Báo cáo Y học: Bax-induced cell death in yeast depends on mitochondrial lipid oxidation doc

Bax-induced cell death in yeast depends on mitochondrial lipid
oxidation
Muriel Priault
1,
*, Jean-Jacques Bessoule
2
, Angela Grelaud-Coq
1
, Nadine Camougrand
1
and Ste
´
phen Manon
1
1
UMR5095 C.N.R.S./Universite
´
de Bordeaux 2, Bordeaux, France;
2
UMR5544 C.N.R.S./Universite
´
de Bordeaux 2, Bordeaux,
France
The oxidant function of pro-apoptotic protein Bax was
investigated through heterologous expression in yeast.
Direct measurements of fatty acid content show that Bax-
expression induces oxidation of mitochondrial lipids. This
effect is prevented by the coexpression of Bcl-x
L
. The oxi-
dation actually could be followed on isolated mitochondria

as respiration-induced peroxidation of polyunsaturated cis-
parinaric acid and on whole cells as the increase in the
amount of thiobarbituric acid-reactive products. Treatments
that increase the unsaturation ratio of lipids, making them
more sensitive to oxidation, increase kinetics of Bax-induced
death. Conversely, inhibitors of lipid oxidation and treat-
ments that decrease the unsaturation ratio of fatty acids
decrease kinetics of Bax-induced death. Taken together,
these results show that Bax-induced mitochondrial lipid
oxidation is relevant to Bax-induced cell death. Conversely,
lipid oxidation is poorly related to the massive Bax-induced
superoxide and hydrogen peroxide accumulation, which
occurs at the same time, as chemical or enzymatic scavenging
of ROS does not prevent lipid oxidation nor has any effects
on kinetics of Bax-induced cell death. Whatever the origin of
mitochondrial lipid oxidation, these data show that it rep-
resents a major step in the cascade of events leading to Bax-
induced cell death. These results are discussed in the light of
the role of lipid oxidation both in mammalian apoptosis and
in other forms of cell death in other organisms.
Keywords: Bax; apoptosis; ROS; lipid oxidation; yeast
mitochondria.
Apoptosis is a cellular death program involved in homeo-
stasis and development of tissues. Numerous inducers
including growth factors deprivation, extracellular death
signal or subnecrotic chemical or physical damages activate
this program. Apoptosis has been well documented in a
wide variety of cellular types and a common set of events
underlies the whole process. Transcriptional activation of
pro-apoptotic genes and repression of antiapoptotic genes

change the balance ratio between pro- and antiapoptotic
proteins among which Bcl-2 family members play a key role
(reviewed in [1,2]). This group of proteins was identified on
the basis of sequence homologies of domains called BH-1–4
(Bcl-2 Homology domain). Three-dimensional structures of
three members of the family (namely Bcl-2, Bcl-x
L
and Bax)
show remarkable conservation although they exhibit
opposite functions [3–5]. A third group of Bcl-2 family
members gathers proteins sharing sequence homology
restricted to only the BH3 domain (e.g. Bid, Bad). These
proteins do not have pro-apoptotic activity by themselves
but do potentiate the pro-apoptotic activity of Bax and
other pro-apoptotic proteins (reviewed in [1]).
The main action site of Bcl-2 family members is mito-
chondria. It is nowwidely acceptedthat, following apoptosis-
induction, the pro-apoptotic protein Bax is translocated
from the cytosol to the outer mitochondrial membrane and
induces the release of several proteins localized in the
intermembrane space to the cytosol, where they exert their
pro-apoptotic activity. The molecular mechanism underlying
this process is still a matter of debate (reviewed in [2]) and
may actually differ, depending on models. Anti-apoptotic
proteins, such as Bcl-2, prevent this release.
Besides their effects on the outer mitochondrial mem-
brane, Bcl-2 family members were shown to act as pro/
antioxidant proteins. Apoptosis is accompanied by an
oxidative burst, with an increase of the intracellular
concentration of reactive oxygen species (reviewed in

[6,7]). Mitochondrial respiratory chain is the main producer
of reactive oxygen species (ROS) and mitochondrial
dysfunctions are known to increase the intracellular con-
centration of namely superoxide and peroxide ions [8]. In
addition to unselective deleterious effects on cellular com-
ponents, ROS may exhibit more specific roles in the
apoptotic process: the intracellular redox state has been
shown to modulate directly permeability transition pore
opening [9], to regulate cell cycle checkpoints [10] by acting
possibly on p53 transactivation [11,12] and to modulate
caspase-3 activity [13–15].
In addition to ROS accumulation, other oxidative path-
ways may play a role in apoptosis. Namely, lipoxygenases
Correspondence to S. Manon, IBGC/CNRS,
1 rue Camille Saint-Sae
¨
ns, F-33077 Bordeaux cedex, France.
Fax: 33 (0)5 56 99 90 51, Tel.: 33 (0)5 56 99 90 45,
E-mail:
Abbreviations:C
18:1
, oleic acid; C
18:2
, linoleic acid; H
2
-DCFDA,
dihydro-dichlorofluorescein diacetate; DHE, dihydroethidium; ROS,
reactive oxygen species; SOD, superoxide dismutase.
*Present address:De
´

partement de Biologie Cellulaire, Sciences III,
Quai Ernest-Ansermet 30, CH-1211 Gene
`
ve 4, Switzerland
(Received 11 June 2002, revised 21 August 2002,
accepted 6 September 2002)
Eur. J. Biochem. 269, 5440–5450 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03234.x
activation has been identified as a possible positive modu-
lator of apoptosis [16–18]. A common regulation between a
lipoxygenase-activating protein and Bcl-2 family member
Bcl-x
L
has also been reported [19]. As the critical function of
Bcl-2 family members occurs at the mitochondrial level,
lipoxygenase-induced oxidation of mitochondrial lipids
could also be a regulatory step of apoptosis.
Yeast has already been a powerful tool to evaluate the
functions of Bcl-2 family members. Heterologous Bcl-2
expression in yeast has been shown to prevent deleterious
effects caused by the inactivation of ROS-scavenging
enzymes superoxide dismutases [20]. Conversely, hydrogen
peroxide-treatment of yeast induces similar phenotypes as
heterologous Bax expression [21]. Bax expressed in yeast is
translocated to the outer mitochondrial membrane [22,23]
and induces cell death [24] accompanied by apoptotic
characteristics such as cytochrome c release [25], phospha-
tidylserine exposure and DNA fragmentation [21,26] and
late maintenance of plasma membrane properties [27]. It has
been used as a tool to demonstrate that Bax is able to induce
these apoptotic characteristics through a process that does

not involve any transition permeability of the inner
membrane [28], nor the voltage-dependent anion channel
[28,29], or the adenine nucleotides carrier [29–31]. Results
obtained with yeast support the hypothesis that Bax acts on
mitochondria by creating a de novo giant channel in the
outer mitochondrial membrane, independent from the
voltage-dependent anion channel [23].
The availability of different mutants and the manipula-
tion of metabolic conditions prompted the use of yeast to
evaluate the participation of a number of factors in Bax-
induced cell death. In the present report, we show that Bax
expression induces lipid oxidation. Manipulation of the
unsaturation degree of fatty acids shows that this oxidation
is involved in Bax-induced cell death. Although ROS
accumulation occurs at the same time as lipid oxidation, the
two phenomena are poorly related and, opposite to lipid
oxidation, preventing ROS accumulation does not have any
positive effect on Bax-induced cell death.
EXPERIMENTAL PROCEDURES
Strains, constructions and cultures
Strains are listed in Table 1. Constructions for Bax and Bcl-
x
L
expression have been described previously [24,31].
Briefly, human Bax gene carrying a C-terminal c-myc tag
was placed under the control of a galactose-inducible
Table 1. Genotypes and phenotypes of strains used in this study.
Strain Genotype Phenotype
W303–1 A Mat a, ade1, his3, leu2, trp1, ura3 Wild-type
W303–1B/50 Mat a, ade1, his3, leu2, trp1, ura3

rho
0
Wild-type
rho
0
WtB1 Mat a, ade1, his3, leu2, trp1, ura3
pCM189(URA3)-bax-cmyc
Bax-expression (tet-promoter)
WtB2 Mat a, ade1, his3, leu2, trp1, ura3
pCM184(TRP1)-bax-cmyc
Bax-expression (tet-promoter)
WtB1-rho
0
Mat a, ade1, his3, leu2, trp1, ura3
rho
0
pCM189(URA3)-bax-cmyc
Bax-expression (tet-promoter)
WtB2-SOD1 Mat a, ade1, his3, leu2, trp1, ura3
pCM184(TRP1)-bax-cmyc
pYES2/GS(URA3)-SOD1-V5-his6
Bax-expression (tet-promoter)
Cytosolic SOD-overexpression
WtB2-SOD2 Mat a, ade1, his3, leu2, trp1, ura3
pCM184(TRP1)-bax-cmyc
pYES2/GS(URA3)-SOD2-V5-his6
Bax-expression (tet-promoter)
Mitochondrial SOD-overexpression
WtB2-CTT1 Mat a, ade1, his3, leu2, trp1, ura3
pCM184(TRP1)-bax-cmyc

pYES2/GS(URA3)-CTT1-V5-his6
Bax-expression (tet-promoter)
Cytosolic Catalase-overexpression
WtB2-CTA1 Mat a, ade1, his3, leu2, trp1, ura3
pCM184(TRP1)-bax-cmyc
pYES2/GS(URA3)-CTA1-V5-his6
Bax-expression (tet-promoter)
Perox. Catalase-overexpression
FG-DSOD1 Mat a, his3, trp1, ura3
sod1::URA3
Cytosolic SOD-deficient
BY4742-DSOD2 Mat a, his3, leu2, lys2, ura3
sod2::kanMX4
Mitochondrial SOD-deficient
HT444 Mat a, his4, leu2, lys2, ura3 Wild-type
HT444/Bi Mat a, his4, leu2, lys2, ura3
GAL1/10-bax-cmyc/LEU2
Bax-expression (Gal-promoter)
HMP1 Mat a, his4, leu2, lys2, ura3
pDP83(URA3)-GAL1/10-Bcl-x
L
Bcl-x
L
-expression (Gal-promoter)
MP1 Mat a, his4, leu2, lys2, ura3
GAL1/10-bax-cmyc/LEU2
pDP83(URA3)-GAL1/10-Bcl-x
L
Bax-expression (Gal-promoter)
Bcl-x

L
-expression (Gal-promoter)
Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur. J. Biochem. 269) 5441
GAL1/GAL10 promoter (strong expression conditions) or a
doxycyclin-repressed tet-off promoter (mild expression
conditions). Dcls1, Dsod2, Dctt1 and Dcta1 strains were
obtained from Euroscarf (Frankfurt, Germany). Dsod1
strain was a gift from Dr Vale
´
rie Prouzet.
SOD1, SOD2 and CTT1 genes were amplified by PCR
using the oligonucleotides pairs 5¢-CCCCAATTGATATC
TATACCTCC-3¢ and 5¢-CTTCAGAGGTTACCAGCA
TCGA-3¢,5¢-CAGGCAAGAAAGATATCGCGC-3¢ and
5¢-ATTAGTTGGTGACCAATGACACC-3¢,5¢-CCTCT
ATTCCAGATATCAATCTTGT-3¢ and 5¢-CAAGTCTT
GGGTTAACCTTCAAG-3¢, respectively. The fragments
contained an EcoRV and a BstEII site for further cloning
between the PvuII/BstEII sites of the pYES2/GS plasmid, in
frame with the sequences of V5 and His6 tags at the
C-terminal end of the proteins (Invitrogen, The Nether-
lands). The same construction with CTA1 gene was directly
purchased from Invitrogen. The pYES2/GS plasmid allows
the inducible expression of the proteins under the control of
a GAL1/GAL10 promoter. All constructions were con-
trolled by PCR and protein expression was checked by
Western blots. Proteins were separated on 12.5% SDS/
PAGE and transferred onto poly(vinylidene difluoride)
(PVDF) membranes (Problott, Perkin-Elmer). The mem-
branes were washed in NaCl/P

i
(10 m
M
sodium phosphate
pH 7.2, 137 m
M
NaCl) containing 0.1% Tween-20 and
saturated with 5% milk powder in NaCl/P
i
/Tween-20.
Primary antihuman Bax rabbit polyclonal antibody N20
(Santa-Cruz, USA) and anti-V5 tag mouse monoclonal
antibody (Invitrogen, The Netherlands) were used at
1 : 2000 and 1 : 5000 dilutions, respectively, and secondary
goat anti-rabbit IgG and goat anti-mouse IgG antibodies
coupled to horse radish peroxidase (Jackson Laboratories,
USA) were used at a 1 : 5000 dilution, and peroxidase
activity was revealed by an Enhanced Chemio-Lumines-
cence kit (Amersham, UK).
Cells were grown in a synthetic medium Yeast Nitrogen
Base (Difco, USA) supplemented with 0.1% KH
2
PO
4
,
0.5% (NH
4
)
2
SO

4
and a mixture of all aminoacids except
tryptophan, plus adenine (Drop-Mix). The carbon source
was 2% lactate, giving a strict respiratory metabolism,
except for the Dsod1 strain which is unable to grow on
lactate, and for which 2% glucose was used. Plasmids used
throughout the study carry URA3 or TRP1 genes and uracil
and tryptophan are thus used as selection markers. For cells
transformed by a tet-off/h-Bax construct, 10 lgÆmL
)1
doxycyclin was added to prevent Bax expression under
repressive conditions. Cells were grown aerobically in
Erlenmeyers (air volume/medium volume ¼ 5), at 28 °C
under rotary shaking (300 r.p.m.). For experiments with
dioctylphtalate, a homemade minimal medium was used,
according to [32], and dioctylphtalate dissolved in ethanol
was added at a final concentration of 100 l
M
.
Bax induction was achieved by adding 1% galactose to
lactate-supplemented cultures (GAL1/GAL10 promoter), or
by washing the cells three times in water to remove
doxycyclin (tet-off promoter).
Spheroplasts and mitochondria preparation
Spheroplasts and mitochondria were isolated from zymol-
yase-treated cells as described previously [33,34]. Protein
concentration was measured using the biuret method.
Bax-induced lethality
Strains were grown under repressive conditions and then
transferred under inductive conditions. At different times,

aliquots of 200 cells were plated on YPD medium (1%
Yeast Extract, 1% Bacto-Peptone, 2% glucose) added with
doxycyclin (10 lgÆmL
)1
). The number of growing colonies
was counted after a 3-day-incubation at 28 °C.
Lipids extraction and fatty acids analysis
Spheroplasts or mitochondria were suspended in methanol
at 10 mg proteinsÆmL
)1
and lipids were extracted twice by a
chloroform/methanol/water (2 : 1 : 1, v/v/v) mixture. The
organic phases were pooled, evaporated to dryness and
resolubilized in 50 lL chloroform/methanol (2 : 1, v/v).
Aliquots were mixed with 900 lL methanol containing
2.5% H
2
SO
4
and 10 lgC
17:0
as an external standard.
Transesterification was carried out at 80 °C for 1 h and,
after cooling on ice, methyl ester fatty acids were extracted
with 1 mL hexane. Separation of methyl ester fatty acids
was perfomed by gas chromatography (Hewlett-Packard
5890 series II) on a 15-m · 0.53 mm Carbowax column
(Alltech, USA). The oven temperature is programmed for
1 min at 160 °C, followed by a 20 °Cmin
)1

ramp up to
190 °C, a 5 °Cmin
)1
ramp up to 210 °C and a final 5 min
at 210 °C. Methyl ester fatty acids were identified and
quantified by comparison of their retention time with those
of standards.
Polar lipids were resolved by one-dimensional thin layer
chromatography on 10 · 10 cm silica gel plates (Merck,
Germany) using the solvent system as described [35]. Lipids
were located by spraying the plates with a solution of
0.001% primuline in 80% acetone and vizualized under UV
light. The silica gel zones corresponding to individual lipids
were scrapped and methyl ester fatty acids were prepared as
above.
Measurements of ROS and lipid oxidation
Aliquots of cells (5 · 10
6
cellsÆmL
)1
) were incubated in the
dark at 28 °C in the presence of 20 l
M
dihydroethidium
(DHE) (Sigma, USA). Following oxidation by ROS,
dihydroethidium is converted to ethidium, which binds to
DNA with a fluorescence increase at 565 nm (excitation at
495 nm). Conditions were adjusted on a strain inactivated
for SOD1 gene, thus producing high amounts of ROS.
F

565nm
increases for 45 min, reaches a plateau and eventu-
ally decreases, probably corresponding to ethidium-induced
death. All further experiments were carried out after a
45-minute incubation.
Aliquots of cells (5 · 10
6
cellsÆmL
)1
) were incubated in
the dark at 28 °C in the presence of 100 l
M
H
2
-DCFDA
(dihydro-dichlorofluorescein diacetate) (Molecular Probes,
The Netherlands). This nonfluorescent compound enters
the cell and is converted to highly fluorescent and nonper-
meant DCF by esterases and chemical oxidation by ROS.
F
525nm
(excitation at 495 nm) increases for 20 min and then
reaches a plateau. All further experiments were carried out
after a 45-minute incubation.
Aliquots of mitochondria (1 mg proteinÆmL
)1
)were
incubated in the dark at 4 °C in the presence of 10 l
M
cis-parinaric acid (Molecular Probes, The Netherlands)

5442 M. Priault et al.(Eur. J. Biochem. 269) Ó FEBS 2002
added as a complex with BSA (0.5 mg of cis-parinaric acid
dissolved in 25 lL dimethylsulfoxide were added to 50 mg
BSA dissolved in 1 mL of a 10-m
M
sodium phosphate
buffer (pH 7.2) containing 137 m
M
NaCl). These conditions
allow the integration of the probe into lipid bilayer [36].
After a 2-h incubation, measurements were carried out at
28 °C (excitation at 324 nm, emission at 420 nm). Ethanol
(20 m
M
) was added as a respiratory substrate.
Fluorescence measurements were performed using a
Xenius spectrofluorometer (Safas, Monaco).
The extent of lipid oxidation was measured according to
[37]. One millilitre of cell suspension (5 · 10
7
cells) was
harvested, washed and added to an equal volume of a
mixture of 15% trichloroacetic acid, 0.375% thiobarbituric
acid in 0.25
M
HCl. After a 15-minute incubation at 90 °C,
samples were centrifuged and the absorbance of the
supernatant was measured at 535 nm and the amounts of
thiobarbituric acid reactive products were determined using
a standard curve built with 1,1,3,3-tetramethoxypropane

solutions treated under the same conditions.
Superoxide dismutase and catalase activities
Cells (100 mL of a culture at A
550nm
¼ 4) were washed
twice and then broken by vigorous shaking for 4 min with
an equal volume of glass beads in a 10-m
M
potassium
phosphate buffer (pH 7.2) containing 0.6
M
mannitol and a
mixture of proteases inhibitors (Complete EDTA-free
TM
,
Boehringer, Germany). Centrifugation (900 g, 10 min)
allows the elimination of pelleted unbroken cells and nuclei.
A second centrifugation (12 000 g, 15 min) allows the
recovery of the pellet containing most mitochondria and
peroxisomes, containing Sod2p and Cta1p. A third centri-
fugation (105 000 g, 30 min) allows the elimination of the
microsomal pellet and the recovery of the cytosolic super-
natant containing Sod1p and Ctt1p.
Superoxide dismutase (SOD) was measured by a reverse
titration method in a double-beam/double wavelength
spectrophotometer in dual mode or directly after separating
proteins by nondenaturating polyacrylamide gel electro-
phoresis.
The reaction mixture for SOD activity was a 50-m
M

sodium carbonate buffer (pH 10.2) containing 0.01%
Triton X-100, 0.1 m
M
EDTA, 0.1 m
M
xanthine, 25 l
M
nitro-blue tetrazolium and the protein extract or bovine
erythrocytes SOD (Boehringer, Germany). The reaction
was started by adding buttermilk xanthine oxidase (Sigma,
USA) and nitro-blue tetrazolium oxidation was followed as
the absorbance difference between 560 and 500 nm. The
amount of xanthine oxidase was chosen so that the variation
in the absorbance difference in the absence of SOD activity
is approximately 2 · 10
)2
absorbance unitsÆmin
)1
and may
change depending on commercial preparations. Under these
conditions, the variation of absorbance difference is linear
for at least one minute. The addition of SOD prevents the
oxidation of nitro-blue tetrazolium by superoxide ions
produced in the xanthine oxidase reaction. A titration curve
of the activity was first built with commercial bovine
erythrocytes SOD so that the activity of SOD in proteic
extract could be calculated.
SOD activity was also qualitatively estimated after separ-
ating the proteins by nondenaturating polyacrylamide gel
electrophoresis. The gel (0.75 mm thick, 10% acrylamide)

was soaked for 5 min under low-light conditions in a
36-m
M
potassium phosphate buffer (pH 7.8) containing
1.5 mgÆmL
)1
nitro-blue tetrazolium and a further 10 min in
the same buffer containing 28 m
M
TEMED and 28 l
M
riboflavine. After a brief washing in water, the gel was
revealed on a light table.
Catalase activity was measured on the same proteic
extracts. Proteins were suspended in a 10-m
M
potassium
phosphate buffer (pH 7.2) containing 0.01% Triton X-100
and dispatched in the cuvettes of a double-beam/double
wavelength spectrophotometer in Split mode. A concentra-
tion range of hydrogen peroxide (1–50 m
M
) was added in
the sample cuvette and the absorbance difference between
the cuvettes was measured at 240 nm. Titration curves with
bovine heart catalase (Sigma, USA) were built to calculate
specific activity in the extracts. All spectrophotometric
measurements were performed with a DW2000 double
beam/double wavelength spectrophotometer (Aminco,
USA).

RESULTS
Bax-expression induces alterations of mitochondrial
lipids
In yeast, as in mammalian cells, unsaturated fatty acids of
phospholipids are primary targets during oxidative stress
(reviewed in [38]). Also, the unsaturation index of fatty acids
is strongly dependent on culture conditions [39] and was
shown to modulate several stress responses, such as heavy
metals-induced stress [40]. We thus investigated a possible
involvement of lipid oxidation in Bax-induced cell death.
Phospholipids were extracted from both whole cells and
isolated mitochondria and fatty acid amount and compo-
sition were measured. When grown on a standard medium,
yeast cells only contain monounsaturated fatty acids and
not polyunsaturated fatty acids. Hydroperoxidation of
monounsaturated fatty acids such as C
18:1
results in the
rupture of the hydrocarbon chain, leading to the appearance
of lower molecular weight compounds (C
8
to C
11
) including
alkanes, alkenes and aldehydes [41]. We thus expected that
lipid oxidation would result in the disappearance of
monounsaturated fatty acids without an increase in satur-
ated fatty acids.
The total amount of fatty acids in whole cells was not
significantly affected (data not shown) but the amount of

fatty acids in isolated mitochondria was markedly decreased
by Bax expression (Fig. 1A). Also, a strong decrease of the
unsaturated/saturated fatty acids ratio was measured on
isolated mitochondria but not on whole cells (Fig. 1B). This
effect is time-dependent, reaching a maximum after 4 h, in
correlation to Bax-induced cell death kinetics ([25] and
Fig. 9). These observations suggest that Bax-expression is
accompanied by mitochondrial lipid oxidation.
Co-expression of antiapoptotic proteins of the Bcl-2
family, such as Bcl-x
L
, was shown to prevent Bax-induced
release of cytochrome c and cell death [25]. Expression of
Bcl-x
L
alone had a slight increasing effect on mitochondrial
fatty acids unsaturation ratio and, most importantly, fully
prevented the effect of Bax (Fig. 2).
To see whether a particular class of phospholipid was
affected, they were separated by thin layer chromatography
and individual fatty acids amount and composition were
Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur. J. Biochem. 269) 5443
determined. It appeared that the alteration of fatty acids
amount (Fig. 3A) and unsaturation index (Fig. 3B) affected
all lipids, but was slightly more marked for cardiolipin.
Although Bax-expression in yeast results in a localization in
the outer mitochondrial membrane and not in the inner
mitochondrial membrane [23], the fact that inner membrane
cardiolipin is altered may suggest that Bax, as in mamma-
lian mitochondria, localizes preferably at the contact sites

between both membranes (reviewed in [42]), where cardio-
lipin plays a crucial role (reviewed in [43]).
To investigate if the alteration of mitochondrial unsatur-
ated fatty acids was actually linked to an oxidation, we first
used the probe cis-parinaric acid. This fluorescent poly-
unsaturated fatty acid can be integrated in vivo or in vitro
into biological membranes, where it supports the same
alterations as native lipids [43–46]. Namely, peroxidation of
this probe induces a decrease of its emission fluorescence
Fig. 1. Effect of Bax-expression on mitochondrial fatty acids amounts
and unsaturation index. HT444/Bi strain was grown in a lactate-sup-
plemented medium and then added with 1% galactose to achieve Bax-
expression. Cells were harvested and spheroplasts and mitochondria
were isolated. Lipids were extracted and analyzed as indicated in the
methods section. (A) Quantification of the different fatty acids in
mitochondria isolated from control (hatched bars) and 4-h Bax-
expressing (white bars) cells. (B) Evolution of the unsaturation index
following Bax-expression of fatty acids in whole spheroplasts (h)and
isolated mitochondria (n). Data are representative of three inde-
pendent experiments.
Fig.2. EffectofBcl-x
L
-expression on mitochondrial fatty acids unsat-
uration index. Strains HT444 (control), HT444/Bi (Bax), HMP1 (Bcl-
x
L
) and MP1 (Bax/Bcl-x
L
) were grown in a lactate-supplemented
medium and then added with 1% galactose to achieve Bax and/or

Bcl-x
L
expressions. After 4 h, cells were harvested and mitochondrial
lipids were analyzed as in Fig. 1.
Fig. 3. Effect of Bax expression on fatty acid amounts and unsaturation
index in the different classes of mitochondrial phospholipid. Growth and
expression conditions (4 h) are similar to Fig. 1. Phospholipids were
extracted from isolated mitochondria and fatty acids were analyzed as
indicated in the methods section. (A) Fatty acid amounts in the dif-
ferent phospholipids extracted from mitochondria of control (hatched
bars) and Bax-expressing (white bars) cells. (B) Unsaturation index of
fatty acids in the different phospholipids extracted from mitochondria
of control (hatched bars) and Bax-expressing (white bars) cells. Data
are representative of two independent experiments.
5444 M. Priault et al.(Eur. J. Biochem. 269) Ó FEBS 2002
intensity. Isolated mitochondria were incubated in the
presence of cis-parinaric acid to allow its integration in
membranes [36]. A respiratory substrate (ethanol) was then
added and the fluorescence of cis-parinaric acid was
monitored. A slow decrease of fluorescence following
ethanol-driven respiration was observed in wild-type mito-
chondria (Fig. 4), showing that normal respiratory chain
activity only induces a marginal oxidation of lipids. The rate
of this decrease was strongly accelerated in mitochondria
isolated from Bax-expressing cells (Fig. 4). This supports
the hypothesis that Bax strongly sensitizes mitochondrial
lipids to respiration-induced oxidation.
The amount of thiobarbituric-acid reactive species is an
indication of the amount of lipid peroxidation products [37].
As shown by Riely et al. [41], the oxidation of monoun-

saturated fatty acids does not normally lead to the formation
of thiobarbituric acid-reactive products and no formation of
such products could be observed in Bax-expressing cells
(Fig. 5). The amount of polyunsaturated fatty acids can be
increased by the addition of C
18:2
, which represses the
expression of D
9
-acyl-coenzyme A desaturase Ole1p, thus
allowing the incorporation of exogenous fatty acids in
phospholipids [47]. Under these conditions, Bax-expression
resulted in a strong increase in the amount of thiobarbituric acid-reactive products (Fig. 5), supporting further the
hypothesis that Bax actually induces lipid peroxidation.
Mitochondrial lipid oxidation is involved in cell death
kinetics
Chatterjee et al. [32] showed that the unsaturation index of
yeast fatty acids could be artificially manipulated by adding
dioctylphtalate to the cultures. Commercial Yeast Nitrogen
Base from Difco contains this contaminating product that
induces a high degree of unsaturation. Bax-expressing cells
were therefore grown in a homemade minimal medium
containing or not dioctylphtalate. We first verified that the
addition of dioctylphtalate did not have significant effect on
cells growth in lactate-supplemented medium (doubling
times of 290 ± 20 and 280 ± 20 min (n ¼ 5) in the
absence and in the presence of 100 l
M
dioctylphtalate,
respectively) as already shown by Chatterjee et al.[32]for

growth in glucose-supplemented medium. In the absence of
Bax expression, dioctylphtalate did not induce any cell
death nor cytochrome c release (data not shown). From
these data, dioctylphtalate had no obvious effects on
mitochondrial metabolism or on cells viability and thus
couldbeassayedonBaxeffects.
The effect of dioctylphtalate on mitochondrial lipid
oxidation was measured. Chatterjee et al.[32]reported
that, on glucose-grown cells, dioctylphtalate induced an
increase of unsaturation index of mitochondrial lipids from
0.75 to 3.30. We found a similar increase on lactate-grown
cells (from 1.54 ± 0.02–6.85 ± 0.06; n ¼ 3), reaching a
value close to that measured in Yeast Nitrogen Base
medium (7.05 ± 0.05; n ¼ 3). Kinetics of Bax-induced cell
death varied accordingly to the extent of Bax-induced
mitochondrial lipid oxidation. In the minimal medium
without dioctylphtalate, Bax-induced cell death kinetics was
much slower than in Yeast Nitrogen Base medium (Fig. 6).
The addition of dioctylphtalate restored cell death kinetics
comparable to experiments in Yeast Nitrogen Base medium,
supporting a role of lipids unsaturation index, and thus
oxidation sensitivity, in Bax-induced cell death.
Fig. 4. Respiration-induced cis-parinaric acid oxidation in isolated
mitochondria. Mitochondria were isolated from control cells and
GAL1/GAL10-driven Bax-expressing cells. cis-Parinaric acid was
incorporated in mitochondrial membranes, and fluorescence meas-
urements were carried out as indicated in the methods section. (A)
Time-course of a typical experiment. (B) Variation of fluorescence
during the first minute after ethanol addition: average from four dif-
ferent mitochondria preparations for each strain ± SD.

Fig. 5. Amount of thiobarbituric acid-reactive species. HT444/Bi strain
grown in lactate-supplemented medium in the absence or in the pres-
ence of 100 l
M
C
18:2
until mid-exponential growth phase. Bax-induc-
tion was achieved by adding 1% galactose. After 2 h, cells were
harvested, washed and resuspended in water. The amount of thio-
barbituric acid-reactive products was determined as described in the
methods section.
Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur. J. Biochem. 269) 5445
The amount of unsaturated fatty acids was also increased
by adding C
18:1
or C
18:2
, which repress the expression of
D
9
-acyl-coenzyme A desaturase Ole1p, thus allowing the
incorporation of exogenous fatty acids in phospholipids [47].
Bax expression was achieved in the presence of these fatty
acids (Fig. 7A): kinetics of cell death significantly increased
in the presence of C
18:2
as compared to C
18:1
and control.
The effects of known inhibitors of lipid oxidation, namely

a-tocopherol and resveratrol were also assayed: both
compounds significantly decreased kinetics of Bax-induced
cell death (Fig. 7B). Taken together, these results strongly
support the hypothesis that Bax-induced lipid oxidation is
directly related to Bax-killing efficiency.
Bax-induced lipid peroxidation is poorly related
to Bax-induced ROS production
Madeo et al. [21] previously reported that mouse Bax
expression in yeast induced a production of ROS, measured
with fluorescent probes DHE and H
2
-DCFDA. Similar
results were observed with human Bax expression under the
control of the strong promoter GAL1/10 (Fig. 8A). How-
ever, under these conditions, yeast supports a massive
cytochrome c relocalization [25], which is likely to interfere
with the response of these probes, namely H
2
-DCFDA, to
ROS [48]. The experiments were therefore reproduced in
yeast cells expressing Bax under the control of the low-
strength promoter tet-off, under conditions where no
massive cytochrome c relocalization is observed [31].
Interestingly, with a system allowing the expression of
Bax under the control of a low-strength promoter, a
difference was observed in the response of the two probes:
no oxidation of DHE could be observed under this
condition (Fig. 8B). Although both probes are oxidizable
by any type of ROS, it has been reported that DHE is more
sensitive to superoxide ion whereas H

2
-DCFDA is more
sensitive to hydrogen peroxide [49]. The difference between
the responses of the two probes to Bax-expression may
indicate that hydrogen peroxide is accumulated more
dramatically than superoxide ion. The response of DHE
under conditions where cytochrome c is massively relocal-
ized to the cytosol probably reflects a secondary accumu-
lation of superoxide ion following the inhibition of
mitochondrial electron transfer after the Bc1 complex [25].
Measurements of ROS-scavenging activities superoxide
dismutases and catalases were performed on whole extracts
from cells expressing Bax under the control of GAL1/10
promoter. Superoxide dismutase activity was decreased by
lessthan10%(±5%SD;n ¼ 4) and catalase activity was
decreased by 55% (± 20% SD, n ¼ 4) in Bax-expressing
cells. This confirms that the accumulation of superoxide ion
depicted when Bax is strongly expressed does not result
Fig. 7. Effect of unsaturated fatty acids and of inhibitors of lipids oxi-
dation on Bax-induced cell death. (A) Wtb1 cells were grown in YNB
medium supplemented with lactate in the presence of doxycyclin, and
added or not with 100 l
M
C
18:1
or 100 l
M
C
18:2
.Att ¼ 0, cells were

washed and resuspended in the same medium with doxycyclin. After 2
or 4 h, the number of colony-forming cells was determined as indicated
in the methods section. (B) Wtb1 cells were grown in YNB medium
supplemented with lactate in the presence of doxycyclin. At t ¼ 0, cells
were washed and resuspended in the same medium in the absence or in
the presence of 200 l
M
a-tocopherol or 100 l
M
resveratrol. After 6 or
14 h, the number of colony-forming cells was determined as indicated
in the methods section.
Fig. 6. Effect of fatty acid-unsaturation-inducer dioctylphtalate on
h-Bax-induced cell death kinetics. WtB1cellsweregrowninacom-
mercial Yeast Nitrogen Base medium (Difco, h), or in a home-made
minimal medium in the absence (j) or in the presence (.) of 100 l
M
dioctylphtalate, all three supplemented with lactate as a carbon source
and in the presence of doxycyclin (Bax-repression). At t ¼ 0, cells were
washed and transferred in the same media in the absence of doxycyclin
(Bax-expression). The number of colony-forming cells was determined
as indicated in the methods section.
5446 M. Priault et al.(Eur. J. Biochem. 269) Ó FEBS 2002
from an inhibition of superoxide dismutase but, more likely,
from the alteration of the redox state of quinones following
the release of cytochrome c.
Is ROS accumulation responsible for lipid oxidation? To
answer this question, ROS accumulation was scavenged by
adding the chemical reducer Tiron to the culture medium:
under this condition, oxidation of H

2
-DCFDA disappeared
(Fig. 9A), but lipid oxidation still occured (Fig. 9B) sug-
gesting that lipid oxidation is not a direct consequence of
ROS accumulation. Consequently, Tiron did not have any
protecting effect on Bax-induced cell death kinetics
(Fig. 9C,D). Identical results were obtained by using
N-acetylcysteine instead of Tiron (data not shown).
In order to confirm this lack of protection of ROS
scavenging against Bax effects, individual overexpressions
of the cytosolic form of yeast superoxide dismutase (Sod1p)
and the two yeast catalases (Cta1p and Ctt1p) were
achieved. Overexpression of the mitochondrial form of
superoxide dismutase (Sod2p) led to a dramatic decrease of
cell growth, even in the absence of Bax (data not shown).
The other enzymes were overexpressed under the form
of hexahistidine/V5-C-terminal-tagged active proteins
(Fig. 10A,B). Overexpression of any of the three enzymes
did not have any positive effect on Bax-induced cell death
kinetics (Fig. 10C,D).
DISCUSSION
Data presented in this paper show that heterologous
expression of Bax alone activates lipid oxidation, and that
preventing lipid oxidation has a significant positive effect on
Bax-induced cell death. Although an indirect effect of ROS
cannot be completely ruled out, it is noteworthy that direct
modulation of lipid oxidation has marked effects on the
kinetics of Bax-induced cell death, while modulation of
ROS accumulation has not.
Bax-induced ROS-accumulation

Numerous reports have demonstrated the occurrence of
oxidative stress as a side effect in apoptosis. As mitochon-
dria are the central effector in apoptosis, it has been
suggested that alterations of the respiratory chain may be
responsible for a dramatic increase of intracellular ROS
concentrations, thus having deleterious effects on biological
constituents. Release of cytochrome c obviously leads to an
increase of the reduced state of ubiquinone, thus favoring
the reduction of molecular oxygen to superoxide ion by the
Bc1 complex [50,51]. Some reports showed that ROS
scavenging induced through overproduction of the mitoch-
ondrial isoform of SOD, might counteract apoptosis, at
least partially [52,53]. Conversely, mitochondrial SOD
deficiency induces apoptosis [54].
Yeast has been a useful tool to test these hypotheses.
Madeo et al. have observed phenotypic similarities between
Bax-expressing yeast cells and H
2
O
2
-treated cells [21] and
that a caspase-like activity is involved in yeast response to
H
2
O
2
-treatment [55]. It has been reported that plant
antimicrobial protein osmotin induced a RAS2-dependent
Ôapoptosis-likeÕ stress response in yeast, prevented by
chemical reducer N-acetylcysteine [56]. Longo et al. [20]

have shown that overexpression of antiapoptotic protein
Bcl-2 protected yeast cells against oxidative stress induced
by SOD1 and SOD2 inactivation: the antioxidant function
of Bcl-2, already shown in mammalian cells [57,58], could
therefore also be depicted in a heterologous model.
Mitochondrial ROS production also appears to be involved
in yeast ageing [59].
Bax-induced lipid oxidation
The present study evidences an additional oxidant function
for Bax on mitochondrial lipids. This effect is prevented by
the coexpression of antiapoptotic Bcl-x
L
, suggesting that it
is relevant to an actual function of Bax in apoptosis.
Different treatments and conditions allowed modulating the
Fig. 8. Measurements of ROS production by Bax-expressing cells.
HT444/Bi (A) or WtB1 (B) were grown aerobically under Bax-
repressive conditions in lactate-supplemented medium (see methods).
At t ¼ 0, cells were transferred under Bax-inductive conditions. At the
indicated times, an aliquot of 1 mL was taken out, diluted in the same
medium to 5 · 10
6
cellsÆmL
)1
, and incubated with 20 l
M
dihydro-
ethidium (h) or 100 l
M
H

2
-DCFDA (n)inthedarkfor45min.
Emission spectra were acquired between 500 and 600 nm (excitation at
495 nm) for both fluorochromes. Y-axis is the ratio of the maximal
emission intensity (at 565 nm for ethidium/DNA complex and 525 nm
forDCF)attimet over the value at t ¼ 0, thus representing the
increase ratio of fluorescence associated to Bax expression. Data are
representative of five experiments for each curve.
Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur. J. Biochem. 269) 5447
unsaturation ratio of fatty acids and, as expected, Bax-
killing effect was modulated by these treatments. In
addition, inhibitors of lipid oxidation, actually slowed down
Bax-induced cell death.
Numerous reports have evidenced a role for fatty
acid oxidation in apoptosis (recently reviewed in [60]).
Although it may occur as a final consequence of ROS
production [61], enzymatic peroxidation by 5-lipoxygenase
and cyclooxygenase has often been demonstrated (reviewed
in [17,61]) enlightning the role of polyunsaturated arachi-
donic acid.
The first crucial observation reported in the present paper
is that, in the absence of a mammalian apoptotic network,
Bax alone is able to activate lipid oxidation. The origin of
Fig. 10. Effect of overexpression of scavenging enzymes on Bax-effects. (A) Control of the overexpression of active Sod1p-V5-his6: (1) Dsod1 strain;
(2) Dsod2 strain; (3) Wtb2-SOD1 grown in glucose + doxycyclin; (4) Wtb2-SOD1 grown in galactose + doxycyclin; (5) Wtb2-SOD1 grown in
glucose ) doxycyclin; (6) Wtb2-SOD1 grown in galactose ) doxycyclin. (A) SOD activities revealed on nondenaturing polyacrylamide gel; (B)
Western-blots with an anti-Bax antibody: (C) Western-blots with an anti-V5-tag antibody. (B) Control of the overexpression of active Cta1p-V5-
his6 and Ctt1p-V5-his6: Wtb2-CTA1 or Wtb2-CTT1 were grown on YNB medium supplemented with glucose or galactose, as indicated, and in the
presence of doxycyclin. Cells were then washed and transferred in the same medium in the absence of doxycyclin. Catalase activity was measured in
cells extracts after 12 h. (C) Effect of overexpression of Sod1p on Bax-induced cell-death. Wtb2 (j)orWtb2-SOD1(m)weregrowninYNB

supplemented with galactose in the presence of doxycyclin. At t ¼ 0, cells were washed and transferred in the same medium in the absence of
doxycyclin. At the indicated times, aliquots of 200 cells were plated on YPD + doxycyclin and the number of growing colonies was counted after
48 h. (D) Effect of overexpression of Cta1p or Ctt1p on Bax-induced cell-death. Wtb2 (j), Wtb2-CTA1 (.)orWtb2-CTT1(r) survival ratio was
measured as in (C).
Fig. 9. Effects of reducer Tiron on Bax-
effects. (A) ROS production by HT444/Bi
following Bax expression was measured as in
Fig. 8 in the absence (black bars) or in the
presence (white bars) of 5 m
M
Tiron. (B)
Unsaturation ratio of mitochondrial fatty
acids was measured as in Fig. 1 on
HT444/Bi following or not Bax expression,
in the absence or in the presence of 5 m
M
Tiron. (C) Survival ratio of HT444/Bi
following Bax expression in the absence (h)
or in the presence (s)of5m
M
Tiron. (D)
Survival ratio of WtB1 following Bax
expression in the absence (h)orinthe
presence (s)of5m
M
Tiron.
5448 M. Priault et al.(Eur. J. Biochem. 269) Ó FEBS 2002
this lipid oxidation can be discussed. Chemical reducers
Tiron and N-acetylcysteine scavenged ROS but did not
prevent lipid oxidation, suggesting that the two effects are

independent. However, one cannot rule out the possibility
that a low concentration of ROS produced even in the
presence of those reducers is still enough to induce
mitochondrial lipid oxidation.
The second crucial observation is that modulation of
Bax-induced lipid oxidation, by acting on the unsaturation
ratio of fatty acids or with inhibitors of oxidation such as
resveratrol, has significant consequences on the rate of Bax-
induced yeast cell death. Conversely, chemical or enzymatic
scavenging of ROS did not have any positive effect on Bax-
induced cell death. From these results, whatever the primary
origin of mitochondrial lipid oxidation (activation of lipid-
oxidizing enzymes or secondary consequence of ROS
production), it is an important step in the cascade of events
leading to Bax-induced cell death.
It should be noted that a similar role of lipid oxidation
was drawn for the reaction of programmed cell death
involved in plant resistance to pathogens and was related to
thepresenceofaselectivelipoxygenase[62].
A role for enzymes catalyzing lipid oxidation might
therefore be a general process, not only in apoptosis, but
also in other forms of programmed cell death, including in
yeast [63].
ACKNOWLEDGEMENTS
This work was supported by grants from the Centre National de la
Recherche Scientifique, the Association pour la Recherche contre le
Cancer, the Conseil Re
´
gional d’Aquitaine and the Universite
´

de
Bordeaux 2 and a fellowship from the Ministe
`
re de la Recherche et de la
Technologie (to M.P.).
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