Báo cáo khoa học: Exogenous oxidative stress induces Ca2+ release in the yeast Saccharomyces cerevisiae potx

Exogenous oxidative stress induces Ca
2+
release in the
yeast Saccharomyces cerevisiae
Claudia-Valentina Popa, Ioana Dumitru, Lavinia L. Ruta, Andrei F. Danet and Ileana C. Farcasanu
Faculty of Chemistry, University of Bucharest, Romania
Introduction
Eukaryotic cells, from yeast to mammals, respond and
adapt to environmental stress by evolutionarily con-
served multicomponent endogenous systems that utilize
a network of signal transduction pathways to regulate
the adaptive and protective phenotype. Changes in the
chemical or physical conditions of the cell that impose
a negative effect on growth demand rapid cellular
responses, which are essential for survival. Molecular
mechanisms induced upon exposure of cells to such
adverse conditions are commonly designated as stress
responses.
Ca
2+
-mediated signaling of stress conditions is used
by virtually every eukaryotic cell to regulate a wide
variety of cellular processes through transient increases
in cytosolic Ca
2+
. Budding yeast (Saccharomyces cere-
visiae) cells use Ca
2+
as a second messenger when they
are exposed to various environmental stress conditions,
such as hypotonic and cold stress [1,2], hyperosmotic

and salt stress [3], b-phenylethylamine-induced intracel-
lular H
2
O
2
generation [4], or high pH [5]. The increase
in cytosolic Ca
2+
can be a consequence of external
Ca
2+
influx via the Cch1p ⁄ Mid1p Ca
2+
channel on
the plasma membrane [1,3,6] or release of vacuolar
Ca
2+
into the cytosol through the vacuole-located
Ca
2+
channel Yvc1p [7,8]. After acting as a second
messenger, cytosolic Ca
2+
is restored to the normal
very low levels through the action of Ca
2+
pumps and
exchangers. Thus, the Ca
2+
-ATPase Pmc1p [9,10] and

the vacuolar Ca
2+
⁄ H
+
exchanger Vcx1p [11,12] inde-
pendently transport cytosolic Ca
2+
into the vacuole,
whereas Pmr1p, the secretory Ca
2+
-ATPase, pumps
cytosolic Ca
2+
into the endoplasmic reticulum and
Keywords
aequorin; cytosolic calcium; oxidative stress;
Saccharomyces cerevisiae; yeast
Correspondence
I. C. Farcasanu, University of Bucharest,
Faculty of Chemistry, Sos. Panduri 90-92,
Bucharest, Romania
Fax: +40 021 410 01 40
Tel: +40 721067169
E-mail:
(Received 16 April 2010, revised 2 July
2010, accepted 28 July 2010)
doi:10.1111/j.1742-4658.2010.07794.x
The Ca
2+
-dependent response to oxidative stress caused by H

2
O
2
or tert-
butylhydroperoxide (tBOOH) was investigated in Saccharomyces cerevisiae
cells expressing transgenic cytosolic aequorin, a Ca
2+
-dependent photopro-
tein. Both H
2
O
2
and tBOOH induced an immediate and short-duration
cytosolic Ca
2+
increase that depended on the concentration of the stres-
sors. Sublethal doses of H
2
O
2
induced Ca
2+
entry into the cytosol from
both extracellular and vacuolar sources, whereas lethal H
2
O
2
shock mobi-
lized predominantly the vacuolar Ca
2+

. Sublethal and lethal tBOOH
shocks induced mainly the influx of external Ca
2+
, accompanied by a more
modest vacuolar contribution. Ca
2+
transport across the plasma membrane
did not necessarily involve the activity of the Cch1p ⁄ Mid1p channel,
whereas the release of vacuolar Ca
2+
into the cytosol required the vacuolar
channel Yvc1p. In mutants lacking the Ca
2+
transporters, H
2
O
2
or tBOOH
sensitivity correlated with cytosolic Ca
2+
overload. Thus, it appears that
under H
2
O
2
-induced or tBOOH-induced oxidative stress, Ca
2+
mediates
the cytotoxic effect of the stressors and not the adaptation process.
Abbreviations

BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N ¢,N ¢-tetraacetic acid; tBOOH, tert-butylhydroperoxide; WT, wild-type.
FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4027
Golgi, and is responsible for Ca
2+
extrusion from the
cell [13,14]. These responses are mediated by the uni-
versal Ca
2+
sensor protein calmodulin, which can bind
and activate calcineurin; this inhibits, at the post-tran-
scriptional level, the function of Vcx1p [11,15,16] and
induces the expression of PMC1 and PMR1 genes via
activation of the Crz1 transcription factor [15,16]. The
release of Ca
2+
from intracellular stores stimulates
extracellular Ca
2+
influx, a process known as capacita-
tive calcium entry [17]. Conversely, the release of vacu-
olar Ca
2+
via Yvc1p can be further stimulated by
Ca
2+
from outside the cell as well as that released
from the vacuole by Yvc1p itself in a positive feedback
process called Ca
2+
-induced Ca

2+
release [8,18–20].
S. cerevisiae is a very useful model and an attractive
alternative to mammalian cell lines for studying the
effect of oxidative stress on the eukaryotic cells and
also an interesting model for studying antioxidants
in vivo [21–28]. Living in an oxidant-rich medium
under natural conditions, it is expected that yeast cells
will respond promptly to variations in the oxidative
state of the environment. How fast the cells respond to
oxidative stress and whether this type of response
is Ca
2+
-mediated are issues that are not clearly
understood.
In this study, evidence is presented that the cytotoxic
effect of the exogenous oxidative stress induced by
H
2
O
2
or by the less hydrophilic oxidant tert-butylhydr-
operoxide (tBOOH) is triggered by transient elevations
in cytosolic Ca
2+
as a result of both rapid influx from
outside the cell and of release from vacuolar stores.
Results
H
2

O
2
induces a transient increase in cytoplasmic
Ca
2+
As a free radical generator, H
2
O
2
is known to have
deleterious effects on cell growth, and exposure to high
concentrations requires an immediate response for cell
survival. To determine whether the cell response to
high concentrations of H
2
O
2
is mediated by Ca
2+
in
S. cerevisiae, the wild-type (WT) strain BY4741 was
transformed with a plasmid expressing the luminescent
Ca
2+
reporter apoaequorin from a constitutive pro-
moter. After reconstitution of functional aequorin by
addition of its cofactor coelenterazine, cells were
exposed to H
2
O

2
shock directly in the luminometer
tube. The cells responded promptly, as H
2
O
2
initiated
immediate and transient luminescence peaks, indicating
sudden elevations in the cytosolic Ca
2+
.Ca
2+
pulses
could be recorded for H
2
O
2
concentrations as low as
0.5 mm, and a sharp rise followed by a rapid fall in
the cytosolic Ca
2+
-caused luminescence was noted for
concentrations of 2 mm and over (Fig. 1A). The pulse
amplitude increased with H
2
O
2
concentration (Fig. 1B).
H
2

O
2
treatment of the cells transformed with the
empty vector under the same conditions did not elicit
any luminescence response (data not shown). The
luminescence spectra were reproducible for the range
of H
2
O
2
concentrations used (0.5–10 mm), which
covered toxic but nonlethal concentrations (0.5–2 mm)
and lethal concentrations (3–10 mm).
AB
Fig. 1. Changes in cytosolic Ca
2+
upon exposure to H
2
O
2
. (A) Effect of H
2
O
2
on the intensity of the Ca
2+
-dependent cell luminescence. WT
BY4147 cells were transformed with the plasmid pYX212–cytAEQ. The cells expressing coelenterazine-reconstituted aequorin were exposed
to various H
2

O
2
concentrations directly in the luminometer tube, as described in Experimental procedures. The arrow indicates the addition
of H
2
O
2
. (B) Maximal response of H
2
O
2
-treated cells. The WT cells transformed with pYX212–cytAEQ were subjected to H
2
O
2
shocks as in
(A) (closed circles). The data are presented as the ratio of the maximum relative light units (RLU) to the average RLU recorded before H
2
O
2
shock. Closed diamonds correspond to the response of control cultures not expressing aequorin (transformed with the empty vector
pYX212). Each determination was repeated at least three times on different days, with no significant variations being seen (P < 0.05).
Oxidative stress and Ca
2+
C V. Popa et al.
4028 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS
Under H
2
O
2

stress, Ca
2+
is mobilized from both
external and internal sources
To further characterize the Ca
2+
response to exoge-
nous H
2
O
2
, we examined whether the Ca
2+
flux comes
from an external or an internal source. In the WT
cells, H
2
O
2
exposure induced sharp and transient lumi-
nescence peaks caused by the increase in cytosolic
Ca
2+
(Fig. 2A). The peak intensity was attenuated by
the presence of the membrane-impermeable Ca
2+
che-
lator 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetra-
acetic acid (BAPTA) (Fig. 2D), suggesting that, at
least in part, the Ca

2+
flux comes from outside the
cells. Following the addition of BAPTA, the pattern of
the luminescence spectrum recorded for the WT strain
changed from a short-duration Ca
2+
pulse (Fig. 2A)
to a prolonged elevation of cytosolic Ca
2+
, which
gradually diminished within 2–3 min (Fig. 2D). Never-
theless, addition of BAPTA did not completely sup-
press the cytosolic Ca
2+
elevation, suggesting that, on
exposure to H
2
O
2
, internal stores are also mobilized.
To test this possibility, the cytosolic Ca
2+
burst was
also monitored in cells lacking the genes encoding the
components of the main plasma membrane Ca
2+
channel, Cch1p ⁄ Mid1p. It was found that, upon expo-
sure to H
2
O

2
, the cch1D and mid1D null mutants
exhibited robust, albeit different, responses. Thus,
whereas the luminescence peak caused by the Ca
2+
flux decreased to approximately half in the cch1D cells
(Fig. 2B), in the mid1D mutant it was (surprisingly)
slightly higher than in the WT cells (Fig. 2C). This
observation suggested that, if involved, Cch1p alone,
independently of Mid1p, may be partially responsible
for the Ca
2+
influx under H
2
O
2
stress. As the differ-
ence between the WT and mid1D cells was small, the
tests described above were repeated on seven different
days, with no obvious variations being seen from day
to day. Statistical analysis showed that mid1D cells
exhibited luminescence maximum peaks that were con-
stantly higher than that recorded for the WT cells,
with an average that was 10 ± 1.6% higher than the
normal luminescence maximum intensity (P < 0.05).
In the parallel study, cch1D cells exhibited lumines-
cence maximum peaks that were constantly lower than
DE
GH
F

AB C
50 s
Fig. 2. Effect of mutations affecting Ca
2+
fluxes in response to H
2
O
2
. Isogenic strains expressing coelenterazine-reconstituted cytAEQ were
exposed to H
2
O
2
(2 mM; a toxic but sublethal concentration) directly in the luminometer tube. When used, the membrane-impermeant Ca
2+
chelator BAPTA was added (5 mM final concentration) 1 min before the H
2
O
2
shock. The arrows indicate the addition of H
2
O
2
. Each determi-
nation was repeated at least three times on different days, with no significant variations being seen (P < 0.05). One typical luminescence
spectrum is presented for each strain. (A) WT strain. (B) Null mutant cch1D strain. (C) Null mutant mid1D strain. (D) WT strain pretreated
with BAPTA. (E) Null mutant cch1D strain pretreated with BAPTA. (F) Null mutant mid1D strain pretreated with BAPTA. (G) Null mutant
yvc1D strain. (H) Null mutant yvc1D strain pretreated with BAPTA.
C V. Popa et al. Oxidative stress and Ca
2+

FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4029
that recorded for the WT cells, with an average that
was 33.5 ± 2.2% smaller than the normal lumines-
cence maximum intensity (P < 0.05).
Interestingly, BAPTA treatment of both cch1D
(Fig. 2E) and mid1D (Fig. 2F) cells attenuated the
Ca
2+
influx to levels comparable to those detected for
the BAPTA-treated WT cells (Fig. 2D), indicating
that, under H
2
O
2
stress, external Ca
2+
may enter the
cytosol via a transporter other than the Cch1p ⁄ Mid1p
channel. Similar results were obtained when BAPTA
was replaced with another Ca
2+
chelator, EGTA (data
not shown).
The addition of BAPTA or other Ca
2+
chelators to
the medium equalized the H
2
O
2

-induced cytosolic
Ca
2+
peak in WT, cch1D and mid1D cells (Fig. 2D–F),
but it still allowed a robust Ca
2+
burst, suggesting
that, on exposure to the oxidant, internal stores are
also mobilized. In yeast, the vacuole is the main stor-
age location for intracellular Ca
2+
, so the H
2
O
2
-
induced Ca
2+
flux was monitored in cells lacking
Yvc1p, the vacuolar membrane channel responsible for
release of Ca
2+
from the vacuole to the cytosol. The
YVC1 gene deletion drastically reduced the amplitude
of the Ca
2+
burst, indicating that most of the Ca
2+
released into the cytosol after H
2

O
2
exposure comes
from the vacuole via the Yvc1p channel (approxi-
mately 93%). However, a luminescence peak was still
noticeable in the yvc1D cells, reaching approximately
1 ⁄ 15 of the normal value (Fig. 2G). This residual peak
was practically abolished by the addition of BAPTA
(Fig. 2H), suggesting that the small Ca
2+
burst seen in
yvc1D cells was the result of Ca
2+
influx from outside
the cell. As BAPTA addition removes  50% of the
signal in the case of WT cells (Fig. 2A,D), it seems
probable that, in the presence of H
2
O
2
, the vacuolar
Yvc1p channel is activated by cytoplasmic Ca
2+
that
may come from outside the cell or be released from
the vacuole by Yvc1p itself in a positive feedback pro-
cess, as previously reported [8,18–20]. Thus, by dele-
tion of YVC1, both the H
2
O

2
-induced component and
the Ca
2+
-induced component are removed. If this is
so, the signal that disappears in BAPTA-treated WT
cells (Fig. 2D) may be interpreted as the signal
through Yvc1p secondarily induced by external Ca
2+
.
Adaptation to H
2
O
2
-induced oxidative stress is
favored under low-Ca
2+
conditions
As the elevation of cytosolic Ca
2+
in response to
oxidative stress was different in the mutant cells
used, growth in media supplemented with H
2
O
2
was
investigated. It was found that the cch1D and yvc1D
cells were more tolerant to H
2

O
2
than the WT cells,
suggesting that the lower level of cytosolic Ca
2+
burst
achieved during oxidative shock may result in a pro-
tective effect on cell growth. In this regard, mid1D
cells, which exhibited the highest Ca
2+
burst when
exposed to H
2
O
2
shock, were slightly more sensitive to
H
2
O
2
than WT cells and considerably more sensitive
than cch1D and yvc1D cells (Fig. 3A, upper right). At
the same time, supplementation of the medium with
the Ca
2+
chelator EGTA augmented cell tolerance to
H
2
O
2

(Fig. 3A, middle), whereas supplementary Ca
2+
augmented sensitivity to H
2
O
2
(Fig. 3A, bottom right).
Paradoxically, yvc1D cells, which exhibited the lowest
cytosolic Ca
2+
burst, were the most tolerant to exoge-
nous H
2
O
2
(Fig. 3B), suggesting that lower cytosolic
Ca
2+
bursts during oxidative shocks may actually
increase the chances of cell survival. To check this pos-
sibility, transgenic cells expressing the YVC1 gene from
a galactose-inducible promoter were monitored in
terms of sensitivity to oxidative stress. WT cells over-
expressing YVC1 are known to release more Ca
2+
into
the cytosol [7] and, indeed, such cells were more sensi-
tive to H
2
O

2
than cells expressing the control vector
(Fig. 3C). Moreover, sensitivity increased with induc-
tion time, probably because of increasing gene expres-
sion induced by activation of the GAL1 promoter by
galactose. In contrast, cells overexpressing the PMC1
gene from a galactose-inducible promoter became
more tolerant to H
2
O
2
than cells expressing the control
vector (Fig. 3C). Pmc1p transports Ca
2+
to the vacu-
ole, and its overproduction probably results in faster
restoration of cytosolic Ca
2+
.
Other mutations that alter cytosolic Ca
2+
were also
investigated. For example, whereas pmc1D and vcx1D
null mutants were both more tolerant than the wild type,
the double mutant pmc1D vcx1D was found to be hyper-
sensitive to H
2
O
2
(Fig. 3B). This double mutant lacks

Ca
2+
in the vacuole, and therefore cannot release Ca
2+
via Yvc1p. Nevertheless, this strain is deficient in restor-
ing cytosolic Ca
2+
levels after a stress burst [12], and is
consequently less tolerant to exogenous oxidants.
Cytosolic Ca
2+
was reported to upregulate the calci-
neurin-dependent degradation of Yap1p [29], the main
transcription factor inducing gene expression in
response to oxidative stress [19,20,30]. If the high cyto-
solic Ca
2+
achieved by cells when they are exposed to
H
2
O
2
can result in calcineurin-dependent Yap1p
degradation and hence sensitivity to oxidants, cells
lacking functional calcineurin should gain a certain
tolerance to H
2
O
2
. To check this possibility, the

growth of cnb1D cells (which lack the gene encoding
the regulatory subunit of calcineurin) under oxidative
stress was tested. It was noted that CNB1 deletion
resulted in an increase of tolerance to H
2
O
2
(Fig. 3B),
Oxidative stress and Ca
2+
C V. Popa et al.
4030 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS
suggesting that the Ca
2+
-mediated toxicity of H
2
O
2
may be also the result of Ca
2+
-mediated degradation
of Yap1p.
Exposure to lethal H
2
O
2
concentrations results in
Ca
2+
mobilization predominantly from the

vacuole
In the experiments described above, Ca
2+
appearance
in the cytosol was monitored in cells exposed to toxic,
but nonlethal, concentrations (as seen from the cell
viability following exposure; data not shown) of H
2
O
2
.
To determine whether cells behaved differently when
subjected to stronger oxidative insults, WT cells were
exposed to a lethal concentration of H
2
O
2
(10 mm). In
this case, it was found that BAPTA pretreatment of
cells did not attenuate the Ca
2+
burst (Fig. 4A,B),
indicating that, under lethal oxidative conditions, cells
rely mainly on internal Ca
2+
stores. Under the same
stress conditions, yvc1D cells exhibited only faint
Ca
2+
-mediated luminescence (Fig. 4A,B), suggesting

that when cells are confronted with higher, lethal con-
centrations of H
2
O
2
, they utilize the vacuole as the
main source for Ca
2+
to be directed to the cytosol.
Alkylhydroperoxide also induces a transient
increase in cytosolic Ca
2+
To determine whether the Ca
2+
-mediated response to
exogenous H
2
O
2
is part of a more general oxidative
stress response mechanism, a less hydrophilic oxidant,
tBOOH, was considered. This substance is largely used
A
B
C
Fig. 3. Effect of H
2
O
2
-induced oxidative

stress on the cell growth of Ca
2+
channel
mutants. (A) Growth properties of Ca
2+
channel mutants. Isogenic strains
(WT, cch1D, mid1 and yvc1D) were spotted
(approximately 4 lL) in 10-fold serial
dilutions (from 10
7
cellsÆmL
)1
, left, to
10
3
cellsÆmL
)1
, right) onto YPD plates
supplemented with the indicated chemicals
(4 m
M H
2
O
2
,20mM EGTA, 10 mM CaCl
2
).
Early exponential growth phase cultures
were used to prepare the diluted cultures,
which were spotted on plates by means of

a replicator. Cells were photographed after
3 days of incubation at 28 °C. The concen-
trations used were 4 m
M H
2
O
2
,20mM
EGTA and 10 mM CaCl
2
. (B) Effect of YVC1
and PMC1 overexpression on sensitivity to
oxidative stress. WT cells transformed with
plasmids pGREG506DSalI (empty vector,
with fragment SalI–SalI excised),
pGAL11–YVC1 and pGAL11–PMC1 were
shifted from a fresh preculture to SG-Ura for
galactose induction of the genes, 6 or 16 h
before being stamped [approximately 4 lL,
in 10-fold serial dilutions from 10
7
cellsÆmL
)1
(left) to 10
3
cellsÆmL
)1
(right)] onto SG-Ura
agar plates containing H
2

O
2
. Time variation
was not observed in the case of the PMC1
galactose-induced phenotype. Similar results
were obtained for tBOOH (1.5 and 2 m
M,
data not shown). (C) H
2
O
2
effect on growth
of mutants with altered Ca
2+
-related
phenotype. The strains were spotted
(approximately 4 lL, 10
6
cellsÆmL
)1
) onto
YPD plates containing the indicated
concentrations of H
2
O
2
.
C V. Popa et al. Oxidative stress and Ca
2+
FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4031

to mimic the oxidative stress that causes formation of
alkyl hydroperoxides (e.g. oxidation of unsaturated
lipid chains). As the minimal inhibitory concentration
for the WT strain is approximately 1 mm,Ca
2+
pulses
were recorded in aequorin-expressing WT cells exposed
to 0.25–1.5 mm tBOOH (Fig. 5A). The pulse ampli-
tude increased with tBOOH concentration (Fig. 5B).
When exposed to toxic, but nonlethal, concentra-
tions of tBOOH, WT cells promptly responded with a
transient elevation in cytosolic Ca
2+
, as seen by the
luminescence spectrum of the aequorin-expressing cells
(Fig. 6A). This observation suggested that, as in the
response to H
2
O
2
, cells utilize Ca
2+
-mediated signaling
to initiate the defense mechanisms. CCH1 deletion
lowered the amplitude of the Ca
2+
burst to approxi-
mately 75% of the normal peak (Fig. 6B), but MID1
deletion resulted in a Ca
2+

cytosolic burst that was
slightly higher than in the WT cells (Fig. 6C); this was
paralleled by an increased sensitivity of the mid1D
mutant to tBOOH (Fig. 7A,C). As the difference
between WT and mid1D cells was small, the tests
described above were repeated on seven different days,
with no major variations being seen from day to day.
Statistical analysis showed that mid1D cells exhibited
luminescence maximum peaks that were consistently
greater than that recorded for WT cells, with an aver-
age that was 12.14 ± 0.8% higher than the normal
luminescence maximum intensity (P < 0.05). In the
parallel study, cch1D cells exhibited luminescence maxi-
mum peaks that were consistently lower than that
recorded for WT cells, with an average that was
25.56 ± 1.2% lower than the normal luminescence
maximum intensity (P < 0.05).
Surprisingly, BAPTA addition prior to exposure to
tBOOH strongly attenuated the Ca
2+
burst to less
than one-quarter of the normal level, both in WT and
mutant cch1D and mid1D cells (Fig. 6D–F). This obser-
vation suggested that, under tBOOH stress, external
Ca
2+
may be predominantly responsible for the cyto-
solic bursts, but that the major route of entry into the
cell may, again, not be through the Cch1p ⁄ Mid1p
channel. The contribution of vacuolar stores could

not, however, be ruled out, because although the
Ca
2+
-dependent luminescence was drastically reduced
by the presence of BAPTA, it still peaked at approxi-
mately one-fifth to one-quarter of the normal
A
B
Fig. 4. Ca
2+
mobilization under lethal H
2
O
2
conditions. Isogenic
strains (WT and yvc1D) expressing coelenterazine-reconstituted
cytAEQ were exposed to 10 m
M H
2
O
2
(lethal) in the absence (A) or
presence (B) of 5 m
M BAPTA directly in the luminometer tube. The
arrow indicates the addition of H
2
O
2
. Each determination was
repeated at least three times on different days, with no significant

variations (P < 0.05) being seen. One typical luminescence
spectrum is presented for each type of experiment. RLU, relative
light units.
AB
Fig. 5. Changes in cytosolic Ca
2+
concentration upon exposure to tBOOH. (A) Effect of tBOOH on the intensity of the Ca
2+
-dependent cell
luminescence. WT cells expressing coelenterazine-reconstituted cytAEQ were exposed to various tBOOH concentrations directly in the lumi-
nometer tube. The arrow indicates the addition of tBOOH. (B) Maximal response of tBOOH-treated cells. The WT cells transformed with
pYX212–cytAEQ were subjected to tBOOH shocks as in (A) (closed squares). The data are presented as ratio of the maximum relative light
units (RLU) to the average RLU recorded before tBOOH shock. Closed diamonds correspond to the response of control cultures not
expressing aequorin (transformed with the empty vector pYX212). Each determination was repeated at least three times on different days,
with no significant variations being seen (P < 0.05).
Oxidative stress and Ca
2+
C V. Popa et al.
4032 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS
maximum, and this was followed by a steady state,
which was observed throughout the recording time. To
determine whether the Ca
2+
also comes from the vacu-
ole, the cytosolic Ca
2+
release under tBOOH stress
was monitored in yvc1D cells. It was seen that, as com-
pared with WT cells, the Ca
2+

burst was attenuated in
yvc1D cells (towards approximately one-quarter of the
normal peak; Fig. 6G), suggesting that the vacuolar
stores may also contribute to the total cytosolic pool.
The burst was completely abolished when yvc1 D cells
were preincubated with BAPTA (Fig. 6H), indicating
that, under tBOOH shock also, the transient elevation
in cytosolic Ca
2+
is the result of cooperative influx
from both outside the cell and from the vacuole, even
though, in this case, the vacuolar contribution seems
to be lower. In terms of growth, cch1D and yvc1D cells
were more tolerant to tBOOH than WT cells, whereas
mid1D cells were more sensitive to tBOOH than WT
cells (Fig. 7A,C). Supplementation of the medium with
the Ca
2+
chelator EGTA slightly augmented cell toler-
ance to tBOOH (Fig. 7A, right), but external Ca
2+
had no obvious effect on cell growth (data not shown).
It was found that the Ca
2+
-mediated cytosol lumi-
nescence increased when the tBOOH concentration
was varied from 0.75 mm (toxic, but nonlethal) to
1.5 mm (lethal) (Fig. 7B, dotted lines), but did not
change when the same conditions were applied to
BAPTA-pretreated cells (Fig. 7B, solid line). This

observation indicated that, unlike the case with H
2
O
2
,
the extent of vacuolar Ca
2+
mobilization may not be
dependent on tBOOH concentration.
The susceptibility to tBOOH of other transgenic
strains with altered Ca
2+
homeostasis was tested. Simi-
lar to what was seen with H
2
O
2
, YAP1 overexpression
reduced, whereas PMC1 overexpression augmented,
the tolerance of WT cells to tBOOH (data not shown).
Other mutations that alter cytosolic Ca
2+
were also
investigated, and the response to tBOOH paralleled
the response to H
2
O
2
. For example, whereas pmc1D
and vcx1D null mutants were both more tolerant than

the wild type, the double mutant pmc1D vcx1D was
found to be hypersensitive to tBOOH (Fig. 7C). Also,
CNB1 deletion resulted in an increase in tolerance to
tBOOH (Fig. 7C). Thus, it seems that Ca
2+
mediates
ABC
DEF
GH
Fig. 6. Effect of mutations affecting Ca
2+
fluxes in response to tBOOH. Isogenic strains expressing coelenterazin-reconstituted cytAEQ
were exposed to tBOOH (0.5 m
M, a toxic but sublethal concentration) directly in the luminometer tube. When used, the membrane-imper-
meant Ca
2+
chelator BAPTA was added (5 mM final concentration) 1 min before the tBOOH shock. The arrows indicate the addition of
tBOOH. Each determination was repeated at least three times on different days, with no significant variations being seen (P < 0.05). One
typical luminescence spectrum is presented for each strain. (A) WT strain. (B) Null mutant cch1D strain. (C) Null mutant mid1D strain. (D)
WT strain pretreated with BAPTA. (E) Null mutant cch1D strain pretreated with BAPTA. (F) Null mutant mid1D strain pretreated with BAPTA.
(G) Null mutant yvc1D strain. (H) Null mutant yvc1D strain pretreated with BAPTA. RLU, relative light units.
C V. Popa et al. Oxidative stress and Ca
2+
FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4033
the cytotoxic effect of exogenous oxidants rather than
the adaptative process.
Discussion
Aerobic cells are continuously exposed to oxidative
insults that have to be perceived and scavenged rapidly
to allow normal growth and development. Studies con-

cerning the antioxidant arsenal of the cell are numer-
ous, and many defense mechanisms have been
elucidated, but how exactly the cell senses the threat of
oxidative species that are above the non-noxious
thresholds is still not clearly understood. Ca
2+
is an
important second messenger in the eukaryotic cell, and
there is increasing evidence that cytosolic Ca
2+
entry
in yeast is critical for survival under a variety of stress
conditions, including hypo-osmotic or hyperosmotic
shock [1,3,5,7], protein-unfolding agents [30], or anti-
fungal drugs [31,32]. The results presented in this study
provide evidence that exogenous oxidative stress
induces a transient increase in cytosolic Ca
2+
that
originates from both outside the cell and from the
A
B
C
Fig. 7. (A) Effect of tBOOH-induced oxida-
tive stress on cell growth. Isogenic strains
(WT, cch1D, mid1D and yvc1D) were
spotted (approximately 4 lL) in 10-fold serial
dilutions [from 10
7
cellsÆmL

)1
(left) to
10
3
cellsÆmL
)1
(right)] onto YPD plates
supplemented with the indicated chemicals
(0.75 m
M tBOOH, 20 mM EGTA). Early
exponential growth phase cultures were
used to prepare the diluted cultures, which
were spotted on plates by means of a repli-
cator. Cells were photographed after 3 days
of incubation at 28 °C. (B) Ca
2+
mobilization
under lethal tBOOH concentrations. WT
cells expressing coelenterazine-reconstituted
cytAEQ were exposed to toxic but nonlethal
(0.75 m
M, left) or lethal (1.5 mM, right)
concentrations of tBOOH directly in the
luminometer tube. The arrow indicates the
addition of tBOOH. When used, BAPTA
was added to a final concentration of 5 m
M.
Each determination was repeated at least
three times on different days, with no
significant variations being seen (P < 0.05).

One typical luminescence spectrum is pre-
sented for each type of experiment. (C) The
effect of tBOOH on growth of mutants with
altered Ca
2+
-related phenotype. The strains
were spotted (approximately 4 lL,
10
6
cellsÆmL
)1
) onto YPD plates containing
the indicated concentrations of tBOOH.
Oxidative stress and Ca
2+
C V. Popa et al.
4034 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS
vacuole. Toxic, but sublethal, concentrations of exoge-
nous H
2
O
2
(1–2 mm) induced very rapid and transient
Ca
2+
bursts. Cytosolic Ca
2+
elevations dropped to
roughly half the normal value when cells were pretreat-
ed with Ca

2+
chelators (BAPTA or EGTA) shortly
before the oxidative shock, so part of the Ca
2+
must
come from outside the cell. However, the external
Ca
2+
seems to be taken up through a system that is
different from the Cch1p ⁄ Mid1p channel, as Ca
2+
bursts were still clearly detectable in cch1D cells and
were higher than normal in mid1D mutants. At the
same time, the process seems to be assisted by the
Yvc1 vacuolar channel, as the H
2
O
2
-triggered cytosolic
Ca
2+
elevation was rather modest in the yvc1D
mutant. The residual Ca
2+
increase observed in the
yvc1D mutant could be explained by the existence of a
low-affinity Ca
2+
system of unknown identity [30,33].
Nevertheless, as the H

2
O
2
concentration increased
from very toxic to lethal doses (3–10 m m), the cells
gradually turned to internal vacuolar stores rather
than external Ca
2+
.
The tolerance to high H
2
O
2
correlated inversely with
the amount of cytosolic Ca
2+
achieved during oxida-
tive shock. Thus, the mid1D mutant, which exhibited
the highest cytosolic Ca
2+
burst, was the least tolerant,
and the yvc1D mutant, with the lowest Ca
2+
load, was
the most tolerant. Excessive or unregulated levels of
Ca
2+
in the cytosol can lead to cell death [34] and,
indeed, there was a good correlation between hyper-
sensitivity to H

2
O
2
or tBOOH and excessive cytosolic
Ca
2+
elevation. The triad Ca
2+
⁄ oxidative stress ⁄ cell
death has already been reported for other systems. In
mammals, for instance, Ca
2+
overload apparently
leads to mitochondrial dysfunction, reactive oxygen
species production and cell death [35]. However, Ca
2+
has also been reported to upregulate the calcineurin-
dependent degradation of Yap1p [29], the transcription
factor that regulates gene expression in response to
oxidative stress in yeast [24,25,36]. In this respect, the
high cytosolic Ca
2+
achieved by the cells when
exposed to lethal concentrations may result in Yap1p
degradation, a situation in which the cells become
hypersensitive to oxidative stress.
The cell response to high H
2
O
2

was paralleled by
the response to the less hydrophilic oxidant tBOOH.
Here also, the cytosolic Ca
2+
transients could be
detected upon exposure, with both external and vacuo-
lar contributions. Again, the mid1D mutant exhibited
the highest Ca
2+
load and the lowest tolerance,
whereas the yvc1D mutant exhibited the lowest Ca
2+
burst, correlating with the highest tolerance to
tBOOH. The only difference seemed to reside in the
prevalence of the external Ca
2+
contribution to the
cytosolic pool, as seen by the low Ca
2+
burst in BAP-
TA-pretreated cells. Release of vacuolar Ca
2+
through
Yvc1p requires activation by Ca
2+
on the cytosolic
side of the vacuolar membrane [8], and the low level of
cytosolic Ca
2+
achieved in BAPTA-pretreated cells

might be accounted for by the inactive Yvc1p. One
can also speculate that tBOOH, being less hydrophilic
than H
2
O
2
, diffuses more slowly in the yeast aqueous
environment, exerting a milder signaling effect. On the
other hand, being unable to synthesize polyunsaturated
fatty acids, the yeast has not evolved special protective
mechanisms against lipid peroxidation, and is less pre-
pared to respond to a stressor that mimics lipid hydro-
peroxides.
Special attention deserves to be paid to the discrep-
ancy between the mid1D and cch1 D mutants. Although
Mid1p and Cch1p cooperate to form a high-affinity
influx system, differences between mid1D and cch1D
mutants have already been reported [4,37]. Among
other things, Mid1p is known to localize not only in
the plasma membrane, but also in the endoplasmic
reticulum membrane [38]. Recently, MID1 mutations
have been reported to cause a defect in the cell wall
structure [39] that might render mid1D cells more per-
meable to Ca
2+
or more susceptible to stressors such
as H
2
O
2

or tBOOH.
The data above suggest that Ca
2+
pulses serve fre-
quently, but not invariably, to transduce an oxidative
burst signal. However, Ca
2+
seems unlikely to be
involved in the adaptation to oxidative stress. Yap1p,
the major yeast transcription factor responsible for the
transcriptional regulation of many genes involved in
the response to oxidative stress, is upregulated directly
by the presence of oxidants [40–43], whereas Ca
2+
mediates its calcineurin-dependent degradation [29].
Thus, it is probable that, under strong oxidative condi-
tions, the cytosolic Ca
2+
elevations actually signal the
ultimate possible way out, which is cell death. This
would explain why, under extreme H
2
O
2
stress, the cell
blocks the import of external Ca
2+
and turns in a last
effort to internal stores.
Experimental procedures

Strains and culture conditions
The S. cerevisiae strains used in this study were isogenic
with the WT parental strain BY4741 (MATa; his3D1;
leu2D0; met15D0; ura3D0) [44]. The deletion mutant strains
used were Y04847 (BY4741, cch1::kanMX4), Y01153
(BY4741, mid1::kanMX4), Y01863 (BY4741, yvc1::kan-
MX4), Y04374 (BY4741, pmc1::kanMX4), Y03825
(BY4741, vcx1::kanMX4), Y13825 (MATa; his3D1; leu2D0;
C V. Popa et al. Oxidative stress and Ca
2+
FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4035
lysD0; ura3D0 vcx1::kanMX4) and Y05040 (BY4741,
cnb1::kanMX4). All strains were obtained from EURO-
SCARF (European S. cerevisiae Archive for Functional
Analysis, Institute of Molecular Biosciences Johann Wolf-
gang Goethe-University Frankfurt, Germany). Strain
VP012 (BY4741, pmc1::kanMX4 vcx1::kanMX4) was
obtained by crossing of strains Y04374 and Y13825 fol-
lowed by diploid sporulation and random spore analysis.
The absence of PMC1 and VCX1 from VP102 cells was
checked by colony PCR with primers specific for PMC1
(forward primer, 5¢-ATGTCTAGACAAGACGAAAAT-3¢;
reverse primer, 5¢-GAAATGACATCACCGACTAA-3¢)
and for VCX1 (forward primer, 5¢-ATGGATGCAACTA-
CCCCACT-3¢; reverse primer, 5¢-GGATAACTCCAATAT-
TTTTC-3¢). Internal control primers for ACT1 (forward
primer, 5¢-GAGGTTGCTGCTTTGGTTAT-3¢; reverse
primer, 5¢-GCGGTTTGCATTTCTTGT-3¢) were included
in all PCR reactions. Cell growth and manipulation were
performed as previously described [45]. Strains were grown

in standard YPD, SD or SG supplemented with the neces-
sary amino acids. For solid media, 2% agar was used.
Plasmid construction
The expression vectors harboring PMC1 or YVC1 were
obtained as follows. The corresponding ORFs were ampli-
fied from the yeast genome by PCR, and were subsequently
cloned into vector pGREG506 [46], which allows for galac-
tose-inducible expression via the GAL1 promoter. Plasmid
pGREG506 was purchased from EUROSCARF. The fol-
lowing primers were used: for PMC1,5¢-CATT
GGAT
CCATGTCTAGACAAGACGAAAAT-3¢ (forward primer,
introducing the BamHI site, underlined) and 5¢-TACT
GTC
GACTTAATAAAAGGCGGTGGACT-3¢ (reverse primer,
introducing the SalI site, underlined); and for YVC1,5¢-TA
CT
GTCGACATGGTATCAGCCAACGGCGA-3¢ (forward
primer, introducing the SalI site, underlined) and 5¢-GAG
A
CTCGAGTTACTCTTTCTTATCCTTTA-3¢ (reverse pri-
mer, introducing the XhoI site, underlined). The PMC1 and
YVC1 ORFs obtained by PCR were purified with a Wizard
SV gel and PCR Clean-up System (Promega), cut with
the appropriate restriction enzymes, and cloned into the
BamHI–SalI and SalI–XhoI sites of pGREG506, respec-
tively, to generate pGAL1–PMC1 and pGAL1–YVC1.As
control vector, pGREG506 with the SalI–SalI fragment
deleted was constructed (pGREG506DSalI). This deletion
removes the HIS3 gene used as a counterselective marker

of pGREG vectors for the in vivo plasmid recombination
[46].
Growth assessment
Overnight precultures were inoculated in fresh media at a
density of 10
5
cellsÆmL
)1
, and cells were then incubated
with shaking (200 r.p.m.) at 28 °C for 2 h before H
2
O
2
,
tBOOH, BAPTA or EGTA was added from sterile stocks
prepared in 0.1 m Mes ⁄ Tris (pH 6.5). The influence of
stressors on cell growth was monitored at time intervals
by determining the attenuance (D) of a cell suspension at
600 nm (Shimadzu UV–visible spectrophotometer, UV
mini 1240). For spot assays, fresh cell cultures of
D
600 nm
 1 were diluted 10-fold, 100-fold, 1000-fold
and 10 000-fold, and stamped on agar plates with a
replicator.
In vivo monitoring of oxidative stress-induced
Ca
2+
pulse
Monitoring of cytosolic Ca

2+
was performed with an apo-
aequorin cDNA expression system [47]. Yeast strains were
transformed with the plasmid pYX212–cytAEQ, containing
the apoaequorin gene under the control of the TPI pro-
moter, generously provided by E. Martegani (Department
of Biotechnology and Biosciences, University of Milano-
Bicocca, Milan, Italy) [48]. Yeast transformation was per-
formed with a modified lithium acetate method [49], and
Ura
+
transformants were selected for growth on SD med-
ium lacking uracil. For luminescence assays, overnight pre-
cultures of cells expressing the apoaequorin gene were
diluted in fresh SD-Ura medium to a density of
5 · 10
6
cellsÆmL
)1
. The cells were incubated with shaking
for another 2 h at 28 °C, before being harvested by centri-
fugation. The cells were washed three times (by centrifuga-
tion at 9167 g, 1 min) and resuspended at a density of
approximately 10
9
cellsÆmL
)1
in 0.1 m Mes ⁄ Tris buffer
(pH 6.5). To reconstitute functional aequorin, 50 lm native
coelenterazine (Sigma; stock solution of 1 lgÆlL

)1
in meth-
anol) was added to the cell suspension, and the cells were
incubated for 2 h at 28 °C in the dark. Aliquots containing
approximately 10
7
cells were harvested, and excess coelen-
terazine was removed by centrifuging three times (9167 g,
30 seconds). The cells were resuspended in 0.1 m Mes ⁄ Tris
buffer (pH 6.5) and transferred to the luminometer tube. A
cellular luminescence baseline was determined by 1 min of
recording at 1 s intervals. The exogenous oxidants H
2
O
2
and tBOOH were added from sterile stocks to give the final
concentrations indicated, and light emission was monitored
with a single-tube luminometer (Turner Biosystems,
20
n
⁄ 20). The light emission was monitored for at least
5 min after the stimulus at 1 s intervals, and reported as
relative luminescence units ⁄ s. When used, the calcium che-
lator BAPTA was added to the cell suspension to the final
concentration of 5 mm 1 min before application of the
stress. To ensure that total reconstituted aequorin was not
limiting in our assay, at the end of each experiment
aequorin expression and activity were checked by lysing
cells with 10% Triton X-100, and only the cells with con-
siderable residual luminescence were considered. Multiple

tests, on different days (a minimum of three for large differ-
ences between transgenic lines; a maximum of seven for
Oxidative stress and Ca
2+
C V. Popa et al.
4036 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS
small differences) were performed for each condition
(strain, ± stressor, ± chelator, concentration), and only
the statistically significant data were considered.
Statistical analysis
Multiple comparisons were performed with Student’s t-test.
The differences were considered to be significant when the
P-value was < 0.05. Data analysis was performed with
spss 15.0 for Windows.
Acknowledgements
The authors acknowledge the contribution of E. Mar-
tegani and R. Tisi (Department of Biotechnology and
Biosciences, University of Milano-Bicocca, Milan,
Italy) to the manuscript. This work was supported by
the Ministry of Education and Research of Romania
through the grant-in-aid PNII Idei_965, 176 ⁄ 2007.
References
1 Batiza AF, Schulz T & Masson PH (1996) Yeast
respond to hypotonic shock with a calcium pulse. J Biol
Chem 271, 23457–23462.
2 Kanzaki M, Nagasawa M, Kojima I, Sato C, Naruse
K, Sokabe M & Iida H (1999) Molecular identification
of a eukaryotic, stretch-activated nonselective cation
channel. Science 285, 882–886.
3 Matsumoto TK, Ellsmore AJ, Cessna SG, Low PS,

Pardo JM, Bressan RA & Hasegawa PM (2002) An
osmotically induced cytosolic Ca
2+
transient activates
calcineurin signaling to mediate ion homeostasis and
salt tolerance of Saccharomyces cerevisiae. J Biol Chem
277, 33075–33080.
4 Pinontoan R, Krystofova S, Kawano T, Mori IC, Tsuji
FI, Iida H & Muto S (2002) Phenylethylamine induces
an increase in cytosolic Ca
2+
in yeast. Biosci Biotechnol
Biochem 66, 1069–1074.
5 Viladevall L, Serrano R, Ruiz A, Domenech G, Giraldo
J, Barcelo A & Arino J (2004) Characterization of the
calcium-mediated response to alkaline stress in Saccha-
romyces cerevisiae. J Biol Chem 279, 43614–43624.
6 Catterall WA (2000) Structure and regulation of voltage-
gated Ca
2+
channels. Annu Rev Cell Dev Biol 16, 521–555.
7 Denis V & Cyert MS (2002) Internal Ca(2+) release in
yeast is triggered by hypertonic shock and mediated by
a TRP channel homologue. J Cell Biol 156, 29–34.
8 Palmer CP, Zhou X, Lin J, Loukin SH, Kung C &
Saimi Y (2001) A TRP homolog in Saccharomyces
cerevisiae forms an intracellular Ca
2+
-permeable
channel in the yeast vacuolar membrane. Proc Natl

Acad Sci USA 98, 7801–7805.
9 Cunningham KW & Fink GR (1994) Calcineurin-
dependent growth control in Saccharomyces cerevisiae
mutants lacking PMC1, a homolog of plasma mem-
brane Ca
2+
ATPases. J Cell Biol 124, 351–363.
10 Cunningham KW & Fink GR (1994) Ca
2+
transport in
Saccharomyces cerevisiae. J Exp Biol 196, 157–166.
11 Cunningham KW & Fink GR (1996) Calcineurin inhib-
its VCX1-dependent H
+
⁄ Ca
2+
exchange and induces
Ca
2+
ATPases in Saccharomyces cerevisiae. Mol Cell
Biol 16, 2226–2237.
12 Miseta A, Kellermayer R, Aiello DP, Fu L & Bedwell
DM (1999) The vacuolar Ca
2+
⁄ H
+
exchanger
Vcx1p ⁄ Hum1p tightly controls cytosolic Ca
2+
levels in

S. cerevisiae. FEBS Lett 451, 132–136.
13 Sorin A, Rosas G & Rao R (1997) PMR1, a Ca
2+
-ATPase in yeast golgi, has properties distinct from
sarco ⁄ endoplasmic reticulum and plasma membrane
calcium pumps. J Biol Chem 272, 9895–9901.
14 Strayle J, Pozzan T & Rudolph HK (1999) Steady-state
free Ca
2+
in the yeast endoplasmic reticulum reaches
only 10 lM and is mainly controlled by the secretory
pathway pump Pmr1. EMBO J 18, 4733–4743.
15 Stathopoulos AM & Cyert MS (1997) Calcineurin acts
through the CRZ1 ⁄ TCN1-encoded transcription factor
to regulate gene expression in yeast. Genes Dev 11,
3432–3444.
16 Matheos DP, Kingsbury TJ, Ahsan US & Cunningham
KW (1997) Tcn1p ⁄ Crz1p, a calcineurin-dependent
transcription factor that differentially regulates gene
expression in Saccharomyces cerevisiae. Genes Dev 11,
3445–3458.
17 Locke EG, Bonilla M, Liang L, Takita Y &
Cunningham KW (2000) A homolog of voltage-gated
Ca
2+
channels stimulated by depletion of secretory
Ca
2+
in yeast. Mol Cell Biol 20, 6686–6694.
18 Zhou XL, Batiza AF, Loukin SH, Palmer CP, Kung C

& Saimi Y (2003) The transient receptor potential
channel on the yeast vacuole is mechanosensitive. Proc
Natl Acad Sci USA 100, 7105–7110.
19 Su Z, Zhou X, Loukin SH, Saimi Y & Kung C (2009)
Mechanical force and cytoplasmic Ca(2+) activate
yeast TRPY1 in parallel. J Membr Biol 227, 141–150.
20 Su Z, Zhou X, Loukin SH, Haynes WJ, Saimi Y &
Kung C (2009) The use of yeast to understand
TRP-channel mechanosensitivity. Pflugers Arch 458,
861–867.
21 Jamieson DJ (1998) Oxidative stress response in the
yeast Saccharomyces cerevisiae. Yeast 14, 1511–1527.
22 Moradas-Fereira P & Costa V (2000) Adaptive response
of the yeast Saccharomyces cerevisiae to reactive oxygen
species: defences, damage and death. Redox Rep 5, 277–
285.
23 Costa V & Moradas-Fereira P (2001) Oxidative stress
and signal transduction in Saccharomyces cerevisiae:
C V. Popa et al. Oxidative stress and Ca
2+
FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4037
insights into ageing, apoptosis and diseases. Mol
Aspects Med 22, 217–246.
24 Moye-Rowley WS (2002) Transcription factors regulat-
ing the response to oxidative stress in yeast. Antioxid
Redox Signal 4, 123–140.
25 Ikner A & Shiozaki K (2005) Yeast signaling pathways
in the oxidative stress response. Mutat Res 569, 13–27.
26 Herrero E, Ros J, Belli G & Cabiscol E (2008) Redox
control and oxidative stress in yeast cells. Biochim

Biophys Acta 1780, 1217–1235.
27 Miller-Fleming L, Giorgini F & Outeiro TF (2008)
Yeast as a model for studying human neurodegenerative
disorders. Biotechnol J 3, 325–338.
28 Shima J & Takagi H (2009) Stress-tolerance of baker’s
yeast (Saccharomyces cerevisiae) cells: stress-protective
molecules and genes involved in stress tolerance.
Biotechnol Appl Biochem 53, 155–164.
29 Yokoyama H, Mizunuma M, Okamoto M, Yamamoto
J, Hirata D & Miyakawa T (2006) Involvement of
calcineurin-dependent degradation of Yap1p in
Ca
2+
-induced G
2
cell-cycle regulation in
Saccharomyces cerevisiae. EMBO Rep 7, 519–524.
30 Bonilla M, Nastase KK & Cunningham KW (2002)
Essential role of calcineurin in response to endoplasmic
reticulum stress. EMBO J 21, 2343–2353.
31 Edlind T, Smith L, Henry K, Katiyar S & Nickels J
(2002) Antifungal activity in Saccharomyces cerevisiae is
modulated by calcium signaling. Mol Microbiol 46,
257–268.
32 Gupta SS, Ton V-K, Beaudry V, Rulli S, Kunningham
KW & Rao R (2003) Antifungal activity of amidarone
is mediated by disruption of calcium homeostasis. J Biol
Chem 31, 28831–28839.
33 Muller EM, Locke EG & Cunningham KW (2001)
Differential regulation of two Ca

2+
influx systems by
pheromone signaling in Saccharomyces cerevisiae.
Genetics 159, 1527–1538.
34 McConkey DJ & Orrenius S (1997) The role of calcium
in the regulation of apoptosis. Biochem Biophys Res
Commun 239, 357–366.
35 Feissner RF, Skalska J, Gaum WE & Sheu SS (2009)
Crosstalk signaling between mitochondrial Ca
2+
and
ROS. Front Biosci 14, 1197–1218.
36 Stephen DW, Rivers SL & Jamieson DJ (1995) The role
of YAP1 and YAP2 genes in the regulation of the
adaptive oxidative stress responses of Saccharomyces
cerevisiae. Mol Microbiol 16, 415–423.
37 Courchesne WE & Ozturk S (2003) Amiodarone
induces a caffeine-inhibited, MID1-dependent rise in
free cytoplasmic calcium in Saccharomyces cerevisiae.
Mol Microbiol 47, 223–234.
38 Yoshimura H, Tada T & Iida H (2004) Subcellular
localization and oligomeric structure of the yeast puta-
tive stretch-activated Ca
2+
channel component Mid1.
Exp Cell Res 293, 185–195.
39 Bormann J & Tudzynski P (2009) Deletion of mid1, a
putative stretch-activated calcium channel in Clavi-
ceps purpurea, affects vegetative growth, cell wall syn-
thesis and virulence. Microbiology 155, 3922–3933.

40 Coleman ST, Epping EA, Steggerda SM &
Moye-Rowley WS (1999) Yap1p activates gene
transcription in an oxidant-specific fashion. Mol Cell
Biol 19, 8302–8313.
41 Kuge S, Arita M, Murayama A, Maeta K, Izawa S,
Inoue Y & Nomoto A (2001) Regulation of the yeast
Yap1p nuclear export signal is mediated by redox
signal-induced reversible disulphide bond formation.
Mol Cell Biol 21, 6139–6150.
42 Delaunay A, Pflieger D, Barrault MB, Vinh J &
Toledano MB (2002) A thiol peroxidase is an H
2
O
2
receptor and redox-transducer in gene activation. Cell
111, 471–481.
43 Azevedo D, Tacnet F, Delaunay A, Rodrigues-Pousada
C & Toledo MB (2003) Two redox centers within Yap1
for H
2
O
2
and thiol-reactive chemical signaling. Free
Radic Biol Med 35, 889–900.
44 Brachmann CB, Davies A, Cost GJ, Caputo E, Li J,
Hieter P & Boeke JD (1998) Designer deletion strains
derived from Saccharomyces cerevisiae S288C: a useful
set of strains and plasmids for PCR-mediated gene
disruption and other applications. Yeast 14, 115–132.
45 Sherman F, Fink GR & Hicks JB (1986) Methods in

Yeast Genetics. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
46 Jansen G, Wu C, Schade B, Thomas DY & Whiteway M
(2005) Drag&Drop cloning in yeast. Gene 344, 43–51.
47 Nakajima-Shimada J, Iida H, Tsuji FI & Anraku Y
(1991) Monitoring of intracellular calcium in Saccharo-
myces cerevisiae with an apoaequorine cDNA expres-
sion system. Proc Natl Acad Sci USA 88, 6878–6882.
48 Tisi R, Baldassa S, Belotti F & Martegani E (2002)
Phospholipase C is required for glucose-induced calcium
influx in budding yeast. FEBS Lett 520, 133–138.
49 Schiestl R & Gietz RD (1989) High efficiency transfor-
mation of intact yeast cells using single stranded nucleic
acid as carrier. Curr Genet 16, 339–346.
Oxidative stress and Ca
2+
C V. Popa et al.
4038 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS