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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 Laboratory of Cell Protective Mechanisms, Institute of Cytology, Russian Academy of Science, Saint Petersburg, Russiab Laboratory of Cell Biology, Institute of Microbiology and Biotechnology, University of Latvia, LV-1586 Riga, Latvia
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 rehydra-
tion 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 andreversible reduction of metabolism and also by a
variety of changes at biochemical and functional levels [1]. The
latter include condensation of chromatin, separation by mem-
branes of rather big parts of nucleus and damaged areas of
cytoplasm [24], synthesis of trehalose and polyols [57],
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
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 [1012]. Protective power of Hsp70 thought to be
linked to its chaperonic activity is proved by studies on hundreds
cell and animal models.
Thegenome ofSaccharomyces cerevisiae yeast contains 14 genes
comprising multigene Hsp70 family proteins [13]. This protein
family includes mitochondrial proteins Ssc1 and Ssc1p [1417],
cytosolicproteins Ssa1, Ssa1p,Ssa2 andSsa4pwhich accumulate in
cell nucleus during yeast starvation[18]. As in other organism in
yeast Hsp70 chaperones facilitate endoplasmic reticulum-asso-
ciated 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;
Process Biochemistry 43 (2008) 11381141
A R T I C L E I N F O
Article history:
Received 20 December 2007
Received in revised form 9 April 2008
Accepted 22 May 2008
Keywords:
Hsp70
Anhydrobiosis
Dehydrationrehydration
Protective reactions
Yeast
Saccharomyces cerevisiae
Debaryomyces hansenii
A B S T R A C T
Yeast is known to experience in natural and industrial conditions cycles of dehydrationrehydration.
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,
andthere wasno marked changein theprotein level at thestationary phaseboth in aerobic andanaerobic
conditions. Interestingly, dehydration of sensitive to this kind of stressS. 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.
2008 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +371 67034891; fax: +371 67227925.
E-mail address: [email protected](A. Rapoport).
Contents lists available atScienceDirect
Process Biochemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o
1359-5113/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.05.012
mailto:[email protected]://www.sciencedirect.com/science/journal/13595113http://dx.doi.org/10.1016/j.procbio.2008.05.012http://dx.doi.org/10.1016/j.procbio.2008.05.012http://www.sciencedirect.com/science/journal/13595113mailto:[email protected]8/10/2019 Deshidratarea drojdiei
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it is also noteworthy that high amount of the chaperone was found
in cells subjectedto deuteriumoxideor 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 yeastS. cerevisiae14 (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. cerevisiaeandD. hansenii) and without shaking
witha great excess of nutrient medium for anaerobicconditions (only S. cerevisiae).
Nutrient medium contained (in g l1): MgSO40.7; NaCl 0.5; (NH4)2SO43.7; KH2PO41.0; K2HPO4 0.13; molasses43 (till finalconcentration of glucose20 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 spectro-
photometric determination of suspensions 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 810%, 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 810%. 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 methodgives 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.
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 disintegrationin
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 fromdisintegrated 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, 50mg, were applied onto lanes of 10% polyacrylamide
gel. Electrophoresis was performed with a voltage gradient of 5 V cm1 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
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.
Theanalysis of Hsp70 expression during the S. cerevisiae growth
was performed in samples taken each 4 h after the cells had been
seededin 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 corre-
sponded 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
growthat time point 18 h after seeding, andthe reductionof Hsp70
level revealed that despite a strong lowering of cellular metabo-
lism 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 theexponential growthphaseand subjectedto
dehydration. It was found that drying led to a complete reductionof Hsp70 level (Fig. 2A). Viability of these cells was also found to be
at very low level14.8 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 toS. cerevisae these cells were found to contain Hsp70 at
the exponential phase of growth and not at the stationary phase
(Fig. 3A). Dehydration of yeastD. 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 comprised5560%. Lastly
dehydration of D. hansenii cells taken from stationary growth
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.
I. Guzhova et al./ Process Biochemistry 43 (2008) 11381141 1139
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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 pre-
conditioning 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
cellsS. cerevisiaebut did not increase cell viability (Fig. 2C). Heat
shock ofD. hanseniicells also did not result in the enhancement of
their survival despite the significant elevation of Hsp70 level
(Fig. 3A and B).
4. Discussion
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 protectionfrom 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 yeastcells to a moderate heat stress. In S. cerevisiaeheat 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 stressedD. 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 ofdehydration 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 shouldbe taken in
mind: yeast dehydration is comparatively long process. At least at
its first stage,whencells are keeping a rest of water and which lasts
approximately 9 h, theprocess is associated with thedestructionof
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
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 heatshockand dehydrationtreatments (A)and viabilityof cellsafter
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.
I. Guzhova et al./ Process Biochemistry 43 (2008) 113811411140
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dehydration, for instance ones taken from exponential phase. One
of possible explanationsmay be that these cells already contain the
amount of Hsp70 family proteins which is completely sufficient for
therealizationof both above mentionedtasksat theinitiative 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) wasexpressed also when tardigrades were subjectedto
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 wasexpected 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 therational 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 chaperoneyeast cells acquire a variety of protective mechanisms andfor some
specific insults they may be much more efficient than Hsp70. It is
worth-mentioning that the thermotolerance of yeast cells over-
expressing 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 anddehydration, however its synthesis can be ratheran
indicator of stress response than a part of protection mechanism.
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