Eur J Biochem 269, 5440–5450 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03234.x Bax-induced cell death in yeast depends on mitochondrial lipid oxidation Muriel Priault1,*, Jean-Jacques Bessoule2, Angela Grelaud-Coq1, Nadine Camougrand1 ´ and Stephen Manon1 UMR5095 C.N.R.S./Universite´ de Bordeaux 2, Bordeaux, France; 2UMR5544 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 Baxexpression induces oxidation of mitochondrial lipids This effect is prevented by the coexpression of Bcl-xL The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cisparinaric 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 treatments 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 represents a major step in the cascade of events leading to Baxinduced 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 Apoptosis is a cellular death program involved in homeostasis 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-xL 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 not have pro-apoptotic activity by themselves but 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 mitochondria It is now widely accepted that, following apoptosisinduction, 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 membrane, 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 concentration of namely superoxide and peroxide ions [8] In addition to unselective deleterious effects on cellular components, 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 pathways may play a role in apoptosis Namely, lipoxygenases Correspondence to S Manon, IBGC/CNRS, rue Camille Saint-Saens, F-33077 Bordeaux cedex, France ă Fax: 33 (0)5 56 99 90 51, Tel.: 33 (0)5 56 99 90 45, E-mail: stephen.manon@ibgc.u-bordeaux2.fr Abbreviations: C18:1, oleic acid; C18:2, linoleic acid; H2-DCFDA, dihydro-dichlorofluorescein diacetate; DHE, dihydroethidium; ROS, reactive oxygen species; SOD, superoxide dismutase ´ *Present address: Departement de Biologie Cellulaire, Sciences III, ` Quai Ernest-Ansermet 30, CH-1211 Geneve 4, Switzerland (Received 11 June 2002, revised 21 August 2002, accepted September 2002) Keywords: Bax; apoptosis; ROS; lipid oxidation; yeast mitochondria Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur J Biochem 269) 5441 activation has been identified as a possible positive modulator of apoptosis [16–18] A common regulation between a lipoxygenase-activating protein and Bcl-2 family member Bcl-xL 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], phosphatidylserine 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 manipulation of metabolic conditions prompted the use of yeast to evaluate the participation of a number of factors in Baxinduced 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 Constructions for Bax and BclxL 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 Genotypes and phenotypes of strains used in this study Strain Genotype Phenotype W303–1 A W303–1B/50 Mat a, ade1, his3, leu2, trp1, ura3 Mat a, ade1, his3, leu2, trp1, ura3 rho0 Mat a, ade1, his3, leu2, trp1, ura3 pCM189(URA3)-bax-cmyc Mat a, ade1, his3, leu2, trp1, ura3 pCM184(TRP1)-bax-cmyc Mat a, ade1, his3, leu2, trp1, ura3 rho0 pCM189(URA3)-bax-cmyc Mat a, ade1, his3, leu2, trp1, ura3 pCM184(TRP1)-bax-cmyc pYES2/GS(URA3)-SOD1-V5-his6 Mat a, ade1, his3, leu2, trp1, ura3 pCM184(TRP1)-bax-cmyc pYES2/GS(URA3)-SOD2-V5-his6 Mat a, ade1, his3, leu2, trp1, ura3 pCM184(TRP1)-bax-cmyc pYES2/GS(URA3)-CTT1-V5-his6 Mat a, ade1, his3, leu2, trp1, ura3 pCM184(TRP1)-bax-cmyc pYES2/GS(URA3)-CTA1-V5-his6 Mat a, his3, trp1, ura3 sod1::URA3 Mat a, his3, leu2, lys2, ura3 sod2::kanMX4 Mat a, his4, leu2, lys2, ura3 Mat a, his4, leu2, lys2, ura3 GAL1/10-bax-cmyc/LEU2 Mat a, his4, leu2, lys2, ura3 pDP83(URA3)-GAL1/10-Bcl-xL Mat a, his4, leu2, lys2, ura3 GAL1/10-bax-cmyc/LEU2 pDP83(URA3)-GAL1/10-Bcl-xL Wild-type Wild-type rho0 Bax-expression (tet-promoter) WtB1 WtB2 WtB1-rho0 WtB2-SOD1 WtB2-SOD2 WtB2-CTT1 WtB2-CTA1 FG-DSOD1 BY4742-DSOD2 HT444 HT444/Bi HMP1 MP1 Bax-expression (tet-promoter) Bax-expression (tet-promoter) Bax-expression (tet-promoter) Cytosolic SOD-overexpression Bax-expression (tet-promoter) Mitochondrial SOD-overexpression Bax-expression (tet-promoter) Cytosolic Catalase-overexpression Bax-expression (tet-promoter) Perox Catalase-overexpression Cytosolic SOD-deficient Mitochondrial SOD-deficient Wild-type Bax-expression (Gal-promoter) Bcl-xL-expression (Gal-promoter) Bax-expression (Gal-promoter) Bcl-xL-expression (Gal-promoter) Ó FEBS 2002 5442 M Priault et al (Eur J Biochem 269) 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 Valerie 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 Netherlands) 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 controlled 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 membranes were washed in NaCl/Pi (10 mM sodium phosphate pH 7.2, 137 mM NaCl) containing 0.1% Tween-20 and saturated with 5% milk powder in NaCl/Pi/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 : 2000 and : 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 : 5000 dilution, and peroxidase activity was revealed by an Enhanced Chemio-Luminescence kit (Amersham, UK) Cells were grown in a synthetic medium Yeast Nitrogen Base (Difco, USA) supplemented with 0.1% KH2PO4, 0.5% (NH4)2SO4 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 lgỈmL)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 lM 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 zymolyase-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 lgỈmL)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 proteinsỈmL)1 and lipids were extracted twice by a chloroform/methanol/water (2 : : 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% H2SO4 and 10 lg C17:0 as an external standard Transesterification was carried out at 80 °C for h and, after cooling on ice, methyl ester fatty acids were extracted with 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 at 160 °C, followed by a 20 °C min)1 ramp up to 190 °C, a °C min)1 ramp up to 210 °C and a final 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 · 106 cellsỈmL)1) were incubated in the dark at 28 °C in the presence of 20 lM 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 F565nm increases for 45 min, reaches a plateau and eventually decreases, probably corresponding to ethidium-induced death All further experiments were carried out after a 45-minute incubation Aliquots of cells (5 · 106 cellsỈmL)1) were incubated in the dark at 28 °C in the presence of 100 lM H2-DCFDA (dihydro-dichlorofluorescein diacetate) (Molecular Probes, The Netherlands) This nonfluorescent compound enters the cell and is converted to highly fluorescent and nonpermeant DCF by esterases and chemical oxidation by ROS F525nm (excitation at 495 nm) increases for 20 and then reaches a plateau All further experiments were carried out after a 45-minute incubation Aliquots of mitochondria (1 mg proteinỈmL)1) were incubated in the dark at °C in the presence of 10 lM cis-parinaric acid (Molecular Probes, The Netherlands) Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur J Biochem 269) 5443 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 mL of a 10-mM sodium phosphate buffer (pH 7.2) containing 137 mM 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 mM) 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 · 107 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 was soaked for under low-light conditions in a 36-mM potassium phosphate buffer (pH 7.8) containing 1.5 mgỈmL)1 nitro-blue tetrazolium and a further 10 in the same buffer containing 28 mM TEMED and 28 lM 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-mM 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 concentration range of hydrogen peroxide (1–50 mM) 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 Superoxide dismutase and catalase activities Cells (100 mL of a culture at A550nm ¼ 4) were washed twice and then broken by vigorous shaking for with an equal volume of glass beads in a 10-mM potassium phosphate buffer (pH 7.2) containing 0.6 M mannitol and a mixture of proteases inhibitors (Complete EDTA-freeTM, 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 centrifugation (105 000 g, 30 min) allows the elimination of the microsomal pellet and the recovery of the cytosolic supernatant 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 electrophoresis The reaction mixture for SOD activity was a 50-mM sodium carbonate buffer (pH 10.2) containing 0.01% Triton X-100, 0.1 mM EDTA, 0.1 mM xanthine, 25 lM 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 · 10)2 absorbance unitsỈmin)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 separating the proteins by nondenaturating polyacrylamide gel electrophoresis The gel (0.75 mm thick, 10% acrylamide) 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 composition 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 C18:1 results in the rupture of the hydrocarbon chain, leading to the appearance of lower molecular weight compounds (C8 to C11) 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 saturated 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 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-xL, was shown to prevent Bax-induced release of cytochrome c and cell death [25] Expression of Bcl-xL 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 5444 M Priault et al (Eur J Biochem 269) Ó FEBS 2002 Fig Effect of Bcl-xL-expression on mitochondrial fatty acids unsaturation index Strains HT444 (control), HT444/Bi (Bax), HMP1 (BclxL) and MP1 (Bax/Bcl-xL) were grown in a lactate-supplemented medium and then added with 1% galactose to achieve Bax and/or Bcl-xL expressions After h, cells were harvested and mitochondrial lipids were analyzed as in Fig Fig Effect of Bax-expression on mitochondrial fatty acids amounts and unsaturation index HT444/Bi strain was grown in a lactate-supplemented medium and then added with 1% galactose to achieve Baxexpression 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 Baxexpressing (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 independent experiments 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 mammalian mitochondria, localizes preferably at the contact sites between both membranes (reviewed in [42]), where cardiolipin plays a crucial role (reviewed in [43]) To investigate if the alteration of mitochondrial unsaturated fatty acids was actually linked to an oxidation, we first used the probe cis-parinaric acid This fluorescent polyunsaturated 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 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 Phospholipids were extracted from isolated mitochondria and fatty acids were analyzed as indicated in the methods section (A) Fatty acid amounts in the different 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 Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur J Biochem 269) 5445 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 mitochondria (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 monounsaturated 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 C18:2, which represses the expression of D9-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 Fig Amount of thiobarbituric acid-reactive species HT444/Bi strain grown in lactate-supplemented medium in the absence or in the presence of 100 lM C18:2 until mid-exponential growth phase Bax-induction was achieved by adding 1% galactose After h, cells were harvested, washed and resuspended in water The amount of thiobarbituric acid-reactive products was determined as described in the methods section acid-reactive products (Fig 5), supporting further the hypothesis that Bax actually induces lipid peroxidation Mitochondrial lipid oxidation is involved in cell death kinetics Fig 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 measurements 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 different mitochondria preparations for each strain ± SD 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 (n ¼ 5) in the absence and in the presence of 100 lM 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 could be assayed on Bax effects 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 5446 M Priault et al (Eur J Biochem 269) Ó FEBS 2002 Fig Effect of fatty acid-unsaturation-inducer dioctylphtalate on h-Bax-induced cell death kinetics WtB1 cells were grown in a commercial Yeast Nitrogen Base medium (Difco, h), or in a home-made minimal medium in the absence (j) or in the presence (.) of 100 lM 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 The amount of unsaturated fatty acids was also increased by adding C18:1 or C18:2, which repress the expression of D9-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 C18:2 as compared to C18:1 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 H2-DCFDA Similar results were observed with human Bax expression under the control of the strong promoter GAL1/10 (Fig 8A) However, under these conditions, yeast supports a massive cytochrome c relocalization [25], which is likely to interfere with the response of these probes, namely H2-DCFDA, to ROS [48] The experiments were therefore reproduced in yeast cells expressing Bax under the control of the lowstrength 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 H2-DCFDA is more Fig Effect of unsaturated fatty acids and of inhibitors of lipids oxidation 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 lM C18:1 or 100 lM C18:2 At t ¼ 0, cells were washed and resuspended in the same medium with doxycyclin After or 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 lM a-tocopherol or 100 lM resveratrol After or 14 h, the number of colony-forming cells was determined as indicated in the methods section 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 relocalized to the cytosol probably reflects a secondary accumulation 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 less than 10% (± 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 Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur J Biochem 269) 5447 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 Fig Measurements of ROS production by Bax-expressing cells HT444/Bi (A) or WtB1 (B) were grown aerobically under Baxrepressive 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 mL was taken out, diluted in the same medium to · 106 cellsỈmL)1, and incubated with 20 lM dihydroethidium (h) or 100 lM H2-DCFDA (n) in the dark for 45 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 for DCF) at time t 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 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 H2-DCFDA disappeared (Fig 9A), but lipid oxidation still occured (Fig 9B) suggesting 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) Numerous reports have demonstrated the occurrence of oxidative stress as a side effect in apoptosis As mitochondria 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 mitochondrial 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 H2O2-treated cells [21] and that a caspase-like activity is involved in yeast response to H2O2-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-xL, suggesting that it is relevant to an actual function of Bax in apoptosis Different treatments and conditions allowed modulating the 5448 M Priault et al (Eur J Biochem 269) Ó FEBS 2002 Fig Effects of reducer Tiron on Baxeffects (A) ROS production by HT444/Bi following Bax expression was measured as in Fig in the absence (black bars) or in the presence (white bars) of mM Tiron (B) Unsaturation ratio of mitochondrial fatty acids was measured as in Fig on HT444/Bi following or not Bax expression, in the absence or in the presence of mM Tiron (C) Survival ratio of HT444/Bi following Bax expression in the absence (h) or in the presence (s) of mM Tiron (D) Survival ratio of WtB1 following Bax expression in the absence (h) or in the presence (s) of mM Tiron 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-V5his6 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) or Wtb2-SOD1 (m) were grown in YNB 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 (.) or Wtb2-CTT1 (r) survival ratio was measured as in (C) unsaturation ratio of fatty acids and, as expected, Baxkilling 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 arachidonic 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 Ó FEBS 2002 Bax-induced oxidation of mitochondrial lipids (Eur J Biochem 269) 5449 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 Baxinduced yeast cell death Conversely, chemical or enzymatic scavenging of ROS did not have any positive effect on Baxinduced cell death From these results, whatever the primary origin of mitochondrial lipid oxidation (activation of lipidoxidizing 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 the presence of a selective lipoxygenase [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 Regional d’Aquitaine and the Universite de ` Bordeaux and a fellowship from the Ministere de la Recherche et de la Technologie (to M.P.) 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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... 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