’ proteins [19]. It is known also that the cytosolic yeast Hsp70 supervises proteins involved in the response to stress and protein synthesis [20]. Loss of mitochondrial Hsp70 (Ssc1p) function causes aggregation of mitochondrial polypeptides in yeast cells [21]. S. cerevisiae cells with Hsp70 knockout demonstrate abnormal nuclear distribution and aberrant microtubule formation in M-phase [22]. A few factors inducing Hsp70 expression in yeast include heat shock and oxidative stress;
I. Guzhova et al. / Process Biochemistry 43 (2008) 1138–1141
it is also noteworthy that high amount of the chaperone was found in cells subjected to deuterium oxide or genetically resistant to low temperatures [23]. The expression and role of Hsp70 in conditions of dehydration and rehydration remains unexplored. The aim of this study was to analyze the possible function of pre-established Hsp70 in cells of two yeast strains subjected to drying as well as to understand if dehydration stress itself leads to the synthesis of Hsp70. 2. Materials and methods 2.1. Yeast strains and cultivation conditions In this study we used yeast S. cerevisiae 14 (Collection of the Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia) and Debaryomyces hansenii (generous gift from Prof. L. Adler, Geteborg University, Sweden). The latter strain was earlier found to be significantly more resistant to dehydration. Yeast cells were cultivated in 750 ml flasks at 30 8C using shaker (180 rpm) for aerobic conditions (S. cerevisiae and D. hansenii) and without shaking with a great excess of nutrient medium for anaerobic conditions (only S. cerevisiae). Nutrient medium contained (in g l 1): MgSO4 0.7; NaCl 0.5; (NH4)2SO4 3.7; KH2PO4 1.0; K2HPO4 0.13; molasses 43 (till final concentration of glucose 20 g l 1). pH value of nutrient medium was adjusted to pH 5.0 using H2SO4. 2.2. Biomass harvesting and dehydration Yeast cells at the exponential phase (for yeast grown in aerobic conditions) and stationary phase (for yeast grown both in aerobic and anaerobic conditions) were collected. To establish time points for the harvesting of the biomass (data not present) direct counting of cell amount in Goryaev chamber and spectrophotometric determination of suspension’s optical density at 600 nm were performed. Harvested biomass was washed and compressed with the aid of vacuum filtration unit. A part of yeast biomass was used in further experiments as ‘‘native’’ counterpart, the second part was dehydrated by convective method at 30 8C to residual humidity of 8–10%, and the third portion of biomass was used for the experiments on heat shock. This part was subjected to heat shock and also was dehydrated till the residual humidity of 8–10%. Biomass relative humidity was measured of its weight after drying at 105 8C during 48 h. 2.3. Determination of cells viability Viability of native and dehydrated cells was measured with the help of fluorescent microscopy using specific probe primuline [24]. The use of this method gives the possibility to reveal live organisms in which only cell wall fluoresces and dead yeast which have bright green fluorescence of the whole cell. 2.4. Heat shock Compressed biomass was put in 250 ml flask. 75 ml of pre-heated till 42 8C filtered cultural liquid was added to the flask. Procedure of heat shock was made at 42 8C during 30 min. After heat stress yeast cells were transferred to fresh nutrient medium in which they were kept 1 h at 30 8C.
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protein can protect yeast cells from the deleterious effect of dehydration as well as to understand if dehydration stress leads to the synthesis of Hsp70 proteins in yeast. To establish the conditions of Hsp70 accumulation we studied the protein level in control and stressed yeast cells. The analysis of Hsp70 expression during the S. cerevisiae growth was performed in samples taken each 4 h after the cells had been seeded in nutrient medium. This study was performed with the aid of Western blotting and showed that the level of Hsp70 was strongly elevated during first 8 h after inoculation that corresponded to exponential growth phase (Fig. 1). Twelve hours after inoculation the level of Hsp70 began to decline and 12 h later the signal fully disappeared. The yeast entered stationary phase of growth at time point 18 h after seeding, and the reduction of Hsp70 level revealed that despite a strong lowering of cellular metabolism the protein is subjected to proteolysis. Thus, the highest level of Hsp70 can be attained in the middle of exponential phase and this point was selected for further experiments on pre-conditional stress designed to increase Hsp70 amount in cells. Since dehydration by itself can induce stress response, we measured Hsp70 amount in S. cerevisiae cells grown in aerobic conditions, taken at the exponential growth phase and subjected to dehydration. It was found that drying led to a complete reduction of Hsp70 level (Fig. 2A). Viability of these cells was also found to be at very low level—14.8 1.15% (Fig. 2C). Dehydration of the same yeast taken at the stationary phase did not cause expression of Hsp70 (Fig. 2A). In these experiments viability of dehydrated cells was 65.4 0.65% that is ordinary value for this yeast grown and dehydrated in ‘‘standard’’ conditions in our previous studies of anhydrobiosis [1]. Finally, dehydration of yeast grown in anaerobic conditions and taken from stationary growth phase led to the accumulation synthesis of Hsp70 family proteins (Fig. 2B). It is necessary to mention that this yeast was extremely sensitive to dehydration and the maximal viability did not exceed 1%. To check whether the same response to stress is typical for various yeast species, we studied profile of Hsp70 expression in D. hansenii cells that are extremely resistant to dehydration [26]. Similar to S. cerevisae these cells were found to contain Hsp70 at the exponential phase of growth and not at the stationary phase (Fig. 3A). Dehydration of yeast D. hansenii taken from exponential growth phase led to the reduction of Hsp70 content (Fig. 3A). As suggested the viability of dehydrated D. hansenii remained high enough in contrast with S. cerevisiae, and comprised 55–60%. Lastly dehydration of D. hansenii cells taken from stationary growth
2.5. Quantification of Hsp70 by immunoblotting To measure Hsp70 content the method of Western blotting was employed using protocol of Towbin et al. [25]. Briefly, yeast cells were subjected to disintegration in 0.1 M K-potassium buffer (pH 7.0) with glass beads (diameter 300 mkm) during 10 min at 4000 rpm with refrigeration using the disintegrator SCP-100-MRE, Innomed-Konsult AB, Sweden. The samples of total protein extract from disintegrated yeast cells and were mixed with sodium dodecylsulfate (SDS) and 2-mercaptoethanol to give final concentration 2% and 15 mM, respectively. Equal amounts of the total protein, 50 mg, were applied onto lanes of 10% polyacrylamide gel. Electrophoresis was performed with a voltage gradient of 5 V cm 1 and currency 30 mA per gel slab. After the electrophoresis protein bands were transferred onto Immobilon nitrocellulose membrane with the aid of the semi-dry blotting apparatus (GE Healthcare, Russia) according to standard protocol [25]. The bands of Hsp70 were stained with the use of SPA-822 monoclonal antibody known to recognize inducible component of the yeast Hsp70 family (StressGen, Canada).
3. Results The major goal of this study was to elucidate whether heat precondition accompanying with the accumulation of Hsp70 stress
Fig. 1. Hsp70 protein content in the cells of Saccharomyces cerevisiae during its growth in aerobic conditions: (A) Hsp70 protein content at different phases of culture growth; (B) yeast culture growth curve.
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phase was not accompanied with the synthesis of Hsp70 family proteins (Fig. 3A). To check whether enhanced amount of Hsp70 due to heat preconditioning can cause the increase of the resistance of yeast cells to dehydration we subjected yeast cells to heat shock at 42 8C 30 min prior drying. In both aerobic (Fig. 2A) or anaerobic culture (Fig. 2B) conditions heat shock led to elevation of Hsp70 in yeast cells S. cerevisiae but did not increase cell viability (Fig. 2C). Heat shock of D. hansenii cells also did not result in the enhancement of their survival despite the significant elevation of Hsp70 level (Fig. 3A and B). 4. Discussion
Fig. 2. Hsp70 protein content in the cells of S. cerevisiae after heat shock and dehydration treatments and viability of cells after dehydration: (A) yeast was grown in aerobic conditions; (B) yeast was grown in anaerobic conditions and was taken at stationary growth phase; (C) viability of yeast cells after dehydration without heat shock ( ) and after heat shock (+). Exp, exponential growth phase; Stat, stationary growth phase; C, control (yeast which has not been subjected to heat shock); HS, yeast subjected to heat shock; Compr, compressed (intact) yeast; Dry, yeast subjected to dehydration.
Fig. 3. Hsp70 protein content in the Debaryomyces hansenii cells grown in aerobic condition after heat shock and dehydration treatments (A) and viability of cells after dehydration without heat shock ( ) and after heat shock (+) (B). Exp, exponential growth phase; Stat, stationary growth phase; C, control (yeast which has not been subjected to heat shock); HS, yeast subjected to heat shock; Compr, compressed (intact) yeast; Dry, yeast subjected to dehydration.
Systematic investigations of main factors able to positively influence yeast viability during its transition to the state of anhydrobiosis reveal a number of intracellular protective systems that function in these conditions. These systems can work at the ultrastructural level as well as they can be associated with synthesis of a number of protective substances. The latter include Hsp70 chaperone whose protective activity in a variety of organisms is convincingly established. In this study we questioned whether enhanced level of Hsp70 can contribute to the protection from the deleterious effect of dehydration. First we studied profiles of Hsp70 expression in control and stressed S. cerevisiae and D. hansenii cells. The results show that Hsp70 is synthesized over exponential phase of growth in both yeast strains. The specific feature of this stage of growth is the active metabolism and intensive synthesis of different proteins. Since one of the most important roles of Hsp70 is chaperonic activity one can suggest that this property must be useful at this particular stage of yeast growth [10]. Subsequently, the reduction of total cellular protein synthesis at the stationary phase does not demand a necessity in Hsp70 synthesis. Probably it is the main reason why Hsp70 expression was not found in S. cerevisiae cells in aerobic and anaerobic conditions and in aerobic D. hansenii cells at the stationary growth phase. We further analyzed the reaction of yeast cells to a moderate heat stress. In S. cerevisiae heat shock at 42 8C induced Hsp70 in both anaerobic and aerobic conditions however in the latter case only at the exponential phase of growth. We demonstrated Hsp70 induction in heat stressed D. hansenii taken from both exponential and stationary growth phases. Since dehydration is also a strong stressor, we checked whether it can induce Hsp70 expression. The data show that this induction occurs only in S. cerevisae living in anaerobic conditions and taken from stationary growth phase. It is worth-mentioning that the cells in these conditions are extremely sensitive to drying. Therefore one can speculate that the synthesis of Hsp70 occurs only in surviving part of cells that comprises about 1% of the whole cell population but we suppose that it would be much more probable that these proteins are synthesized in the cells at the early stages of dehydration when cells are still viable. It can be concluded that unfortunately also this protective reaction does not help them to increase their viability rate. Discussing these results one significant thing should be taken in mind: yeast dehydration is comparatively long process. At least at its first stage, when cells are keeping a rest of water and which lasts approximately 9 h, the process is associated with the destruction of a number of unnecessary proteins, and this is also a part of the program ‘‘preparation of the cells to dehydration’’ [1]. Taking into account chaperonic function of Hsp70 we assume that it participates in the degradation of intracellular proteins at the early stages of drying process and simultaneously in prevention of total demolition of cells. Certainly, one can ask why there was no Hsp70 synthesis in other S. cerevisiae yeast probes subjected to
I. Guzhova et al. / Process Biochemistry 43 (2008) 1138–1141
dehydration, for instance ones taken from exponential phase. One of possible explanations may be that these cells already contain the amount of Hsp70 family proteins which is completely sufficient for the realization of both above mentioned tasks at the initiative stage of yeast drying. The changes of Hsp70 content in organisms that experience anhydrobiosis in natural conditions were reported for tardigrades Milnesium tardigradum. It was shown that three isoforms (isoforms 1, 2, 3) of this protein were expressed at the stage of their restoration from the anhydrobiosis, however only one of these (isoform 2) was expressed also when tardigrades were subjected to drying, whereas being in the active state tardigrades contained extremely low quantity of this Hsp70 [27]. Similar results were obtained using another tardigrades species, Richtersius coronifer. Total amount of Hsp70 family proteins in these organisms was low before their transfer into anhydrobiosis conditions and increased during the first hour after beginning of rehydration [28]. Generally, these results resemble our data with the only notice: we have not observed dehydration-induced Hsp70 expression but it is still possible that such phenomenon can take place at the stage of yeast reactivation and it has to be studied in the further investigations. Major goal of this study was to analyze the reaction of yeast cells with enhanced Hsp70 level on dehydration stress. It was expected that a moderate heat shock would contribute to the increase of Hsp70 and cells would be more resistant to deleterious effect of drying. However, the data show that there was no difference in viability between cells pretreated with heat shock and untreated, see Figs. 2 and 3. The lack of Hsp70-mediated protection can be explained by two reasons. First is that the amount of chaperone can be insufficient to meet the demands of a proper cell response to dehydration. As was shown above for anaerobic S. cerevisae responding to drying at the stationary phase, only a few remaining alive cells keep the rational amount of Hsp70. The same can be in case of cells that experience two sequential stresses, heat precondition and drying: only a small part of cell population can survive that is able to keep its protective resources including Hsp70 chaperone. Secondly, besides Hsp70 chaperone yeast cells acquire a variety of protective mechanisms and for some specific insults they may be much more efficient than Hsp70. It is worth-mentioning that the thermotolerance of yeast cells overexpressing different members of SSA gene family was not higher than in their control counterpart [29]. In summary we show that Hsp70 can be induced in yeast by two environmental stressors, heat shock and dehydration, however its synthesis can be rather an indicator of stress response than a part of protection mechanism. References [1] Beker MJ, Rapoport AI. Conservation of yeasts by dehydration. Advances in biochemical engineering/biotechnology, vol. 35. Berlin/Heidelberg/New York/ London/Paris/Tokyo: Springer-Verlag; 1987. pp. 127–71. [2] Rapoport AI. Rejection of areas of damaged cytoplasm by microorganisms in a state of anabiosis. Microbiology 1973;42:317–8. [3] Rapoport AI, Kostrikina NA. Cytological investigation of an anabiosis state of yeast organisms. Proc Acad Sci USSR Biology 1973;5:770–3.
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[4] Rapoport AI, Birjusova VI, Meissel MN. The structure of the yeast cell nucleus in an anabiotic state. Rep Acad Sci USSR 1973;213:708–10. [5] Rapoport AI, Puzyrevskaya OM, Saubenova MG. Polyols and resistance of yeasts to dehydration. Microbiology 1988;57:269–71. [6] Panek AD. Trehalose metabolism—new horizons in technological applications. Braz J Med Biol Res 1995;28:169–81. [7] Pereira EJ, Panek AD, Eleutherio ECA. Protection against oxidation during dehydration of yeast. Cell Stress Chaperones 2003;8:120–4. [8] Leslie SB, Teter SA, Crowe LM, Crowe JH. Trehalose lowers membrane phase transitions in dry yeast cells. Biochim Biophys Acta 1994;1192:7–13. [9] Franca MB, Panek AD, Eleutherio ECA. The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae. Cell Stress Chaperones 2003;10:167–70. [10] Gething MJ. Guidebook to molecular chaperones and protein folding catalysts. Oxford: Oxford University Press; 1997. [11] Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992;355:33–45. [12] Craig EA, Gambill BD, Nelson RJ. Heat shock proteins: molecular chaperones of protein biogenesis. Microbiol Rev 1993;57:402–14. [13] Matsumoto R, Akama K, Rakwal R, Iwahashi H. The stress response against denatured proteins in the deletion of cytosolic chaperones SSA1/2 is different from heat-shock response in Saccharomyces cerevisiae. BMC Genom 2005;6: 141. [14] Craig E, Kramer J, Kosic-Smithers J. SSC1, a member of the 70 kDa heat shock protein multigene family of Saccharomyces cerevisiae is essential for growth. Proc Natl Acad Sci USA 1987;84:4156–60. [15] Craig EA, Kramer J, Shilling J, Werner-Washburne M, Holmes S, Kosic-Smithers J, et al. SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. Mol Cell Biol 1989;9:3000–8. [16] Gambill B, Voos W, Kang P, Miao B, Langer T, Craig E, et al. A dual role for mitochondrial heat shock protein-70 in membrane translocation of preproteins. J Cell Biol 1993;123:109–17. [17] Kang P-J, Ostermann J, Shilling J, Neupert W, Craig EA, Pfanner N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 1990;356:683–9. [18] Chughtai ZS, Rassadi R, Matusiewicz N, Stochaj U. Starvation promotes nuclear accumulation of the HSP70 Ssa4p in yeast cells. J Biol Chem 2001; 23:20261–6. [19] Zhang Y, Nijbroek G, Sullivan ML, McCracken AA, Watkins SC, Michaelis S, et al. Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Mol Biol Cell 2001;12:1303–14. [20] Matsumoto R, Rakwal R, Agrawal GK, Jung YH, Jwa NM, Yonekura M, et al. Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes SSA1 and SSA2. Mol Cell 2006; 21:381–8. [21] Kawai A, Nishikawa S, Hirata A, Endo T. Loss of mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells. J Cell Sci 2001;114:3565–74. [22] Oka M, Nakai M, Endo T, Lim CR, Kimata Y, Kohno K. Loss of Hsp70-Hsp40 chaperone activity causes abnormal nuclear distribution and aberrant microtubule formation in M-phase of Saccharomyces cerevisiae. J Biol Chem 1998;273:29727–3. [23] Unno K, Kishido T, Morioka M, Okada S, Oku N. Increased expression of Hsp70 for resistance to deuterium oxide in a yeast mutant cell line. Biol Pharm Bull 2003;26:799–802. [24] Rapoport AI, Meissel MN. Survival rates of yeast organisms after dehydration as determined by fluorescence microscopy. Microbiology 1985;54:53–5. [25] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979. Biotechnology 1992;24:145–9. [26] Khroustalyova GM, Adler L, Rapoport AI. Exponential growth phase cells of the osmotolerant yeast Debaryomyces hansenii are extremely resistant to dehydration stress. Process Biochem 2001;36:1163–6. [27] Schill RO, Steinbruck GHB, Kohler HR. Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages. J Exp Biol 2004;207:1607–13. [28] Jonsson KI, Schill RO. Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer. Comp Biochem Physiol B 2007;146:456–60. [29] Weitzel G, Li GC. Thermal response of yeast cells overexpressing hsp70 genes. Int J Hyperthermia 1993;9:783–97.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Dehydration of yeast: Changes in the intracellular content of Hsp70 family proteins Irina Guzhova a, Irina Krallish b, Galina Khroustalyova b, Boris Margulis a, Alexander Rapoport b,* a b
Laboratory of Cell Protective Mechanisms, Institute of Cytology, Russian Academy of Science, Saint Petersburg, Russia Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia, LV-1586 Riga, Latvia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 December 2007 Received in revised form 9 April 2008 Accepted 22 May 2008
Yeast is known to experience in natural and industrial conditions cycles of dehydration–rehydration. Several molecular mechanisms can be triggered in response to this and other environmental stressors and to rescue yeast cells of the cytotoxic effect. Since heat shock proteins constitute one of the most important systems of the response to stress we studied whether the pre-induced major stress protein, Hsp70, can cope with yeast cell drying. To induce Hsp70 expression the cells of two yeast species, Saccharomyces cerevisiae and Debaryomyces hansenii, were subjected to non-lethal heat shock. It was found that during yeast culture growth Hsp70 accumulation occurred at the exponential growth phase, and there was no marked change in the protein level at the stationary phase both in aerobic and anaerobic conditions. Interestingly, dehydration of sensitive to this kind of stress S. cerevisiae grown in anaerobic conditions led to the increase of Hsp70 expression; to our knowledge this finding was presented for the first time. Dehydration of yeast taken from the stationary growth phase did not cause the induction of Hsp70 expression. Irrespective of the inducer, Hsp70 did not rescue yeast cells from dehydration stress damages. This result well coincides with data of other groups found that Hsp70 in yeast possesses chaperonic activity, and the latter does not impact to an increase in protective power of the protein demonstrated in many other organisms. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Hsp70 Anhydrobiosis Dehydration–rehydration Protective reactions Yeast Saccharomyces cerevisiae Debaryomyces hansenii
1. Introduction Yeasts as many other types of microorganisms can be subjected to significant changes of humidity in nature. As the result they can undergo multiple cycles of dehydration and subsequent rehydration in their life. During evolution they worked out a variety of mechanisms which help to maintain their viability at their transfer into ‘‘non-active’’ state of anhydrobiosis. This state is characterized by a transient and reversible reduction of metabolism and also by a variety of changes at biochemical and functional levels [1]. The latter include condensation of chromatin, separation by membranes of rather big parts of nucleus and damaged areas of cytoplasm [2–4], synthesis of trehalose and polyols [5–7], stabilization of molecular organization of intracellular membranes [8], maintenance of redox homeostasis [9] and other. Heat shock proteins belonging to Hsp70 family are established to be the ubiquitous stress-responsive system in all living organisms. The accumulation of Hsp70 signals that a cell or tissue
* Corresponding author. Tel.: +371 67034891; fax: +371 67227925. E-mail address: [email protected] (A. Rapoport). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.05.012
respond to an environmental or xenobiotic harmful factor, and in most cases the increase of Hsp70 expression renders cells more resistant to repetitive stressors. Intracellular functions of Hsp70 are based on its chaperonic activity that implies assembly, folding, intracellular localization, secretion, and degradation of cellular polypeptides [10–12]. Protective power of Hsp70 thought to be linked to its chaperonic activity is proved by studies on hundreds cell and animal models. The genome of Saccharomyces cerevisiae yeast contains 14 genes comprising multigene Hsp70 family proteins [13]. This protein family includes mitochondrial proteins Ssc1 and Ssc1p [14–17], cytosolic proteins Ssa1, Ssa1p, Ssa2 and Ssa4p which accumulate in cell nucleus during yeast starvation [18]. As in other organism in yeast Hsp70 chaperones facilitate endoplasmic reticulum-associated degradation of ‘‘defective’’ proteins [19]. It is known also that the cytosolic yeast Hsp70 supervises proteins involved in the response to stress and protein synthesis [20]. Loss of mitochondrial Hsp70 (Ssc1p) function causes aggregation of mitochondrial polypeptides in yeast cells [21]. S. cerevisiae cells with Hsp70 knockout demonstrate abnormal nuclear distribution and aberrant microtubule formation in M-phase [22]. A few factors inducing Hsp70 expression in yeast include heat shock and oxidative stress;
I. Guzhova et al. / Process Biochemistry 43 (2008) 1138–1141
it is also noteworthy that high amount of the chaperone was found in cells subjected to deuterium oxide or genetically resistant to low temperatures [23]. The expression and role of Hsp70 in conditions of dehydration and rehydration remains unexplored. The aim of this study was to analyze the possible function of pre-established Hsp70 in cells of two yeast strains subjected to drying as well as to understand if dehydration stress itself leads to the synthesis of Hsp70. 2. Materials and methods 2.1. Yeast strains and cultivation conditions In this study we used yeast S. cerevisiae 14 (Collection of the Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia) and Debaryomyces hansenii (generous gift from Prof. L. Adler, Geteborg University, Sweden). The latter strain was earlier found to be significantly more resistant to dehydration. Yeast cells were cultivated in 750 ml flasks at 30 8C using shaker (180 rpm) for aerobic conditions (S. cerevisiae and D. hansenii) and without shaking with a great excess of nutrient medium for anaerobic conditions (only S. cerevisiae). Nutrient medium contained (in g l 1): MgSO4 0.7; NaCl 0.5; (NH4)2SO4 3.7; KH2PO4 1.0; K2HPO4 0.13; molasses 43 (till final concentration of glucose 20 g l 1). pH value of nutrient medium was adjusted to pH 5.0 using H2SO4. 2.2. Biomass harvesting and dehydration Yeast cells at the exponential phase (for yeast grown in aerobic conditions) and stationary phase (for yeast grown both in aerobic and anaerobic conditions) were collected. To establish time points for the harvesting of the biomass (data not present) direct counting of cell amount in Goryaev chamber and spectrophotometric determination of suspension’s optical density at 600 nm were performed. Harvested biomass was washed and compressed with the aid of vacuum filtration unit. A part of yeast biomass was used in further experiments as ‘‘native’’ counterpart, the second part was dehydrated by convective method at 30 8C to residual humidity of 8–10%, and the third portion of biomass was used for the experiments on heat shock. This part was subjected to heat shock and also was dehydrated till the residual humidity of 8–10%. Biomass relative humidity was measured of its weight after drying at 105 8C during 48 h. 2.3. Determination of cells viability Viability of native and dehydrated cells was measured with the help of fluorescent microscopy using specific probe primuline [24]. The use of this method gives the possibility to reveal live organisms in which only cell wall fluoresces and dead yeast which have bright green fluorescence of the whole cell. 2.4. Heat shock Compressed biomass was put in 250 ml flask. 75 ml of pre-heated till 42 8C filtered cultural liquid was added to the flask. Procedure of heat shock was made at 42 8C during 30 min. After heat stress yeast cells were transferred to fresh nutrient medium in which they were kept 1 h at 30 8C.
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protein can protect yeast cells from the deleterious effect of dehydration as well as to understand if dehydration stress leads to the synthesis of Hsp70 proteins in yeast. To establish the conditions of Hsp70 accumulation we studied the protein level in control and stressed yeast cells. The analysis of Hsp70 expression during the S. cerevisiae growth was performed in samples taken each 4 h after the cells had been seeded in nutrient medium. This study was performed with the aid of Western blotting and showed that the level of Hsp70 was strongly elevated during first 8 h after inoculation that corresponded to exponential growth phase (Fig. 1). Twelve hours after inoculation the level of Hsp70 began to decline and 12 h later the signal fully disappeared. The yeast entered stationary phase of growth at time point 18 h after seeding, and the reduction of Hsp70 level revealed that despite a strong lowering of cellular metabolism the protein is subjected to proteolysis. Thus, the highest level of Hsp70 can be attained in the middle of exponential phase and this point was selected for further experiments on pre-conditional stress designed to increase Hsp70 amount in cells. Since dehydration by itself can induce stress response, we measured Hsp70 amount in S. cerevisiae cells grown in aerobic conditions, taken at the exponential growth phase and subjected to dehydration. It was found that drying led to a complete reduction of Hsp70 level (Fig. 2A). Viability of these cells was also found to be at very low level—14.8 1.15% (Fig. 2C). Dehydration of the same yeast taken at the stationary phase did not cause expression of Hsp70 (Fig. 2A). In these experiments viability of dehydrated cells was 65.4 0.65% that is ordinary value for this yeast grown and dehydrated in ‘‘standard’’ conditions in our previous studies of anhydrobiosis [1]. Finally, dehydration of yeast grown in anaerobic conditions and taken from stationary growth phase led to the accumulation synthesis of Hsp70 family proteins (Fig. 2B). It is necessary to mention that this yeast was extremely sensitive to dehydration and the maximal viability did not exceed 1%. To check whether the same response to stress is typical for various yeast species, we studied profile of Hsp70 expression in D. hansenii cells that are extremely resistant to dehydration [26]. Similar to S. cerevisae these cells were found to contain Hsp70 at the exponential phase of growth and not at the stationary phase (Fig. 3A). Dehydration of yeast D. hansenii taken from exponential growth phase led to the reduction of Hsp70 content (Fig. 3A). As suggested the viability of dehydrated D. hansenii remained high enough in contrast with S. cerevisiae, and comprised 55–60%. Lastly dehydration of D. hansenii cells taken from stationary growth
2.5. Quantification of Hsp70 by immunoblotting To measure Hsp70 content the method of Western blotting was employed using protocol of Towbin et al. [25]. Briefly, yeast cells were subjected to disintegration in 0.1 M K-potassium buffer (pH 7.0) with glass beads (diameter 300 mkm) during 10 min at 4000 rpm with refrigeration using the disintegrator SCP-100-MRE, Innomed-Konsult AB, Sweden. The samples of total protein extract from disintegrated yeast cells and were mixed with sodium dodecylsulfate (SDS) and 2-mercaptoethanol to give final concentration 2% and 15 mM, respectively. Equal amounts of the total protein, 50 mg, were applied onto lanes of 10% polyacrylamide gel. Electrophoresis was performed with a voltage gradient of 5 V cm 1 and currency 30 mA per gel slab. After the electrophoresis protein bands were transferred onto Immobilon nitrocellulose membrane with the aid of the semi-dry blotting apparatus (GE Healthcare, Russia) according to standard protocol [25]. The bands of Hsp70 were stained with the use of SPA-822 monoclonal antibody known to recognize inducible component of the yeast Hsp70 family (StressGen, Canada).
3. Results The major goal of this study was to elucidate whether heat precondition accompanying with the accumulation of Hsp70 stress
Fig. 1. Hsp70 protein content in the cells of Saccharomyces cerevisiae during its growth in aerobic conditions: (A) Hsp70 protein content at different phases of culture growth; (B) yeast culture growth curve.
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phase was not accompanied with the synthesis of Hsp70 family proteins (Fig. 3A). To check whether enhanced amount of Hsp70 due to heat preconditioning can cause the increase of the resistance of yeast cells to dehydration we subjected yeast cells to heat shock at 42 8C 30 min prior drying. In both aerobic (Fig. 2A) or anaerobic culture (Fig. 2B) conditions heat shock led to elevation of Hsp70 in yeast cells S. cerevisiae but did not increase cell viability (Fig. 2C). Heat shock of D. hansenii cells also did not result in the enhancement of their survival despite the significant elevation of Hsp70 level (Fig. 3A and B). 4. Discussion
Fig. 2. Hsp70 protein content in the cells of S. cerevisiae after heat shock and dehydration treatments and viability of cells after dehydration: (A) yeast was grown in aerobic conditions; (B) yeast was grown in anaerobic conditions and was taken at stationary growth phase; (C) viability of yeast cells after dehydration without heat shock ( ) and after heat shock (+). Exp, exponential growth phase; Stat, stationary growth phase; C, control (yeast which has not been subjected to heat shock); HS, yeast subjected to heat shock; Compr, compressed (intact) yeast; Dry, yeast subjected to dehydration.
Fig. 3. Hsp70 protein content in the Debaryomyces hansenii cells grown in aerobic condition after heat shock and dehydration treatments (A) and viability of cells after dehydration without heat shock ( ) and after heat shock (+) (B). Exp, exponential growth phase; Stat, stationary growth phase; C, control (yeast which has not been subjected to heat shock); HS, yeast subjected to heat shock; Compr, compressed (intact) yeast; Dry, yeast subjected to dehydration.
Systematic investigations of main factors able to positively influence yeast viability during its transition to the state of anhydrobiosis reveal a number of intracellular protective systems that function in these conditions. These systems can work at the ultrastructural level as well as they can be associated with synthesis of a number of protective substances. The latter include Hsp70 chaperone whose protective activity in a variety of organisms is convincingly established. In this study we questioned whether enhanced level of Hsp70 can contribute to the protection from the deleterious effect of dehydration. First we studied profiles of Hsp70 expression in control and stressed S. cerevisiae and D. hansenii cells. The results show that Hsp70 is synthesized over exponential phase of growth in both yeast strains. The specific feature of this stage of growth is the active metabolism and intensive synthesis of different proteins. Since one of the most important roles of Hsp70 is chaperonic activity one can suggest that this property must be useful at this particular stage of yeast growth [10]. Subsequently, the reduction of total cellular protein synthesis at the stationary phase does not demand a necessity in Hsp70 synthesis. Probably it is the main reason why Hsp70 expression was not found in S. cerevisiae cells in aerobic and anaerobic conditions and in aerobic D. hansenii cells at the stationary growth phase. We further analyzed the reaction of yeast cells to a moderate heat stress. In S. cerevisiae heat shock at 42 8C induced Hsp70 in both anaerobic and aerobic conditions however in the latter case only at the exponential phase of growth. We demonstrated Hsp70 induction in heat stressed D. hansenii taken from both exponential and stationary growth phases. Since dehydration is also a strong stressor, we checked whether it can induce Hsp70 expression. The data show that this induction occurs only in S. cerevisae living in anaerobic conditions and taken from stationary growth phase. It is worth-mentioning that the cells in these conditions are extremely sensitive to drying. Therefore one can speculate that the synthesis of Hsp70 occurs only in surviving part of cells that comprises about 1% of the whole cell population but we suppose that it would be much more probable that these proteins are synthesized in the cells at the early stages of dehydration when cells are still viable. It can be concluded that unfortunately also this protective reaction does not help them to increase their viability rate. Discussing these results one significant thing should be taken in mind: yeast dehydration is comparatively long process. At least at its first stage, when cells are keeping a rest of water and which lasts approximately 9 h, the process is associated with the destruction of a number of unnecessary proteins, and this is also a part of the program ‘‘preparation of the cells to dehydration’’ [1]. Taking into account chaperonic function of Hsp70 we assume that it participates in the degradation of intracellular proteins at the early stages of drying process and simultaneously in prevention of total demolition of cells. Certainly, one can ask why there was no Hsp70 synthesis in other S. cerevisiae yeast probes subjected to
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dehydration, for instance ones taken from exponential phase. One of possible explanations may be that these cells already contain the amount of Hsp70 family proteins which is completely sufficient for the realization of both above mentioned tasks at the initiative stage of yeast drying. The changes of Hsp70 content in organisms that experience anhydrobiosis in natural conditions were reported for tardigrades Milnesium tardigradum. It was shown that three isoforms (isoforms 1, 2, 3) of this protein were expressed at the stage of their restoration from the anhydrobiosis, however only one of these (isoform 2) was expressed also when tardigrades were subjected to drying, whereas being in the active state tardigrades contained extremely low quantity of this Hsp70 [27]. Similar results were obtained using another tardigrades species, Richtersius coronifer. Total amount of Hsp70 family proteins in these organisms was low before their transfer into anhydrobiosis conditions and increased during the first hour after beginning of rehydration [28]. Generally, these results resemble our data with the only notice: we have not observed dehydration-induced Hsp70 expression but it is still possible that such phenomenon can take place at the stage of yeast reactivation and it has to be studied in the further investigations. Major goal of this study was to analyze the reaction of yeast cells with enhanced Hsp70 level on dehydration stress. It was expected that a moderate heat shock would contribute to the increase of Hsp70 and cells would be more resistant to deleterious effect of drying. However, the data show that there was no difference in viability between cells pretreated with heat shock and untreated, see Figs. 2 and 3. The lack of Hsp70-mediated protection can be explained by two reasons. First is that the amount of chaperone can be insufficient to meet the demands of a proper cell response to dehydration. As was shown above for anaerobic S. cerevisae responding to drying at the stationary phase, only a few remaining alive cells keep the rational amount of Hsp70. The same can be in case of cells that experience two sequential stresses, heat precondition and drying: only a small part of cell population can survive that is able to keep its protective resources including Hsp70 chaperone. Secondly, besides Hsp70 chaperone yeast cells acquire a variety of protective mechanisms and for some specific insults they may be much more efficient than Hsp70. It is worth-mentioning that the thermotolerance of yeast cells overexpressing different members of SSA gene family was not higher than in their control counterpart [29]. In summary we show that Hsp70 can be induced in yeast by two environmental stressors, heat shock and dehydration, however its synthesis can be rather an indicator of stress response than a part of protection mechanism. References [1] Beker MJ, Rapoport AI. Conservation of yeasts by dehydration. Advances in biochemical engineering/biotechnology, vol. 35. Berlin/Heidelberg/New York/ London/Paris/Tokyo: Springer-Verlag; 1987. pp. 127–71. [2] Rapoport AI. Rejection of areas of damaged cytoplasm by microorganisms in a state of anabiosis. Microbiology 1973;42:317–8. [3] Rapoport AI, Kostrikina NA. Cytological investigation of an anabiosis state of yeast organisms. Proc Acad Sci USSR Biology 1973;5:770–3.
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