Effect of amiodarone on thermotolerance and Hsp104p synthesis in the yeast Saccharomyces cerevisiae.
ABSTRACT Amiodarone (AMD) is known to induce a transient increase in cytosolic Ca2+ level in cells of the yeast Saccharomyces cerevisiae. In the present study the effect of AMD on the thermotolerance and Hsp104p synthesis of the yeast was studied. AMD induced Hsp104p synthesis and increased survival of the yeast after a severe heat shock (50°C). The development of thermotolerance to a considerable extent depended on the presence of Hsp104p. The same effect was achieved by treatment with the classical uncoupler CCCP, which is also known to increase the cytosolic Ca2+ level. It is supposed that the change in intracellular Ca2+ concentration plays an important role in activation of the HSP104 gene expression and in increasing the thermotolerance of the yeast. The possible link between mitochondrial activity and calcium homeostasis is discussed.
- SourceAvailable from: Eugene Rikhvanov[Show abstract] [Hide abstract]
ABSTRACT: Heat stress in plants elevates the potential across the inner mitochondrial membrane (mtΔψ) and activates the expression of heat shock proteins (HSPs). The treatment of Saccharomyces cerevisiae cells with amiodarone (AMD) elevated the cytosolic Ca2+ level ([Ca2+]cyt) in parallel with (mtΔψ) increase and led to the induction of Hsp104 synthesis. The hyperpolarization was presumably due to the increase in [Ca2+]cyt. In the present study the effects of AMD (0–100 μM) on cell viability, HSP expression, mtΔψ, and [Ca2+]cyt were investigated using the cell culture of Arabidopsis thaliana (L.) Heynh. The treatment of cultured cells with AMD led to the elevation of [Ca2+]cyt, which was accompanied by the increase in mtΔψ and by activation of HSP101 expression. The increase in [Ca2+]cyt and expression of HSP101 were also observed upon the treatment with the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone, 4 μM) known to diminish mtΔψ. The results suggest that plant cell mitochondria modulate the cytosolic Ca2+ level by changing the potential at the inner mitochondrial membrane and, thereby, participate in the retrograde regulation of HSP101 expression.Russian Journal of Plant Physiology 01/2014; 61(1). · 0.62 Impact Factor
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ABSTRACT: Mild heat stress induces the expression of heat shock proteins (HSPs) that protect plants from death during damaging heat treatments. It was assumed that the appearance in the cell of denatured proteins triggers the expression of HSP; however, recent results show that protein denaturation is not a prerequisite for this process. In this work we discuss a hypothetical mechanism for activation under heat stress of HSP expression promoted by short-term elevation of cytosolic Ca2+ level. According to our hypothesis, a prolonged elevation of Ca2+ has a negative influence on HSP expression. Therefore, calcium is transported from the cytosol into intracellular compartments, including mitochondria. The Ca2+ entry into mitochondria is accompanied by hyperpolarization of the inner mitochondrial membrane and by the increased production of reactive oxygen species (ROS). The increased ROS production contributes to the activation of HSP expression under mild heat stress but leads to plant death under severe heat shock. Thus, mitochondria and, possibly, other organelles play the crucial role in determining life or death fate of heat-treated plant cells by controlling the cytosolic Ca2+ content and ROS production.Russian Journal of Plant Physiology 01/2014; 61(2). · 0.62 Impact Factor
In all studied species, mild heat shock itself does not
cause damage, but induces heat shock protein (Hsp) syn?
thesis followed by development of resistance to subse?
quent more severe heat shock. This phenomenon is called
acquired (induced) thermotolerance . Many Hsp act as
molecular chaperones to repair cell proteins damaged by
heat . In Saccharomyces cerevisiae the protein
Hsp104p, a member of Hsp100/ClpB (caseinolytic pro?
tease B) family, is shown to play the crucial role in
acquired thermotolerance . Under the heat shock con?
ditions the HSP104 gene expression is regulated by heat
shock transcription factors Hsf1p and Msn2/Msn4p
(multicopy suppressor of SNF1 mutation), which bind
with regulatory HSE (heat shock element) and STRE
(stress responsive element), respectively, localized in its
Transcriptional activity of Msn2/Msn4p factors in S.
cerevisiae cells is downregulated by both cAMP?depend?
ent protein kinase A and the TOR (targets of rapamycin;
a rapamycin?sensitive protein kinase) signaling system
. The system elevating expression of HSP genes by
transcriptional activation of Hsfp is present in all studied
species. It was thought for a long time that the Hsfp tran?
scriptional activity in the absence of heat shock is sup?
pressed due to downregulation caused by interaction
between Hsp70p (heat shock protein 70) and Hsp90p
(heat shock protein 90). This hypothesis supposed that
the level of denatured proteins in the cell increases under
the heat shock. The proteins Hsp70p and Hsp90p are
released from their complex with Hsfp to react with dena?
tured proteins, which results in activation of Hsfp .
However, recent data suggests that this hypothesis is too
simple to describe all the diversity of factors influencing
transcription activity of Hsfp . It becomes obvious that
reactive oxygen species (ROS)  and Ca2+ can have
a significant effect on Hsfp activation and, hence, on
HSP expression in mammalian and plant cells.
In plant  and mammalian  cells heat shock
causes a short?term increase in cytosolic Ca2+level. One
can expect that a similar effect can be also observed in S.
cerevisiae . Earlier, we demonstrated that exogenous
ISSN 0006?2979, Biochemistry (Moscow), 2012, Vol. 77, No. 1, pp. 78?86. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © I. V. Fedoseeva, D. V. Pjatricas, N. N. Varakina, T. M. Rusaleva, A. V. Stepanov, E. G. Rikhvanov, G. B. Borovskii, V. K. Voinikov, 2012, published
in Biokhimiya, 2012, Vol. 77, No. 1, pp. 99?109.
Abbreviations: AMD, amiodarone (2?butyl?3?(3,5?diiodo?4?
diethylaminoethoxybenzoyl)?benzofuran); CCCP, carbonyl
cyanide 3?chlorophehylhydrazone; FDA, fluorescein diacetate;
Hsf, heat shock factor; Hsp, heat shock protein; mt∆ψ, trans?
membrane potential on the inner mitochondrial membrane;
PCD, programmed cell death; PI, propidium iodide; ROS,
reactive oxygen species.
* To whom correspondence should be addressed.
Effect of Amiodarone on Thermotolerance and Hsp104p
Synthesis in the Yeast Saccharomyces cerevisiae
I. V. Fedoseeva*, D. V. Pjatricas, N. N. Varakina, T. M. Rusaleva,
A. V. Stepanov, E. G. Rikhvanov, G. B. Borovskii, and V. K. Voinikov
Siberian Institute of Plant Physiology and Biochemistry, Siberian Division of the Russian Academy of Sciences,
ul. Lermontova 132, 664033 Irkutsk, Russia; fax: (3952) 517?054; E?mail: firstname.lastname@example.org
Received June 7, 2011
Revision received July 19, 2011
Abstract—Amiodarone (AMD) is known to induce a transient increase in cytosolic Ca2+level in cells of the yeast
Saccharomyces cerevisiae. In the present study the effect of AMD on the thermotolerance and Hsp104p synthesis of the yeast
was studied. AMD induced Hsp104p synthesis and increased survival of the yeast after a severe heat shock (50°C). The
development of thermotolerance to a considerable extent depended on the presence of Hsp104p. The same effect was
achieved by treatment with the classical uncoupler CCCP, which is also known to increase the cytosolic Ca2+level. It is sup?
posed that the change in intracellular Ca2+concentration plays an important role in activation of the HSP104 gene expres?
sion and in increasing the thermotolerance of the yeast. The possible link between mitochondrial activity and calcium
homeostasis is discussed.
Key words: amiodarone, Saccharomyces cerevisiae, thermotolerance, Hsp104p, mitochondrion
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Ca2+induces Hsp104p synthesis in yeast cells and elevates
their thermotolerance . This suggests that in yeasts,
like in plants and mammals, Ca2+plays an important role
in both activation of HSP gene expression and develop?
ment of acquired thermotolerance. To test this hypothe?
sis, here we studied the effect of amiodarone (AMD), a
substance elevating Ca2+level at ordinary temperatures,
on HSP synthesis and thermotolerance in the yeast S.
cerevisiae. As previously reported [12?14], AMD causes a
short?term elevation of Ca2+level in the yeast cell cytosol.
MATERIALS AND METHODS
Strains and growth conditions. The yeast S. cerevisiae,
parent type strain Ψ?74?D694 (MATa, ade1?14(UGA),
trp1?289(UAG), his3∆?200, ura3?52, leu2?3, 112 [psi–])
and its isogenic mutant Ψ?74?D694::hsp∆?1L (MATa,
ade1?14(UGA), trp1?289(UAG), his3∆?200, ura3?52,
HSP104::LEU2), were kindly provided by Susan L.
Lindquist (Whitehead Institute for Biomedical Research,
USA); we also used the strain W303?1A (MATa, ade2?1,
ura3?1, his3?11, 15 leu2?3, 112 trp1?1, can1?100, SUC2).
The yeast cells were maintained at 30°C on the YEPD
(yeast extract, peptone, dextrose) medium containing
0.5% yeast extract, 1% peptone, 2% glucose, and 1.5%
agar?agar. The cells were grown at 30°C in 100?ml
Erlenmeyer flasks with 25 ml of liquid YEPD or YEPE
(yeast extract, peptone, ethanol; 2% ethanol was added
instead of glucose), and cells in logarithmic growth phase
were used in experiments.
Fluorescence microscopy. The count rates of live and
dead cells were determined from the fluorescence of flu?
orescein diacetate (FDA) and propidium iodide (PI) fol?
lowing incubation of the cells for 2 min with these dyes
taken at concentrations of 50 µM and 10 µg/ml, respec?
tively. The inner mitochondrial membrane potential was
qualitatively visualized using the potential?dependent
cationic dye TMRM (tetramethylrhodamine methyl and
ethyl esters) at the final concentration of 5 µM. The data
were recorded following 10?min incubation of the cells
with the dye. Fluorescence microscopy was carried out
using an AxioObserverZ1
(Germany) equipped with an AxioCamMRm3 digital
Counting of colony?forming units (CFU). To count
CFU, serial dilutions of yeast cells were seeded on
agarized YEPD medium. Following 24?48?h incubation
at 30°C, the number of colonies was counted.
Isolation of total protein and immunoblotting. The
cells were centrifuged, washed thrice with distilled water,
and stored at –70°C before experiments. The cells were
thawed, resuspended in isolation buffer (0.1 M Tris?HCl,
pH 7.4?7.6, containing 3 mM SDS and 1 mM β?mercap?
toethanol), frozen in liquid nitrogen, and ground with
and software package
quartz sand. Crude cell components were removed by
centrifugation (15,000g for 15 min), and the protein was
treated with three volumes of cold acetone. The pellet was
washed thrice with acetone and dissolved in sample buffer
(0.625 M Tris?HCl, pH 6.8, containing 8 mM SDS,
0.1 M β?mercaptoethanol, 10% glycerol, and 0.001%
bromophenol blue). Protein concentration was deter?
mined by the Lowry method. Following SDS?PAGE in
12% polyacrylamide gel, immunoblotting was carried out
using antibodies against Hsp104p (SPA?8040; Stressgen,
USA) and against Hsp60p (US Biological H1830?77B,
All experiments were repeated no less than three
times. The data were statistically processed for the mean
values and standard deviations.
Yeast cell death under mild heat shock following the
treatment with AMD. AMD, which is used in clinical
practice as an antiarrhythmic drug, is a potent fungicide.
Taken at micromolar concentrations, it inhibits growth
and causes death of S. cerevisiae cells [12?14]. To deter?
mine toxic effect of AMD on the yeast cells, we estimat?
ed their survival by counting colonies on a solid medium
after 48?h growth at 30°C (Fig. 1a) as well as by staining
with FDA and PI directly after incubation with AMD
(Fig. 1b). FDA and PI stain live and dead cells, respec?
In accordance with the literature, 60?min treatment
with AMD resulted in death of S. cerevisiae W303?1A
cells (Fig. 1), the level increasing with increasing AMD
concentrations. Estimations of cell survival by CFU
counting 48 h after the treatment (Fig. 1a) and by count?
ing FDA?positive and PI?negative cells directly after the
treatment (Fig. 1b) gave similar results. Some difference
between these data was observed at AMD concentrations
of 20 and 50 µM.
It was reported elsewhere  that AMD caused
programmed cell death (PCD) of yeast cells due to
increase in ROS production, DNA fragmentation, and
cytochrome c release from mitochondria. Damaging heat
shock can also lead to PCD of cultured plant cells. The
hallmark of PCD in plants is time course of viability
decrease [16, 17]. The cells remained alive directly after
the heat shock and died after 24?48 h incubation at the
normal temperature. So, to compare the character of cell
death after treatment with AMD and under heat shock
conditions, we studied yeast cell survival after heat shock
of varied intensity.
The live cell count by the two methods described
above demonstrated that incubation at both 39 and 42°C
for 60 min has no effect on the yeast cell survival. Cell
death began to be observed when the temperature was
increased to 45°C (Fig. 2, a and b). However, in this case
80FEDOSEEVA et al.
BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012
some difference was seen in the data obtained by two
methods. The CFU counting demonstrated that only
10% of the cells formed colonies (Fig. 2a), whereas >50%
were stained with FDA as live directly after the heating
(60 min at 45°C) (Fig. 2b). These data suggest the time
progression of cell death induced by the heat shock at
45°C. To verify this, we subjected the S. cerevisiae Ψ?74?
D694 cells to mild heat shock (45°C, 30 min) and moni?
Fig. 1. Characteristics of S. cerevisiae cell death following treatment with AMD. Survival of the cells was estimated by CFU counting 48 h after
the treatment with AMD (a) and by counting FDA?positive and PI?negative cells directly after the treatment (b).
5 1020 50100
FDA+, PI–, %
Fig. 2. Effect of different temperatures on survival of S. cerevisiae W303?1A cells. Cells cultured on YEPD medium were incubated for 60 min
at 30, 39, 42, or 45°C. Survival was determined by CFU counting 24?48 h after the incubation (a) and by staining with FDA and PI (b). c)
Saccharomyces cerevisiae Ψ?74?D694 cells were incubated at 45°C for 30 min, and survival was determined by staining with FDA and PI 0, 2,
8, 24, and 96 h after further incubation at 30°C.
39 42 4530 39
FDA+, PI–, %
FDA+, PI–, %
EFFECT OF AMIODARONE ON S. cerevisiae THERMOTOLERANCE81
BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012
tored percentage of live and dead cells during incubation
(2?96 h) at 30°C by staining with FDA and PI. Directly
after the heat shock 100% of the cells were stained as live
(Fig. 2c). The same result was obtained 2 h after incuba?
tion at 30°C. Dead cells only appeared after 8 h incuba?
tion, and their share reached 90% after 24 h. Following
96?h incubation the level of live cells began to increase,
suggesting that the cells outliving the stress had begun to
divide. Thus, the data has shown that, like cultured plant
cells [16, 17], yeast cells die with time following mild heat
chock at 45°C.
AMD induces synthesis of Hsp104p. Mild heat shock
causes a short?term elevation of Ca2+level in cells of
plants  and animals . It is likely that a similar event
also occurs under heat shock in S. cerevisiae . It is
arguable that elevation of intracellular Ca2+is in causal
connection with expression of the HSP genes [8, 9]. It is
known that AMD leads to increase in Ca2+concentration
in the yeast cell cytosol [12?14]. The treatment with
AMD at 30°C for 60 min induced Hsp104p synthesis that
was more prominent with increase in concentration of
AMD (Fig. 3a). A distinct increase in amount of Hsp104p
was observed at AMD concentrations of 20, 50, and
100 µM. The induction of Hsp104p synthesis following
heat shock at 39°C was very much more than that follow?
ing the treatment with AMD taken at any of the tested
concentrations (Fig. 3a).
Despite the fact that AMD produced lethal effect on
the yeast cells (Fig. 1), it induced Hsp104p synthesis
under the same conditions (Fig. 3a). Hence, the yeast
cells dying from AMD continue to express Hsps. As we
showed earlier on cultured Arabidopsis thaliana cells,
induction of various Hsps, such as Hsp101p, Hsp70p, and
Hsp17.6p (class I) and Hsp17.6p (class II) was only
observed on heating causing no negative effect on survival
. This suggests that activation of HSP expression and
death under severe heat shock are mutually exclusive. To
make sure this suggestion is correct for the yeast cell, we
performed a similar experiment. One can see in Fig. 3b
that Hsp104p synthesis is induced at 39 and 42°C, while
elevation of the temperature to 45°C inhibits synthesis of
this protein. The data presented in Fig. 2a indicates that
the temperatures 39 and 42°C had no effect on the yeast
cell survival, whereas incubation at 45°C significantly
decreased it. Thus, induction of Hsp104p synthesis in
yeast is suppressed when the temperature high enough to
affect their survival.
AMD elevates yeast thermotolerance that depends on
the presence of Hsp104p. Protein Hsp104p plays a crucial
role in development of yeast cell thermotolerance .
Since AMD induces Hsp104p synthesis, we studied how
this substance influences the yeast cell ability for oppos?
ing the effect of severe heat shock. To do this, we used the
parent strain Ψ?74?D694 and its isogenic HSP104 mutant
(hsp104∆) characterized by absence of Hsp104p synthesis
at 39°C (Fig. 4a). In these experiments AMD was used at
concentration 20 µM. The yeast cells were treated with
AMD at 30°C for 30 min, washed, and subjected to severe
heat shock at 50°C. Previously, we had found that AMD
itself did not affect the cell viability of the tested strains
(data not shown). As follows from Fig. 4b, pretreatment
with AMD led to drastic increase in thermotolerance of
the parental strain. Under the same conditions, thermo?
tolerance of hsp104∆ mutant also increased, but to a far
less extent. Thus, both AMD and mild heat stress protect
the yeast cells from death under severe heat shock.
Moreover, this effect depends on the presence of
CCCP induces synthesis of Hsp104p and elevates
thermotolerance of yeast cells. We previously reported that
under heat stress mitochondrial inhibitors and uncouplers
suppressed both induction of Hsp synthesis and develop?
ment of acquired thermotolerance in plant  and yeast
 cells. However, at the ordinary temperature these
inhibitors in some cases could elevate thermotolerance
and induce synthesis of Hsps [17, 18]. Like AMD, the
classical uncoupler CCCP (carbonyl cyanide 3?
chlorophehylhydrazone) is known to elevate Ca2+con?
centration in S. cerevisiae cells .
Taken at concentration of 20 µM, CCCP inhibited
induction of Hsp104p synthesis caused by mild heat stress
(39°C) (Fig. 5a), thus supporting previously reported data
Fig. 3. Effects of AMD and different temperature on Hsp104p synthesis. a) Induction of Hsp104p synthesis following treatment with AMD.
Saccharomyces cerevisiae W303?1A cells were treated with 0?100 µM AMD at 30°C for 60 min (K30 – control). For comparison, synthesis of
Hsp104p and Hsp60p at 39°C for 60 min (K39) is shown on the right. b) Synthesis of Hsp104p and Hsp60p following the incubation of yeast
cells for 60 min at different temperatures.
К30 5 10 20 50 100 К39
30°C 39°C 42°C 45°C
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BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012
. At 30°C both 20 µM CCCP and 20 µM AMD taken
separately considerably elevated Hsp104p synthesis, but
their combined effect was negligible. A similar result was
obtained when we studied thermotolerance of yeasts
treated with CCCP and AMD at 30°C and then subjected
to severe heat shock at 50°C. CCCP and AMD taken sep?
arately considerably increased survival of the yeast cells,
whereas their combination decreased thermotolerance
below that of the control (Fig. 5b).
AMD elevates fluorescence of TMRM. It was shown
using cultured cells of mammals , plants , and
yeasts  that mild heat stress elevates the transmem?
brane potential on the inner mitochondrial membrane
(mt∆ψ). AMD caused a similar increase in mt∆ψ in S.
cerevisiae cells as determined from change in fluores?
cence of potential?dependent probes TMRM  and
JC?1 . In accordance with the literature data [12, 20],
the treatment of S. cerevisiae W303?1A cells with AMD
for 10 min at 30°C led to increase in TMRM fluores?
cence, and this was more prominent as the AMD con?
centration was increased (Fig. 6a). These data suggest
that AMD elevates mt∆ψ in S. cerevisiae cells.
Saccharomyces cerevisiae is a facultative aerobe that
meets ~75% of its energy requirements by fermentation
(when grown on glucose). Under these conditions its res?
piration is lowered, although not completely repressed.
When grown on non?fermentable carbon source, particu?
larly ethanol, yeast cells obtain energy only by oxidative
phosphorylation . Because of this TMRM fluores?
cence of yeast cells was significantly lower when grown on
Fig. 4. Effect of AMD on synthesis of Hsp104p and thermotolerance of S. cerevisiae cells. a) Synthesis of Hsp104p and Hsp60p under mild
heat stress (39°C). b) Survival of the cells after a severe heat shock (50°C). The Ψ?74?D694 (parental type) and hsp104∆ mutant cells were incu?
bated with 20 µM AMD for 30 min at 30°C, washed free from AMD, and subjected to the heat shock (50°C).
30°C 39°C 30°C 39°C 30°C 39°C
W303?1A ψ?74?D694 hsp104∆
K AMD K AMD
Fig. 5. Effect of AMD and CCCP on thermotolerance and synthesis of Hsp104p in S. cerevisiae. Cells were treated for 30 min at 30 or 39°C
either in the absence of additives (K) or in the presence of 20 µM AMD, 20 µM CCCP (C), or 20 µM AMD plus 20 µM CCCP (AMD + C).
a) Synthesis of Hsp104p and Hsp60p. b) Survival after heat shock (50°C) determined from CFU number.
К AMD С AMD + С К С
AMD + С
EFFECT OF AMIODARONE ON S. cerevisiae THERMOTOLERANCE83
BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012
Fig. 6. Effect of AMD on TMRM fluorescence in yeast cells. a) Dependence of TMRM fluorescence level in S. cerevisiae W303?1A cells on
concentration of AMD. The cells were treated with 0?100 µM AMD for 60 min at 30°C on YEPD medium. b) Intensity of TMRM fluores?
cence (c) following incubation of the cells in presence of 20 µM AMD or without it at 30°C (K30) or 39°C (K39) in YEPD (containing glu?
cose) or YEPE (containing ethanol) medium. DIC, differential interference contrast microscopy. d, e) Effects of AMD, antimycin A, and
CCCP on the TMRM fluorescence of yeast cells depending on the energy source. Saccharomyces cerevisiae W303?1A cells were incubated for
10 min at 30 or 39°C in the absence (K30 and K39) or in the presence of 20 µM AMD (Am), 10 µM antimycin A (A30), 20 µM CCCP (C30),
20 µM AMD and 10 µM antimycin A (Am + A), or 20 µM AMD plus 20 µM CCCP (Am + C).
510 20 50 100
Fluorescence, arbitrary units
Fluorescence, % of control
К30 AMD К39 К30 AMD К39
К30 AMDК39 К30AMD
Fluorescence, % of control
К30 А30 С30 Аm Аm+А Аm+СК39
К30 А30 С30 Аm Аm+А Аm+СК39
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BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012
the medium with glucose than on the medium with
ethanol (Fig. 6, b and c). Similar data were reported ear?
lier by other authors who showed that fluorescence of
another potential?dependent dye, rhodamine 123,
depends on whether oxidative or fermentative metabo?
lism is occurring in yeasts . During fermentation,
mt∆ψ in yeasts can be maintained due to the electrogenic
transport of (ATP)4–in exchange for (ADP)3–across the
inner mitochondrial membrane by adenine nucleotide
translocator . Elevation of TMRM fluorescence in
yeast cells grown on ethanol as the energy source suggests
that alteration of TMRM fluorescence correctly reflects
alteration of mt∆ψ.
AMD and heat stress elevated TMRM fluorescence
independently of the energy metabolism type. AMD at
concentration 20 µM caused about the same elevation as
mild heat stress at 39°C (Fig. 6, b and c). The
protonophore CCCP effectively suppressed elevation of
TMRM fluorescence induced by AMD independently of
the energy metabolism type (Fig. 6, d and e). However,
antimycin A insignificantly inhibited elevation of TMRM
fluorescence on glucose?containing medium (Fig. 6d),
but its inhibitory effect was the same as that of CCCP on
ethanol?containing medium (Fig. 6e). Thus, we suppose
that mechanisms of mt∆ψ elevation in response to AMD
differ depending on the energy metabolism type. Earlier,
we obtained a similar result when we studied the effect of
heat shock on mt∆ψ .
In accordance with the literature [12?14], AMD
decreases yeast viability (Fig. 1). The mechanism of
AMD toxicity is not completely understood. AMD is
known to induce a short?term elevation of Ca2+concen?
tration in yeast cell cytosol [12?14]. Since excessive eleva?
tion of internal Ca2+level can result in cell death, the tox?
icity of AMD is supposed to be associated with the eleva?
tion in concentration of cytosolic Ca2+up to its danger?
ous level . In fact, a direct correlation has been shown
between the ability of AMD to elevate cytosolic Ca2+level
and its lethal effect on the yeast cell [13, 14]. However,
this point of view is in disagreement with the results sug?
gesting substantial elevation of AMD toxic effect on yeast
due to inhibition of Ca2+influx into the cytosol by Ca2+
chelators, and on the contrary, a protective effect of
exogenous Ca2+. Hence, when cells are treated with
AMD the elevation of Ca2+content in their cytosol is
necessary for elevation of yeast resistance against the
stress. It is very likely that both points of view are correct.
Calcium ion plays a dual role in the cells of any organism
. The elevation of Ca2+concentration above its cer?
tain critical level leads to cell death. On the other hand,
Ca2+induces an adaptive reaction and development of
It is obvious that, under the conditions of our exper?
iments, the mechanism of yeast cell death following the
treatment with AMD differs from that under mild heat
shock. The used AMD concentrations caused rapid cell
death. This is supported by the data on cell viability after
24?h incubation (Fig. 1a) and immediately after the treat?
ment with AMD (Fig. 1b). However, yeast cell death after
mild heat shock develops with time (Fig. 2c).
Despite its toxic effect, AMD induces synthesis of
Hsp104p in S. cerevisiae cells (Fig. 3a). On the other hand,
heat shock inducing the yeast cell death (45°C) did not
result in induction of Hsp104p synthesis (Fig. 3b).
Arabidopsis thaliana cells also did not synthesize Hsps
when elevation in heat treatment intensity decreased their
viability . Thus, in the case of treatment with AMD a
reverse feedback is absent between Hsp synthesis in yeast
cells and their death. Possible causes of this difference
might be the different dynamics of cell death under treat?
ment with AMD (Fig. 1) and heat shock (Fig. 2).
Nevertheless, the treatment with AMD at a concentration
that does not lead to cell death not only induced the syn?
thesis of Hsp104p, but also increased thermotolerance of
the cells. Development of thermotolerance under AMD
treatment also depended on the presence of functional
Hsp104p (Fig. 4b), additionally supporting the leading role
of this protein in development of yeast thermotolerance.
Treatment with exogenous Ca2+induces Hsp synthe?
sis and elevates thermotolerance in yeasts  and plants
. A similar effect can be achieved by treatment of the
yeast with AMD or the typical uncoupler CCCP (Fig. 5).
AMD is well known to induce a short?term elevation of
cytosolic Ca2+content in S. cerevisiae [12?14]. CCCP has
a similar effect . Thus, the results support the suppo?
sition  that calcium can play an important role in acti?
vation of HSP104 expression and development of yeast
ability to resist the damaging effect of heat shock.
As we mentioned above, HSP104 gene expression
under heat stress is regulated by Hsfp and Msn2/Msn4p
transcriptional factors . It was shown that exogenous
Ca2+and AMD activate expression of 54 genes in S. cere?
visiae cells, most of them being under the control of
Msn2/Msn4p . We suppose that the ability of AMD to
induce Hsp104p synthesis (Figs. 3a and 5a) depends on
Msn2/Msn4p functioning, and the activity of these tran?
scriptional factors depends on the presence of Ca2+. The
latter supposition is confirmed by data demonstrating the
activation of Msn2/Msn4?dependent expression of the S.
cerevisiae GPX1 gene encoding glutathione peroxidase
under treatment with CaCl2.
AMD causes transient elevation in the level of yeast
cytosolic Ca2+[12?14]. AMD also induces TMRM fluo?
rescence increase in yeast cells, which may suggest the
elevation of mt∆ψ (Fig. 6). The ability of AMD to hyper?
polarize the yeast inner mitochondrial membrane has
been demonstrated earlier [12, 20]. The authors of those
works supposed that elevated Ca2+content stimulates
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BIOCHEMISTRY (Moscow) Vol. 77 No. 1 2012
activity of respiratory enzymes and elevates the coupling
of respiration and energy production, thus resulting in
elevation of mt∆ψ.
As a rule, the increase in cytosolic Ca2+in mam?
malian cells is accompanied by the elevation of mt∆ψ
. This is because mitochondria serve as a store for
intracellular Ca2+in mammalian cells. Transport of Ca2+
into the mitochondria is determined by the value of
mt∆ψ. Being inside the mitochondria, Ca2+activates
pyruvate dehydrogenase, 2?oxoglutarate dehydrogenase,
and NAD+?isocitrate dehydrogenase. This, in turn, stim?
ulates respiration, increases ATP production, and, as a
result, elevates mt∆ψ. In contrast, none of the listed
enzymes are activated by Ca2+in S. cerevisiae .
Moreover, the system of active transport of Ca2+was not
found at all in S. cerevisiae mitochondria . Hence, it
remains unknown whether elevation of Ca2+level in the
S. cerevisiae cell can influence activity of mitochondria.
So, the data on the effect of AMD on elevation of mt∆ψ
in S. cerevisiae cells require further careful consideration
and confirmation by other studies.
Nonetheless, some literature data suggest that an
association is still exists between alteration of intracellu?
lar Ca2+level and mitochondrial function in S. cerevisiae
cells. As we noted above, the classical mitochondrial
uncoupler CCCP caused elevation of Ca2+concentration
in yeast cytosol. Glycerol had the same effect, and
antimycin A inhibited the effect of glycerol on the Ca2+
level . Deletion of the PMR1 gene encoding
Ca2+/Mn2+ATPase increased intracellular Ca2+concen?
tration  and simultaneously affected respiration .
It is likely that in S. cerevisiae cells Ca2+can indirectly
influence the mitochondrial activity by modulating the
TOR signaling system. This system is known to influence
activity of mitochondria in yeasts and depend on the
presence of Ca2+. This supposition is confirmed by
the fact that the patterns of S. cerevisiae gene expression
in response to AMD, exogenous Ca2+, and rapamycin (an
inhibitor of the TOR signaling system) are quite similar
. When analyzing mechanisms by which the intracel?
lular Ca2+can influence the activity of yeast mitochon?
dria, one must take into account possible contacts
between the mitochondrion and endoplasmic reticulum,
which can facilitate transduction of Ca2+?signal .
Thus, we cannot exclude that elevation of Ca2+in
response to AMD might influence indirectly rather than
directly the activity of yeast mitochondria and, corre?
spondingly, the change in mt∆ψ.
Like AMD, heat stress at 39°C leads to increase in
TMRM fluorescence in S. cerevisiae cells (Fig. 3, see
), as well as in cultured cells of A. thaliana .
Similar data were obtained using mammalian cell culture
. The authors of work  showed that both heat shock
and agents causing lipid alterations in plasma membrane
lead to elevation of Ca2+level in cytosol and hyperpolar?
ization of the inner mitochondrial membrane, which is
accompanied by expression of HSP genes. It was hypoth?
esized [9, 17, 18] that elevation of mt∆ψ can be the early
response of the cell to stress, which leads to further acti?
vation of HSP gene expression.
The data of the present work confirm this hypothesis.
There is a direct correlation between elevation of TMRM
fluorescence and induction of Hsp104p synthesis in S.
cerevisiae cells under treatment with AMD and under
heat shock. Elevation of mt∆ψ in mitochondria of mam?
malian  and yeast  cells correlates with elevation
of ROS production. At the same time, it is likely that ele?
vation of ROS production is an important element in
activation of HSP gene expression in yeasts  and
plants . The elevation of Ca2+level might indirectly
stimulate activity of yeast mitochondria and elevate
mt∆ψ. Elevation of mt∆ψ leads to enhancement of ROS
production, which, in turn, activates HSP gene expres?
sion. It is worth noting that a tight association exists
between the change in intracellular Ca2+level and ROS
production in the living cell .
This study was supported by the Russian Foundation
for Basic Research grant No. 10?04?00921?a.
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