Abstract

The yeast transcriptional response to murine Bax expression was compared with the changes induced by H2O2 treatment via microarray technology. Although most of the Bax-responsive genes were also triggered by H2O2 treatment, OYE3, ICY2, MLS1 and BTN2 were validated to have a Bax-specific transcriptional response not shared with the oxidative stress trigger. In knockout experiments, only deletion of OYE3, coding for yeast Old yellow enzyme, attenuated the rate of Bax-induced growth arrest, cell death and NADPH decrease. Lipid peroxidation was completely absent in ΔOYE3 expressing Bax. However, the absence of OYE3 sensitized yeast cells to H2O2-induced cell death, and increased the rate of NADPH decrease and lipid peroxidation. Our results clearly indicate that OYE3 interferes with Bax- and H2O2-induced lipid peroxidation and cell death in Saccharomyces cerevisiae.

Introduction

Apoptosis is an evolutionarily conserved cell death process that eliminates unwanted cells that are damaged, aged, infected or useless, and plays important roles in embryonic development and in maintenance of tissue homeostasis [1]. Apoptosis is characterized by a number of morphological changes, including cell rounding and shrinkage, chromatin condensation, oligonucleosomal DNA cleavage, plasma membrane ruffling, and packaging of the contents of a dying cell into small membrane-bound vesicles [2],[3]. Another typical feature of apoptosis is the exposure of phosphatidylserine on the outer leaflet of the plasma membrane [4]. Many of the typical biochemical and morphological changes in apoptotic cells are mediated by the proteolytic action of cysteine-dependent aspartate-specific proteases, designated caspases [5],[6].

In yeast, apoptosis-like cell death is observed (i) in cells with a defective cell cycle [7], (ii) upon inactivation of Asf1 [8], Cdc13 [9], or Stm1 [10], (iii) in aged cells [11]–[13], (iv) after treatment with H2O2[14], acetic acid [15], high salt [16], osmotin [17], α-factor [18], UV irradiation [19], and synthetic compounds [20]–[22], as well as (v) when there is nutrient imbalance [23]. Furthermore, the caspase-like protein Yca1 and the nuclear serine protease Nma111 are involved in yeast apoptosis-like cell death [24],[25].

Members of the Bcl-2 (B-cell lymphoma) family of proteins are important regulators of apoptosis. This family is composed of anti-apoptotic members (such as Bcl-2 and Bxl-XL), pro-apoptotic members (such as Bax and Bak), and a large group of BH3-only death proteins that trigger apoptosis or sensitize for it. The ratio between anti- and pro-apoptotic proteins determines the susceptibility of a particular cell to apoptosis [26]–[28]. Hyperexpression of the pro-apoptotic protein Bax is sufficient to induce apoptosis in mammalian cells [29]. In addition, ectopic overexpression of Bax is toxic to Saccharomyces cerevisiae[30],[31], inducing a variety of morphological abnormalities, including loose chromatin condensation, mitochondrial swelling, DNA strand breaks and plasma membrane blebbing [31],[32]. However, there is no evidence for compact chromatin condensation or oligonucleosomal DNA degradation [31], probably due to the absence of linker DNA between yeast nucleosomes [33]. Bax toxicity is also observed in other organisms [34]–[38].

In mammalian and S. cerevisiae cells, Bax is targeted to mitochondria [31],[39], inserts into their membranes [40],[41], triggers cytochrome c release [42],[43], and induces the production of reactive oxygen species [14],[29]. Its expression induces mitochondrial damage [44],[45], and leads to mitochondrial dysfunction [43],[46], probably due to the formation of a channel in the outer mitochondrial membrane [47] by direct pore formation [48],[49]. The precise mechanism of mitochondrial permeabilisation remains unclear [50] and different molecular mechanisms have been proposed [51]. In mammalian cells, Bax-mediated alterations in mitochondrial and endoplasmic reticulum Ca2+ levels serve as upstream signals for cytochrome c release [52]–[55].

Several yeast mutants impaired in Bax-induced yeast cell death have been selected [46],[56]–[62] and several suppressors of Bax-induced yeast cell death have been discovered [63]–[70]. Together, these mutants and suppressors implicate mitochondrial physiology, oxidative stress, vesicular transport and autophagy in determining the final effect of Bax toxicity.

To focus on the mechanism by which oxidative stress is elicited following Bax protein expression, one has to theoretically subtract from the cellular effects of Bax those effects that are caused by the oxidative stress associated with its expression. In the present study, the transcriptional response to Bax expression in S. cerevisiae was compared with that elicited by an equally toxic H2O2 treatment, using microarray technology.

Materials and methods

Cloning of the mouse Bax coding sequence

cDNA encoding the mouse Bax-α protein (EMBL L22472) was amplified from an EL4/13.18 mouse thymoma cDNA library (BCCM™/LMBP-LIB15) by PCR (Polymerase Chain Reaction) using Pfu DNA polymerase (Stratagene, La Jolla, CA) and the primers 5′-ATGGACGGGTCCGGGAGCAG-3′ and 5′-TCAGCCCATCTTC-TTCCAGATGGTGAG-3′. The resultant PCR product was cloned using standard procedures in a HincII-opened pUC19 plasmid [71], to produce pUC19B.

Plasmid constructions

The 2-mu ori and the URA3 marker gene were consecutively excised from pUT332 [72] by digestion followed by self-ligation using ClaI and BglII, respectively, to produce YIpUT332. A BamHI–HindIII fragment containing the GAL1 promoter was then ligated into the BglII–HindIII-opened YIpUT332 plasmid, thereby obtaining YIpUTGAL1p. This plasmid was linearized with HindIII, end-blunted, digested with XbaI, and ligated to an FspI–XbaI fragment containing the FLP1 terminator, creating plasmid YIpUT. Insertion of a Ty δ element as a blunted-ended EcoRI–BsaAI fragment in the KpnI–AatII-opened and blunted YIpUT resulted in YIpUTy. Subsequent insertion of the LEU2 marker gene, as a blunted BsaAI–BsrGI fragment, in the BamHI-opened and blunted YIpUTy created plasmid YIpUTyL (pSCTyGAL1-L) (LMBP 4913). Mouse bax cDNA was excised from pUC19B by digestion with XbaI and HindIII and subcloned into the XbaI–HindIII-opened plasmids pSCTyGAL1-L and p415GALL [73] (LMBP 4032), obtaining pSCTyGAL1mBAX-L (LMBP 3871) as an integrative expression plasmid for GAL1-driven Bax expression, and p415GALLmBAX (LMBP 4575) as a centromeric plasmid for GALL-driven Bax expression. Plasmids indicated with an LMBP number were deposited at BCCM (http://www.dmbr.Ugent.be/lmbp).

Yeast strains and growth conditions

The S. cerevisiae strains used were INVSc1 (MATαhis3Δ1 leu2-3, 112 trp1-289 ura3-52) (Invitrogen, Gaithersburg, MD), BY4742 (MATαhis3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), and their knockout derivatives (Euroscarf, Frankfurt, Germany). Yeast strains were grown at 30 °C and 250 rpm in rich YPD-medium (2% glucose, 2% Bacto-peptone, 1% yeast extract) or SD-medium (2% glucose, 0.67% yeast nitrogen base, 0.5% ammonium sulphate), either minimally supplemented (minimal SD-medium) with amino acids (0.2 mM leucine, 0.2 mM lysine, 0.1 mM histidine, 0.1 mM tryptophan) and 0.2 mM uracil, or fully supplemented (synthetic SD-medium) with a complete amino acid mix containing uracil. For transformant selection, leucine was dropped out (selective SD-medium) where appropriate. SG-medium, used for the induction of the galactose regulated promoters, differs from SD-medium only by the replacement of glucose with 2% galactose as carbon source. To prepare plates, media were solidified with 2% agar. The S. cerevisiae strains were transformed by the lithium acetate method [74], and selected on leucine-deficient SD-plates. The pSCTyGAL1-L and pSCTyGAL1mBAX-L plasmids were linearized at the Ty δ element with XhoI prior to transformation to ensure targeted integration. INVSc1 and BY4742 strains were used for integrative and episomal transformation, respectively.

RNA preparation following Bax expression or H2O2 treatment

Induction of Bax expression – precultures of strains

INVSc1::pSCTyGAL1mBAX-L and INVSc1::pSCTyGAL1-L were grown overnight in selective minimal SD-medium. They were diluted at least 500-fold in the same growth medium and grown to an OD600 of 1.0. Subsequently, the cells were washed three times with water, resuspended in 100 ml selective minimal SG-medium, and incubated for 1 h.

Challenge with H2O2

The yeast strain INVSc1::pSCTyGAL1-L was grown as described above, but the selective minimal SG-medium was supplemented with different concentrations of H2O2 ranging from 0.1 to 0.5 mM.

Total RNA extraction

After a 1 h of Bax protein induction, H2O2-treatment, or mock-treatment, about 109 cells were harvested by centrifugation at 2200g for 5 min at 4 °C, and washed with ice-cold sterile water. Cells were then combined with 1 ml RNApure™ reagent (Genhunter Corporation, Nashville, NY) and 1 g of glass beads (0.5 mm diameter), and broken by thorough mixing. The lysate was combined with 150 μl chloroform and centrifuged at 20,000g for 10 min at 4 °C. The RNA in the supernatant was precipitated with an equal volume of isopropanol for 10 min on ice, pelleted at 20,000g for 10 min at 4 °C, and washed with 70% ice-cold ethanol. The RNA was resuspended in 50 μl RNAse-free water. Typically, about 1 mg total RNA could be extracted from 109 cells. The purity and quality of the RNA preparations were checked by spectrophotometry (A260/A280 and A260/A230) and by an RNA assay using the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany).

Gene expression profiling using microarray technology

Construction of the microarrays

The yeast microarrays consisted of 70-mer oligos (Qiagen, Hilden, Germany) spotted on Type VII silane-coated slides, and represented 6307 well-characterized yeast genes with high specificity. The oligos were designed for optimal discriminatory potential between homologous genes. The microarrays were kindly provided by the Microarray Department of the Swammerdam Institute for Life Sciences at the University of Amsterdam.

RNA amplification and labeling

RNA was amplified in duplicate using a modified version of the in vitro transcription procedure [75]. Briefly, 5 μg total RNA was converted to double stranded cDNA using 5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-T24(ACG)-3′ (Eurogentec, Seraing, Belgium) as anchored oligo-dT containing the T7 promoter. RNA was transcribed from this cDNA using T7-in vitro transcriptase to produce 10–30 μg amplified RNA. From the latter, 1 μg was labeled with the CyScribe Direct™ mRNA labeling kit (Amersham Biosciences, Piscataway, NJ) in either Cy3 or Cy5 (Amersham Biosciences), and purified using the CyScribe™ GFX™ purification kit (Amersham Biosciences). The resultant probes were analyzed for amplification yield and incorporation efficiency by measuring the RNA concentration at 280 nm, Cy3 incorporation at 550 nm, and Cy5 incorporation at 650 nm using a Nanodrop spectrophotometer (NanoDrop Technologies, Rockland, DE). Typically, a labeling density of 44 pmol CyDye μg−1 was obtained, corresponding to an incorporation rate of one fluorochrome each 70 nucleotides. For each probe, 40 pmol of incorporated Cy5 or Cy3 were added to 210 μl hybridization solution containing 50% formamide and 0.1% SDS in 1 × Hybridization Buffer (Amersham BioSciences).

Array hybridization and post-hybridization processes

Hybridization and post-hybridization washings were performed at 37 °C in an Automated Slide Processor (Amersham BioSciences, Piscataway, NJ). Post-hybridization washing was performed in 1 × SSC, 0.1% SDS, followed by 0.1 × SSC, 0.1% SDS, and finally 0.1 × SSC. Arrays were scanned at 532 and 635 nm using the Agilent DNA MicroArray scanner (Agilent Technologies, Waldbronn, Germany). Images were analyzed with ArrayVision™ (Imaging Research Inc, Ont., Canada), and spot intensities were measured as median intensities corrected for local background (sMedianDensity). Data were normalized for dye intensity differences using a Lowess-procedure [76] between ratio log2 (Cy5/Cy3) and average signal intensity log2(Cy5 × Cy3). Signals from non-yeast controls could not be distinguished from background. Subsequently, between-slide normalization was performed with the MARAN web application (http://www.esat.kuleuven.ac.be/maran) using a generic model for sequential analysis of variance [77]. Regulated gene candidates were selected when expression level changes were at least 2-fold, the coefficient of variation between the ratios was <0.5, and the signal to background ratios were at least 10.

Subtractive gene expression profiling strategy

The normalized data of the Bax-responsive genes were compared with the normalized data obtained after H2O2 treatment in order to eliminate the common gene expression changes. The Bax-specific genes were defined as those Bax-responsive genes for which the expression level due to Bax was at least 2-fold more manifest or I in the opposite sense compared with the expression difference due to H2O2.

Real-time quantitative polymerase chain reaction

Total RNA (5 μg of the same pool used in primary gene expression profiling) was incubated with 2 units of RQ1 RNAse-free DNAse (Promega, Madison, WI) at 37 °C for 30 min. The volume was adjusted to 200 μl with RNAse-free water and the mixture desalted using Microcon-YM100 (Millipore, Billerica, MA). The eluate, typically about 5 μl, was added to 2 μg oligo-dT, the volume adjusted to 10 μl with RNAse-free water, and the mixture incubated at 70 °C for 10 min. After cooling on ice, the following components were added: first-strand buffer (Life Technologies, Paisley, UK), 3.3 mM dithiothreitol, 40 units RNase Block (Stratagene, La Jolla, CA), 1 mM of each dNTP (Amersham Pharmacia Biotec, Uppsala, Sweden), and 200 units Superscript II reverse transcriptase (Life Technologies). The 25 μl mixture was incubated for 1 h at 42 °C. The resulting first-strand cDNA was used as a template for the subsequent real-time quantitative polymerase chain reaction (RT Q-PCR), which was performed in 1 × SYBR Green PCR buffer containing ROX as passive reference (Eurogentec, Seraing, Belgium), 3.5 mM MgCl2, 0.2 mM of each dNTP, 300 nM primers and 0.025 units of AmpliTaq Gold® DNA polymerase. SYBR Green was used at a 1:66,000 dilution. RT Q-PCR amplification was performed in triplicate using the following conditions: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The gradual increase in fluorescence due to the formation of a complex between SYBR Green and double-stranded DNA was monitored in real-time using an ABI prism 7700 (Perkin–Elmer Applied Biosystems, Foster City, CA). Primers were designed with the assistance of the PrimerExpress™ software (Perkin–Elmer Applied Biosystems). The following forward and reverse gene-specific primers were used: 5′-ACGTTTACGATATCAGGAAGGATTGT-3′ and 5′-AGCTTCTTTGACGTAGTCCTGGTT-3′ (PRC1); 5′-GAATGCTCCTGTGAACACCTATATGA-3′ and 5′-TGATCGCGTCGTAGAGATTAACTT-3′ (MLS1); 5′-GGCTGTTGGTTTGAGTGACTCTTT-3′ and 5′-CGACCATATCTCTCAACAACGTAAA-3′ (HXT7); 5′-AGACAGGAGTAAAAGAAGAAGGTTCAAA-3′ and 5′-CCGGTGTCAGACCAGGATATCTAT-3′ (YIL127C); 5′-GGTAAGCCAGGCGATGAGATT-3′ and 5′-TTGTTCATGCGATTGCAAATTT-3′ (STF2); 5′-AAACGAGGTGTCAGCAGTGTCATA-3′ and 5′-CTTTACCTGACCTGAATGTCTACCAA-3′ (CYC7); 5′-GAGTCAGAATGCACGCACACTAA-3′ and 5′-GCGGCGTTGCCGTTAA-3′ (ICY2); 5′-GGCTGCTCAAAAGTCTTTCAGAAT-3′ and 5′-TCCAGTTTCTTCTCTTAGCGTTGTAA-3′ (RPL39); 5′-CGCCGATGGTGTAGAAATTCAT-3′ and 5′-CGATCGTTCCGCCGTATT-3′ (OYE3); 5′-CCAGTGAGCTATTATCCAGAATGTAAA-3′ and 5′-GCATACCTCAAAGGTTCTGAACAA-3′ (BTN2); 5′-TGAAGCCGAAAATTGTGTTGTT-3′ and 5′-GTATATAGCTTCAAAAGCTTGGTAAATTTCT-3′ (TBP1); and 5′-AATATATCATCAGTGACAGGTGCCATT-3′ and 5′-AGCGCCTGTAGCGAAATCA-3′ (UBC1). TBP1 and UBC1 were used as reference genes for normalization. Specificity of the amplicons generated during the PCR reactions was confirmed by melting curve generation using Dissociation Curves 1.0 supplied by the manufacturer (Perkin–Elmer). Gene expression was quantified on the basis of the threshold cycle values (Ct) at which a statistically significant increase in ROX-normalized fluorescence intensity was first detected. The Ct-value (as the mean of three independent RT Q-PCR reactions) for the gene of interest (target) was normalized to both reference genes within each sample, and this normalized target value was compared between the samples of interest (Bax expression or H2O2 treatment) and the control sample (untreated), generating a ΔΔCt value. The normalized level of target mRNA in the sample relative to the control was expressed as 2−ΔΔCt.

Growth assay

Precultures were grown overnight in selective synthetic SD-medium, diluted at least 500-fold in the same growth medium, and grown to early logarithmic phase. The cultures were washed three times and resuspended at 5 × 107 cells ml−1 in sterile water. A sample of 5 μl from each suspension (250,000 cells) was then inoculated in 195 μl selective synthetic SD- or SG-medium (Honeywell plate format). Cells were grown at 30 °C for 72 h with intermittent shaking for 10 s at 10 min intervals. Growth was assayed using a Bioscreen C (Labsystems, Helsinki, Finland), measuring optical densities every hour. The logistic growth curve was chosen for modeling purposes [78], and the growth rate of each culture was calculated based on the parameters of the fitted growth equation (R2 > 0.999).

Clonogenic survival assay

Precultures were grown overnight in synthetic SD-medium (leucine drop-out in the case of transformants), diluted at least 500-fold in the same growth medium, and grown to early logarithmic phase. The cells were washed three times with sterile water if the carbon source had to be switched from glucose to galactose. For induction of Bax expression, cells were resuspended in selective synthetic SG-medium. After specific time intervals, 1000 colony-forming units, estimated from measurement of OD600, were plated on synthetic semi-solid SD-medium (leucine-deficient in the case of transformants) using 4-mm diameter sterile glass beads. The plates were incubated for 3 days and colonies were counted automatically with the Scanalyzer (LemnaTec, Würselen, Germany). For each time point, percentage survival was calculated with reference to the mock treatment.

Immunological detection of Bax protein

About 108 cells were harvested by centrifugation at 2200g and washed with sterile water. They were broken with 0.5 g glass beads (0.5 mm) by vigorous vortexing for 5 min in 200 μl ice-cold lysis buffer (50 mM Tris–HCl, pH 8, 0.1% Triton X-100, 0.5% SDS) containing 1 × Complete™ protease inhibitor (Roche, Basel, Switzerland). The lysate was then centrifuged at 6000g for 5 min at 4 °C to remove cell debris and glass beads. Protein concentrations were determined with the Bicinchonic Acid Protein assay (Pierce, Rockford, IL). Samples (30 μg) were fractionated by SDS–PAGE (15% acrylamide) and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, FRG). Membranes were blocked overnight with 2% skimmed milk in PBS supplemented with 0.05% Tween-20, and probed with a mouse anti-Bax monoclonal antibody (B9, sc-7480) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by horseradish-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO). The protein bands were visualized by chemiluminescence using Western Lightning™ (Perkin–Elmer Life Sciences, Boston, MA).

NADPH assay

BY4742 and ΔOYE3 yeast cells transformed with p415GALLmBAX were grown in selective synthetic SD-medium. Samples were taken prior to the carbon switch (0 h) and at three additional time points (6, 12 and 24 h) after resuspension in selective synthetic SG-medium. To extract the pyridine nucleotides from the yeast, about 108 cells were harvested, washed three times with ice-cold water, and resuspended in 250 μl ice-cold extraction buffer (100 mM Tris–HCl, pH 8, 10 mM EDTA and 0.05% Triton X-100). Cells were broken with 0.5 g glass beads (0.5 mm) by vigorous vortexing for 5 min at 4 °C. The lysates were centrifuged at 6000g for 5 min at 4 °C, and the supernatants were analyzed immediately. NADPH was assayed spectrophotometrically (Shimadzu, Kyoto, Japan) as described [79] and NADPH concentrations were normalized to the protein contents of the samples.

Assays for oxidative protein and DNA damage

BY4742 p415GALLmBAX transformants were grown in selective synthetic SD-medium and reference samples were taken prior to the carbohydrate switch (0 h). Upon resuspension in selective synthetic SG-medium, cells were harvested at additional time points (12 and 24 h).

Assay for oxidative protein damage

Approximately 3 × 108 cells were harvested, washed with sterile water, and broken with 0.5 g glass beads (0.5 mm) in 200 μl ice-cold breaking buffer (15% glycerol, 2 mM EDTA) containing 1 × Complete™ protease inhibitor (Roche, Basel, Switzerland). Beads and cell debris were removed by centrifugation, and protein concentrations were determined. Oxidative protein damage was determined [80] by reaction with 2,4-dinitrophenylhydrazine (DNPH) [81] using the OxyBlot™ Oxidized Protein Detection Kit (Intergen, Purchase, NY). DNP-derivatized proteins were detected using a rabbit anti-DNP antibody and horseradish-conjugated goat anti-rabbit IgG. The protein dots were visualized by chemiluminescence using Western Lightning™ (Perkin–Elmer Life Sciences, Boston, MA), and the signals were analyzed with the LumiAnalysis™ image analysis software (Roche, Basel, Switzerland). The dot blots were then washed extensively, and the proteins were stained with Amido Black (0.1% in 45% methanol/10% acetic acid) for 15 min. After destaining with 45% methanol/10% acetic acid, the signal was analyzed using the Quantity One® program (BioRad, Richmond, CA), and the immunostaining signals were normalized to the total protein content of the dot.

Assay for oxidative mitochondrial DNA damage

Total DNA was prepared from 3 × 108 cells using MasterPure™ (Epicentre, Madison, WI) according to the guidelines provided by the manufacturer. DNA concentration was determined using the PicoGreen® dsDNA Quantitation Kit (Molecular Probes, Eugene, OR) and the CytoFluor fluorescence reader (PerSeptive Biosystems, Framingham, MA), employing λ DNA as standard. Oxidative mitochondrial DNA damage was measured by quantitative PCR, as the degree by which the amplification of a large (error-prone) amplicon (6.9 kb) was hampered relative to a short (error-resistant) amplicon (0.3 kb) of the mitochondrial COX1 gene [82].

Assay for lipid peroxidation

BY4742 and ΔOYE3 yeast cells transformed with p415GALLmBAX were grown in selective synthetic SD-medium. Samples were taken prior to the carbohydrate switch (0 h) and at two additional time points (12 and 24 h) after resuspension in selective synthetic SG-medium. BY4742 and ΔOYE3 yeast cells were grown in synthetic SD-medium and treated with 0.5, 1.0 or 2.0 mM H2O2 for 30 min. The extent of lipid peroxidation was measured by thiobarbituric acid (TBA) reactivity with malondialdehyde (MDA). About 5 × 108 cells were washed with sterile water and resuspended in 125 μl 25% HCl. Next, 125 μl of 1% (w/v) TBA in 50-mM NaOH were added. Tubes were incubated at 85 °C for 30 min and cooled. The chromogen was extracted by the addition of 250 μl 1-butanol, vigorous vortexing, and centrifugation for 1 min at 6000g. The upper organic phase was used for fluorescence analysis (excitation: 530 nm, emission: 590 nm) using a CytoFluor fluorescence reader (PerSeptive Biosystems, Framingham, MA). To determine the MDA concentration in the samples, 1,1,3,3-tetramethoxypropane, an MDA precursor in mild acidity [83], was used as a standard at concentrations ranging from 2 to 200 μM.

Results

Achieving equal toxicity with Bax protein expression and H2O2 treatment

S. cerevisiae cells (INVSc1 strain) were transformed with the expression plasmid pSCTyGAL1mBAX-L or with the parental plasmid pSCTyGAL1-L. The Ty δ element present in both plasmids allowed stable single-copy integration in the INVSc1 genome, as confirmed by Southern analysis (data not shown).

Induction of Bax expression resulted in a time-dependent increase in the amount of dead cells, with 10% of the cells dying within 1 h of Bax induction. For comparison of Bax-induced with oxygen stress-induced cell death, we optimized the H2O2 dose so that, under the same growth conditions, a 1-h treatment would be as lethal as 1 h of Bax induction. Yeast cells were challenged with increasing concentrations of H2O2 and the percentage of cell death after 1 h was determined by survival assay. Incubation in 0.1 mM H2O2 for 1 h resulted in the same level of mortality observed after 1 h of Bax induction (data not shown).

Comparison of the transcriptional responses upon Bax expression and H2O2 treatment

Total RNA was extracted after a 1-h Bax-induction, a 1-h mock (control) or H2O2 treatment (0.1 mM), and used for microarray transcriptional profiling. The experiment had a triangular loop design comprising control (green) versus Bax (red), Bax (green) versus H2O2 (red), and H2O2 (green) versus control (red). Digital images of the fluorescent signals from the hybridized slides were normalized for dye intensity differences by Lowess fitting and for between-slide normalization using MARAN for variance analysis. Inspection of the signals revealed that 75 genes had mRNA steady-state levels that changed at least 2-fold after 1 h of Bax induction (Table 1).

1

Classification of the 75 Bax-responsive genes determined by microarray transcriptional profiling

ORF Gene Description Fold change 
Carbohydrate metabolism    
YNL117W MLS1 Malate synthase (glyoxylate cycle) +2.09 
YBR218C PYC2 Gluconeogenic pyruvate carboxylase, NADPH regeneration −2.08 
YMR169C ALD3 Aldehyde dehydrogenase, induced by oxidative shock −2.50 
Cell polarity    
YKR055W RHO4 Ras homolog involved in actin filament organisation −2.70 
Degradation    
YMR297W PRC1 Carboxypeptidase Y (Proteinase C) for vacuolar protein catabolism +2.29 
YLL039C UBI4 Ubiquitin, transcriptionally induced in stress conditions +2.10 
YBR208C DUR80 Urea amidolyase, degrades urea to CO2 and NH3 −2.00 
YBR058C UBP14 Ubiquitin-specific protease, negative regulator of gluconeogenesis −2.04 
YLR351C NIT3 Hydrolase acting on carbon-nitrogen (no peptide) bonds −2.08 
YGR020C VMA7 Subunit f of vacuolar H+-ATPase, for vacuolar acidification −2.17 
DNA synthesis    
YNL102W POL1 DNA polymerase I α-subunit p180 +2.12 
YMR234W RNH1 Ribonuclease H1, involved in DNA replication +2.12 
YJR068W RFC2 Replication factor C, involved in cell cycle checkpoint +2.10 
Energy generation    
YBR039W ATP3 γ-Subunit of the F1 sector of mitochondrial F1F0 ATP synthase +2.18 
YPL078C ATP4 Subunit b of the stator stalk of mitochondrial F1F0 ATP synthase +2.16 
YGL256W ADH4 Alcohol dehydrogenase type IV for glucose fermentation +2.00 
YGR008C STF2 Stabilizing factor of the F1F0 ATP synthase −2.04 
YEL039C CYC7 Cytochrome c isoform 2 involved in mitochondrial electron transport −2.08 
Intracellular transport    
YPL094C SEC62 Membrane component of ER protein cotranslocation apparatus +2.16 
YBL102W SFT2 Protein involved in Golgi-to-endosome transport +2.06 
YMR195W ICY1 Protein involved in nuclear transport −2.13 
YPL250C ICY2 Protein involved in nuclear transport −2.17 
YJR058C APS2 Protein involved in vesicle-mediated transport −2.17 
YNL036W NCE103 Endogenous substrate for nonclassical export −2.27 
YGR142W BTN2 Protein involved in intracellular protein transport −2.50 
YER103W SSA4 Membrane component of ER protein cotranslocation apparatus −3.13 
Lipid metabolism    
YGR157W CHO2 Phosphatidyl-ethanolamine N-methyltransferase −2.13 
YPL171C OYE3 NADPH dehydrogenase, possibly involved in sterol metabolism −2.22 
Protein modifications    
YER123W YCK3 Protein involved in amino acid phosphorylation +2.27 
YKL035W UGP1 UTP-glucose-1-phosphate uridylyltransferase for glycosylation −2.13 
YGR161C RTS3 Protein involved in amino acid dephosphorylation −2.78 
Protein synthesis    
YOL077C BRX1 Essential protein required for biogenesis of the 60S ribosomal subunit +2.55 
YOL040C RPS15 Protein component of the 40S ribosomal subunit −2.00 
YJL189W RPL39 Protein component of the 60S ribosomal subunit −2.22 
YER035W EDC2 RNA-binding protein, activates mRNA decapping −2.56 
Small molecule transport    
YDR342C HXT7 Glucose, fructose and mannose transporter activity +2.06 
YDR040C ENA1 P-type ATPase sodium pump for sodium ion transport −2.00 
YMR011W HXT2 Glucose, fructose and mannose transporter activity −2.63 
YJR049C UTR1 Protein involved in iron homeostasis −2.70 
YHR094C HXT1 Glucose, fructose and mannose transporter activity −2.94 
Stress response    
YHR106W TRR2 Mitochondrial thioredoxin reductase, response to oxidative stress −2.17 
YDR258C HSP78 Protein involved in folding of some mitochondrial proteins −2.17 
YBR203W COS111 Protein required for resistance to the antifungal ciclopiroxolamine −2.22 
YKR013W PRY2 Protein similar to pathogen related proteins −2.44 
YPL152W RRD2 Protein responding to osmotic stress −2.63 
Transcription factor    
YMR270C RRN9 RNA polymerase I upstream activator subunit +2.15 
YGL209W MIG2 Involved in repression of invertase expression by high glucose −2.13 
YFL021W GAT1 Transcriptional activator involved in nitrogen catabolite repression −2.33 
YGL035C MIG1 Transcription factor involved in glucose repression −2.38 
YML099C ARG81 Regulator of arginine-responsive genes −2.44 
Unknown    
YPR002C-A  Protein of unknown function +2.80 
YDR525W-A SNA2 Protein of unknown function +2.65 
YDR396W  Protein required for cell viability +2.63 
YKL102C  Protein of unknown function +2.39 
YMR046W-A  Protein of unknown function +2.32 
YGL029W CGR1 May contribute to compartmentalization of nucleolar constituents +2.25 
YNL156C NSG2 Potential homolog of mammalian Insig 1 +2.16 
YDR034C-A  Protein of unknown function +2.11 
YMR118C  Protein of unknown function +2.10 
YBL043W ECM13 Protein of unknown function +2.09 
YGR122C-A  Protein of unknown function +2.07 
YDR051C  Protein of unknown function +2.06 
YBR056W  Protein of unknown function +2.04 
YPL282C  Protein of unknown function +2.03 
YDR033W MRH1 Membrane protein related to Hsp30p +2.00 
YPR091C  Protein of unknown function −2.00 
YNL134C  Protein with alcohol dehydrogenase (NADP+) activity −2.00 
YIL127C  Protein of unknown function −2.04 
YLR108C  Protein of unknown function −2.04 
YKR075C  Protein of unknown function; expression regulated by Rgt1 −2.08 
YDR070C FMP16 Found in mitochondrial proteome −2.17 
YFL062W COS4 Subtelomerically encoded protein of unknown function −2.22 
YBR285W  Protein of unknown function −2.27 
YER085C  Protein of unknown function −2.33 
YNR034W-A  Protein of unknown function −2.38 
ORF Gene Description Fold change 
Carbohydrate metabolism    
YNL117W MLS1 Malate synthase (glyoxylate cycle) +2.09 
YBR218C PYC2 Gluconeogenic pyruvate carboxylase, NADPH regeneration −2.08 
YMR169C ALD3 Aldehyde dehydrogenase, induced by oxidative shock −2.50 
Cell polarity    
YKR055W RHO4 Ras homolog involved in actin filament organisation −2.70 
Degradation    
YMR297W PRC1 Carboxypeptidase Y (Proteinase C) for vacuolar protein catabolism +2.29 
YLL039C UBI4 Ubiquitin, transcriptionally induced in stress conditions +2.10 
YBR208C DUR80 Urea amidolyase, degrades urea to CO2 and NH3 −2.00 
YBR058C UBP14 Ubiquitin-specific protease, negative regulator of gluconeogenesis −2.04 
YLR351C NIT3 Hydrolase acting on carbon-nitrogen (no peptide) bonds −2.08 
YGR020C VMA7 Subunit f of vacuolar H+-ATPase, for vacuolar acidification −2.17 
DNA synthesis    
YNL102W POL1 DNA polymerase I α-subunit p180 +2.12 
YMR234W RNH1 Ribonuclease H1, involved in DNA replication +2.12 
YJR068W RFC2 Replication factor C, involved in cell cycle checkpoint +2.10 
Energy generation    
YBR039W ATP3 γ-Subunit of the F1 sector of mitochondrial F1F0 ATP synthase +2.18 
YPL078C ATP4 Subunit b of the stator stalk of mitochondrial F1F0 ATP synthase +2.16 
YGL256W ADH4 Alcohol dehydrogenase type IV for glucose fermentation +2.00 
YGR008C STF2 Stabilizing factor of the F1F0 ATP synthase −2.04 
YEL039C CYC7 Cytochrome c isoform 2 involved in mitochondrial electron transport −2.08 
Intracellular transport    
YPL094C SEC62 Membrane component of ER protein cotranslocation apparatus +2.16 
YBL102W SFT2 Protein involved in Golgi-to-endosome transport +2.06 
YMR195W ICY1 Protein involved in nuclear transport −2.13 
YPL250C ICY2 Protein involved in nuclear transport −2.17 
YJR058C APS2 Protein involved in vesicle-mediated transport −2.17 
YNL036W NCE103 Endogenous substrate for nonclassical export −2.27 
YGR142W BTN2 Protein involved in intracellular protein transport −2.50 
YER103W SSA4 Membrane component of ER protein cotranslocation apparatus −3.13 
Lipid metabolism    
YGR157W CHO2 Phosphatidyl-ethanolamine N-methyltransferase −2.13 
YPL171C OYE3 NADPH dehydrogenase, possibly involved in sterol metabolism −2.22 
Protein modifications    
YER123W YCK3 Protein involved in amino acid phosphorylation +2.27 
YKL035W UGP1 UTP-glucose-1-phosphate uridylyltransferase for glycosylation −2.13 
YGR161C RTS3 Protein involved in amino acid dephosphorylation −2.78 
Protein synthesis    
YOL077C BRX1 Essential protein required for biogenesis of the 60S ribosomal subunit +2.55 
YOL040C RPS15 Protein component of the 40S ribosomal subunit −2.00 
YJL189W RPL39 Protein component of the 60S ribosomal subunit −2.22 
YER035W EDC2 RNA-binding protein, activates mRNA decapping −2.56 
Small molecule transport    
YDR342C HXT7 Glucose, fructose and mannose transporter activity +2.06 
YDR040C ENA1 P-type ATPase sodium pump for sodium ion transport −2.00 
YMR011W HXT2 Glucose, fructose and mannose transporter activity −2.63 
YJR049C UTR1 Protein involved in iron homeostasis −2.70 
YHR094C HXT1 Glucose, fructose and mannose transporter activity −2.94 
Stress response    
YHR106W TRR2 Mitochondrial thioredoxin reductase, response to oxidative stress −2.17 
YDR258C HSP78 Protein involved in folding of some mitochondrial proteins −2.17 
YBR203W COS111 Protein required for resistance to the antifungal ciclopiroxolamine −2.22 
YKR013W PRY2 Protein similar to pathogen related proteins −2.44 
YPL152W RRD2 Protein responding to osmotic stress −2.63 
Transcription factor    
YMR270C RRN9 RNA polymerase I upstream activator subunit +2.15 
YGL209W MIG2 Involved in repression of invertase expression by high glucose −2.13 
YFL021W GAT1 Transcriptional activator involved in nitrogen catabolite repression −2.33 
YGL035C MIG1 Transcription factor involved in glucose repression −2.38 
YML099C ARG81 Regulator of arginine-responsive genes −2.44 
Unknown    
YPR002C-A  Protein of unknown function +2.80 
YDR525W-A SNA2 Protein of unknown function +2.65 
YDR396W  Protein required for cell viability +2.63 
YKL102C  Protein of unknown function +2.39 
YMR046W-A  Protein of unknown function +2.32 
YGL029W CGR1 May contribute to compartmentalization of nucleolar constituents +2.25 
YNL156C NSG2 Potential homolog of mammalian Insig 1 +2.16 
YDR034C-A  Protein of unknown function +2.11 
YMR118C  Protein of unknown function +2.10 
YBL043W ECM13 Protein of unknown function +2.09 
YGR122C-A  Protein of unknown function +2.07 
YDR051C  Protein of unknown function +2.06 
YBR056W  Protein of unknown function +2.04 
YPL282C  Protein of unknown function +2.03 
YDR033W MRH1 Membrane protein related to Hsp30p +2.00 
YPR091C  Protein of unknown function −2.00 
YNL134C  Protein with alcohol dehydrogenase (NADP+) activity −2.00 
YIL127C  Protein of unknown function −2.04 
YLR108C  Protein of unknown function −2.04 
YKR075C  Protein of unknown function; expression regulated by Rgt1 −2.08 
YDR070C FMP16 Found in mitochondrial proteome −2.17 
YFL062W COS4 Subtelomerically encoded protein of unknown function −2.22 
YBR285W  Protein of unknown function −2.27 
YER085C  Protein of unknown function −2.33 
YNR034W-A  Protein of unknown function −2.38 

After a 1-h induction of Bax protein expression, RNA was extracted and used for a comprehensive analysis of the transcript steady-state levels. As a control, the same culture conditions were used for yeast harboring the empty vector. Following RNA extraction, the expression level changes due to Bax expression were determined. Genes for which the expression level changed at least 2-fold due to Bax expression were considered Bax-responsive. Data are represented by the mean of two independent experiments.

Next, the changes in normalized spot intensity due to Bax expression and due to H2O2 treatment were compared, focusing on the 75 Bax-responsive genes. Out of these genes, we could detect nine genes for which the mRNA expression level changed to a higher degree with Bax expression than with H2O2 treatment (PRC1, MLS1, HXT7, YIL127C, STF2, CYC7, ICY2, OYE3, BTN2). We also detected one gene for which the transcriptional level was downregulated by Bax but upregulated by H2O2 (RPL39) (Table 2). These 10 genes were designated as Bax-specific transcriptional response candidates.

2

Validation of the transcript level changes for genes thought to have a Bax-specific transcriptional response

Microarray profiling Microarray results: Fold change RT Q-PCR results: Fold change 
ORF Gene Bax/Ctrl H2O2/Ctrl Bax/Ctrl H2O2/Ctrl 
YMR297W PRC1 +2.29 −1.33 +1.81 −1.78 
YNL117W MLS1 +2.09 −1.22 +5.75 +1.18 
YDR342C HXT7 +2.06 −1.47 +1.02 −2.70 
YIL127C  −2.04 +1.20 +1.89 +3.39 
YGR008C STF2 −2.04 +1.09 −1.14 −4.55 
YEL039C CYC7 −2.08 +1.15 −1.27 +1.25 
YPL250C ICY2 −2.17 −1.06 2.38 +2.02 
YJL189W RPL39 −2.22 +2.31 +1.75 +2.48 
YPL171C OYE3 −2.22 +1.85 2.70 +1.50 
YGR142W BTN2 −2.50 +1.22 2.04 +1.45 
Microarray profiling Microarray results: Fold change RT Q-PCR results: Fold change 
ORF Gene Bax/Ctrl H2O2/Ctrl Bax/Ctrl H2O2/Ctrl 
YMR297W PRC1 +2.29 −1.33 +1.81 −1.78 
YNL117W MLS1 +2.09 −1.22 +5.75 +1.18 
YDR342C HXT7 +2.06 −1.47 +1.02 −2.70 
YIL127C  −2.04 +1.20 +1.89 +3.39 
YGR008C STF2 −2.04 +1.09 −1.14 −4.55 
YEL039C CYC7 −2.08 +1.15 −1.27 +1.25 
YPL250C ICY2 −2.17 −1.06 2.38 +2.02 
YJL189W RPL39 −2.22 +2.31 +1.75 +2.48 
YPL171C OYE3 −2.22 +1.85 2.70 +1.50 
YGR142W BTN2 −2.50 +1.22 2.04 +1.45 

A gene was considered transcriptionally responsive to Bax when the transcriptional change elicited by Bax protein expression was at least 2-fold more manifest or was in the opposite sense compared to the oxidative stress trigger. The results obtained for the microarray profiling were compared with the independent RT Q-PCR data. Out of the 10 candidates, four were finally identified to have a Bax-specific transcriptional response. Bold: Genes for which the Bax-specific transcriptional change was verified. Data are represented by the mean of at least two independent experiments.

The level of expression of the 10 Bax-specific candidate genes was verified by a real-time PCR procedure relative to the genes TBP1 and UBC1, for which the transcript levels remained stable upon Bax-expression or H2O2 addition (data not shown). The primers used for this validation are listed in Section 2. Four of the 10 candidate genes (MLS1, OYE3, ICY2 and BTN2) were confirmed to have a Bax-specific transcriptional regulation (Table 2). We next wanted to determine whether knockout of these genes influenced Bax-induced cell death.

Bax-induced cell death in strains knocked out for genes specifically responsive to Bax

The plasmids p415GALL and p415GALLmBAX were transformed into BY4742, ΔMLS1, ΔOYE3, ΔICY2 and ΔBTN2, and their transformants were analyzed by a growth assay in selective synthetic SG-medium (Fig. 1). For the empty vector transformants, the growth of all four knockout strains did not differ from that of the control BY4742. For p415GALLmBAX transformants, the growth rate of ΔOYE3 was higher than that of BY4742, but none of the other three knockout strains demonstrated any difference. Besides visual inspection, a logistic growth curve [78] was used for modeling purposes. The growth rate was calculated as the first derivative at the inflection point, and the growth rate ratio of the Bax-expressing population to the control transformant was calculated for each strain (Fig. 1). The absence of OYE3 resulted in a 19.5% (standard deviation: 4.0%) improvement in the growth rate ratio of the Bax-expressing population.

1

The rate of Bax-induced growth inhibition is decreased upon deletion of OYE3. Yeast cells were transformed with the plasmids p415GALLmBAX (Bax) or p415GALL (empty), grown in glucose-based medium, and diluted to equal cell densities in galactose-based medium to induce protein expression from the GALL promoter. Growth was monitored automatically over 72 h. Inset: The ratio of the growth rate following Bax protein induction to the control growth rate. Growth in the presence (Bax) and absence (empty) of Bax for (a) ΔMLS1, (b) ΔICY2, (c) ΔOYE3 and (d) ΔBTN2, each compared to wild type. Error bars represent the standard deviation of the mean of three independent experiments. (∗) Statistically significant difference based on the Wilcoxon Rank Sum test (α= 0.05).

1

The rate of Bax-induced growth inhibition is decreased upon deletion of OYE3. Yeast cells were transformed with the plasmids p415GALLmBAX (Bax) or p415GALL (empty), grown in glucose-based medium, and diluted to equal cell densities in galactose-based medium to induce protein expression from the GALL promoter. Growth was monitored automatically over 72 h. Inset: The ratio of the growth rate following Bax protein induction to the control growth rate. Growth in the presence (Bax) and absence (empty) of Bax for (a) ΔMLS1, (b) ΔICY2, (c) ΔOYE3 and (d) ΔBTN2, each compared to wild type. Error bars represent the standard deviation of the mean of three independent experiments. (∗) Statistically significant difference based on the Wilcoxon Rank Sum test (α= 0.05).

In addition, the rate of Bax-induced cell death was decreased in ΔOYE3 (Fig. 2(a)), with a maximal cell death diminution of 18.0% (standard deviation: 4.5%). Bax protein expression in both BY4742 and ΔOYE3 was verified by Western blot and Bax protein levels were found to be identical for both strains (Fig. 3).

2

The absence of OYE3 has different effects on Bax-induced and H2O2-induced cell death. (a) Wild-type BY4742 and ΔOYE3 cells were transformed with the plasmids p415GALLmBAX (Bax) or p415GALL (empty), grown in glucose-based medium, and recultured in galactose-based medium to induce protein expression from the GALL promoter. Clonogenic survival was determined by recovering cells at various times from the galactose-based medium and plating 1000 cells on glucose-based semisolid medium. For each timepoint, the percentage of survival was calculated by dividing the number of colonies surviving the Bax protein induction by the number of colonies appearing after mock-treatment (empty). The absence of OYE3 attenuates Bax-induced cell death. (b) Wild-type BY4742 and ΔOYE3 cells were grown in glucose-based medium. Following the addition of H2O2 to a final concentration of 0.5 mM, cells were recovered at various times and their clonogenic survival determined. The absence of OYE3 leads to a sensitivity towards H2O2. Error bars represent the standard deviation of the mean of three independent experiments. (∗) Statistically significant difference based on the Wilcoxon Rank Sum test (α= 0.05).

2

The absence of OYE3 has different effects on Bax-induced and H2O2-induced cell death. (a) Wild-type BY4742 and ΔOYE3 cells were transformed with the plasmids p415GALLmBAX (Bax) or p415GALL (empty), grown in glucose-based medium, and recultured in galactose-based medium to induce protein expression from the GALL promoter. Clonogenic survival was determined by recovering cells at various times from the galactose-based medium and plating 1000 cells on glucose-based semisolid medium. For each timepoint, the percentage of survival was calculated by dividing the number of colonies surviving the Bax protein induction by the number of colonies appearing after mock-treatment (empty). The absence of OYE3 attenuates Bax-induced cell death. (b) Wild-type BY4742 and ΔOYE3 cells were grown in glucose-based medium. Following the addition of H2O2 to a final concentration of 0.5 mM, cells were recovered at various times and their clonogenic survival determined. The absence of OYE3 leads to a sensitivity towards H2O2. Error bars represent the standard deviation of the mean of three independent experiments. (∗) Statistically significant difference based on the Wilcoxon Rank Sum test (α= 0.05).

3

Bax protein expression is unaffected in yeast cells knocked out for OYE3. Wild-type and ΔOYE3 cells were transformed with the plasmids p415GALLmBAX (Bax) or p415GALL (empty), grown in glucose-based medium, transferred to galactose-based medium and cultured for an additional period of 12 or 24 h. Total protein extracts (30 μg) were subjected to SDS–PAGE and immunoblot analysis, using a monoclonal antibody against murine Bax.

3

Bax protein expression is unaffected in yeast cells knocked out for OYE3. Wild-type and ΔOYE3 cells were transformed with the plasmids p415GALLmBAX (Bax) or p415GALL (empty), grown in glucose-based medium, transferred to galactose-based medium and cultured for an additional period of 12 or 24 h. Total protein extracts (30 μg) were subjected to SDS–PAGE and immunoblot analysis, using a monoclonal antibody against murine Bax.

Correlation of Bax- and H2O2-induced cell death with a drop in NADPH level

OYE3 encodes an NADPH dehydrogenase for which the physiological substrate is unknown to date. Therefore, the correlation between cell death rate and NADPH levels was investigated. BY4742 and ΔOYE3 had the same initial NADPH levels (Fig. 4(a)). During Bax-induced cell death NADPH levels decreased, though this NADPH drop was less pronounced in ΔOYE3 than in BY4742 (Fig. 4(a)). Further, the level of NADPH also decreased during H2O2-induced cell death (Fig. 4(c)). In these experiments, the use of 0.5 mM H2O2 gave the most reproducible and discriminatory results, although the same trend was observed with lower concentrations. However, contrary to the effects observed during Bax-induced cell death, the absence of OYE3 resulted in a more prominent drop in the level of NADPH (Fig. 4(c)). As shown in Fig. 2(b), deletion of OYE3 decreased the survival upon administration of 0.5 mM H2O2.

4

The absence of OYE3 has different effects on Bax- and H2O2-induced NADPH decrease and increase of thiobarbituric acid-reactive species. Wild-type BY4742 and ΔOYE3 cells were transformed with the plasmid p415GALLmBAX (Bax), grown in glucose-based medium, and recultured in galactose-based medium. Cells were recovered at various times and assayed (a) for their NADPH levels and (b) for lipid peroxidation breakdown products. The decrease in NADPH level, seen during Bax-induced cell death in wild type, is clearly less pronounced in the strain knocked-out for OYE3. The increase in thiobarbituric acid-reactive species, seen during Bax-induced cell death in wild type, is completely absent from the strain knocked out for OYE3. (c) Wild-type BY4742 and ΔOYE3 cells were grown in glucose-based medium. Following the addition of H2O2 to a final concentration of 0.5 mM, cells were recovered at various times and assayed for their NADPH levels. The decrease in NADPH level, seen during H2O2-induced cell death in wild type, is clearly more pronounced in the strain knocked-out for OYE3. (d) Wild-type BY4742 and ΔOYE3 cells were grown in glucose-based medium. Cells were recovered 1 h following the addition of various concentrations of H2O2, and assayed for lipid peroxidation. The increase in thiobarbiturc acid-reactive species, seen during H2O2-induced cell death in wild type, is more pronounced in the strain knocked-out for OYE3. Error bars represent the standard deviation of the mean of three independent experiments. (∗) Statistically significant difference based on the Wilcoxon Rank Sum test (α= 0.05).

4

The absence of OYE3 has different effects on Bax- and H2O2-induced NADPH decrease and increase of thiobarbituric acid-reactive species. Wild-type BY4742 and ΔOYE3 cells were transformed with the plasmid p415GALLmBAX (Bax), grown in glucose-based medium, and recultured in galactose-based medium. Cells were recovered at various times and assayed (a) for their NADPH levels and (b) for lipid peroxidation breakdown products. The decrease in NADPH level, seen during Bax-induced cell death in wild type, is clearly less pronounced in the strain knocked-out for OYE3. The increase in thiobarbituric acid-reactive species, seen during Bax-induced cell death in wild type, is completely absent from the strain knocked out for OYE3. (c) Wild-type BY4742 and ΔOYE3 cells were grown in glucose-based medium. Following the addition of H2O2 to a final concentration of 0.5 mM, cells were recovered at various times and assayed for their NADPH levels. The decrease in NADPH level, seen during H2O2-induced cell death in wild type, is clearly more pronounced in the strain knocked-out for OYE3. (d) Wild-type BY4742 and ΔOYE3 cells were grown in glucose-based medium. Cells were recovered 1 h following the addition of various concentrations of H2O2, and assayed for lipid peroxidation. The increase in thiobarbiturc acid-reactive species, seen during H2O2-induced cell death in wild type, is more pronounced in the strain knocked-out for OYE3. Error bars represent the standard deviation of the mean of three independent experiments. (∗) Statistically significant difference based on the Wilcoxon Rank Sum test (α= 0.05).

Thus, NADPH levels correlated with the rate of cell death for both cell death triggers.

Lipid peroxidation during Bax-induced cell death is not present in the OYE3 knockout

Based on the observation that the rate of Bax-induced cell death decreases and the rate of H2O2-triggered cell death increases upon deletion of OYE3, it could be argued that the absence of Oye3 somehow hampered the generation of oxygen stress, providing a protective mechanism against Bax-induced cell death. Therefore, we determined the extent of oxidative damage during Bax-induced cell death in ΔOYE3 compared to BY4742.

Neither oxidative protein damage nor oxidative mitochondrial DNA damage could be detected following Bax induction (data not shown), indicating that either these molecules are not damaged by the oxidative insult accompanying Bax expression, or else that they are rapidly and efficiently repaired or removed.

Lipid peroxidation, however, was found to be associated with Bax-induced cell death in BY4742 (Fig. 4(b)), but we could not observe any lipid peroxidation in ΔOYE3 during Bax-induced cell death. These findings implicate Old yellow enzyme Oye3, directly or indirectly, in Bax-induced lipid peroxidation.

Lipid peroxidation was also observed during H2O2-induced cell death (Fig. 4(d)), the extent of which was related to the H2O2 dose. However, oxidative lipid damage following H2O2 treatment was higher in ΔOYE3 (Fig. 4(d)), in line with the increased H2O2 sensitivity (Fig. 2(b)) and the more severe NADPH drop (Fig. 4(c)) upon H2O2 addition compared to the wild type.

Overall, these results show that the deletion of OYE3 clearly interferes with Bax-induced cell death as demonstrated by Bax tolerance, and augments H2O2-induced cell death. The different phenotypes induced by the two cell death triggers were reflected in the extent of NADPH decrease and lipid peroxidation in ΔOYE3.

Discussion

In the present study, we wanted to determine whether the Bax-induced transcriptional responses were different from those elicited by oxidative stress, and if so, whether these differences could explain how Bax expression actually generates a cellular oxidative stress.

First, we performed microarray expression profiling of the early transcriptional responses to Bax expression, and identified 75 Bax-responsive genes. Second, we optimized a H2O2 challenge to be as lethal as Bax expression. Due to the high membrane permeability of H2O2, its addition to the culture medium was thought to mimic the intracellular ROS generated as a consequence of expressed Bax protein. The transcript profiling of the H2O2 responses was in agreement with data published by others [84],[85]. Third, the transcriptional responses of the Bax-responsive genes to Bax induction and H2O2 treatment were compared. Ten of the 75 Bax-responsive genes showed a Bax-associated change that was at least 2-fold more manifest or was in the opposite sense compared to the corresponding H2O2-induced change. Fourth, we independently validated the transcriptional responses of these 10 genes by quantitative real-time PCR, and four genes (MLS1, OYE3, ICY2 and BTN2) were confirmed to have a Bax-specific transcriptional response. Homologues to these genes (MLS2, OYE2 and ICY1) did not demonstrate Bax-associated transcriptional changes in this study. Thus, the genes MLS1, OYE3, ICY2 and BTN2 could constitute part of the mechanism either by which Bax elicits oxygen stress or by which cells protect themselves against Bax toxicity. Particularly, the selection of four genes with a Bax-specific response reinforced our view about the difference between Bax expression and H2O2 treatment in eliciting yeast cell death.

We analyzed the phenotypic outcome of Bax expression in yeast strains individually knocked out for each of these four genes. We observed that only the absence of OYE3 (Old yellow enzyme) increased the tolerance to Bax-induced cell death. However, the ΔOYE3 strain clearly demonstrated H2O2 sensitivity. The difference in phenotypes of ΔOYE3 upon oxidative stress and Bax expression was interpreted as a reflection of the differential transcriptional response of this gene towards the two stresses (Bax: 2.70-fold down, H2O2: 1.50-fold up). Moreover, deletion of OYE3 per se did not increase generation time.

Old yellow enzyme (OYE; EC 1.6.99.1) was originally isolated from brewer's bottom yeast as “das gelbe Ferment”[86], and was shown to be composed of a colorless apoprotein and a redox-active flavin mononucleotide prosthetic group that gives it its distinctive yellow color [87]. Old yellow enzyme reduces the olefinic bond of α, β-unsaturated aldehydes and ketones [88]–[90], with NADPH as the physiological reductant for the enzyme-bound flavin [91],[92].

As Oye3 is an NADPH dehydrogenase, we wondered whether an increased intrinsic NADPH level, due to the absence of an NADPH-converting enzyme in ΔOYE3, could be responsible for the increased Bax tolerance. However, the decreased rate of Bax-induced cell death in the absence of OYE3 could not be attributed to an intrinsically augmented redox buffering capacity. Further, Bax-induced and H2O2-induced cell death were accompanied by a drop in NADPH level proportionate to the cell death rate (Fig. 5). Our observation of NADPH consumption during Bax-induced cell death further strengthens the position of oxygen stress as a general regulator of cell death [14], and extends the observation that intracellular glutathione is shifted to the more oxidized state during Bax expression [64].

5

Old yellow enzyme Oye3 interferes with Bax toxicity. Both Bax expression and H2O2 treatment induce lipid peroxidation and NADPH consumption, and these changes are part of both cell death processes. A drop in cellular NADPH will lead to redox buffer shortage and oxidative burden. Peroxidized lipids could affect membrane physiology or may interfere with an unknown signaling mechanism. The absence of Oye3 sensitizes yeast cells to H2O2-induced cell death but attenuates Bax-induced cell death. The absence of Oye3 is thought to increase H2O2 sensitivity due to the loss of its NADPH dehydrogenase activity, the substrates of which are likely to be lipid peroxidation breakdown products. Oye3 may be involved in the mechanism by which Bax expression leads to oxidative stress. For details, please refer to text.

5

Old yellow enzyme Oye3 interferes with Bax toxicity. Both Bax expression and H2O2 treatment induce lipid peroxidation and NADPH consumption, and these changes are part of both cell death processes. A drop in cellular NADPH will lead to redox buffer shortage and oxidative burden. Peroxidized lipids could affect membrane physiology or may interfere with an unknown signaling mechanism. The absence of Oye3 sensitizes yeast cells to H2O2-induced cell death but attenuates Bax-induced cell death. The absence of Oye3 is thought to increase H2O2 sensitivity due to the loss of its NADPH dehydrogenase activity, the substrates of which are likely to be lipid peroxidation breakdown products. Oye3 may be involved in the mechanism by which Bax expression leads to oxidative stress. For details, please refer to text.

The decreased rate of Bax-induced cell death and the increased rate of H2O2-triggered cell death upon deletion of OYE3 led us to investigate the extent of oxidative damage during Bax-induced cell death in ΔOYE3 compared to the wild type. Surprisingly, neither protein nor mitochondrial DNA oxidative damage was observed following Bax expression in wild type.

It has been proposed that the prime physiological substrates for Old yellow enzyme may be lipid peroxidation breakdown products [92], which can be toxic to cells [93], and are known to be potent apoptotic triggers [94]–[97]. Lipid peroxidation was found to be associated with Bax-induced cell death, in agreement with another study [44], and with H2O2-induced cell death (Fig. 5). Due to the absence of OYE3, no lipid peroxidation was observed during Bax-induced cell death, but lipid peroxidation breakdown products accumulated to a higher level following H2O2 treatment. The latter result is in agreement with the observation that OYE3 is part of the Yap1 regulon [85],[98]–[100].

In conclusion, our results clearly indicate that Oye3 is involved, directly or indirectly, in Bax-induced lipid peroxidation and the drop in NADPH level. This suggests Oye3 may interfere with the mechanism by which Bax expression leads to oxidative stress, being a part of the yeast-specific response to Bax-induced cell death.

Acknowledgements

The authors wish to thank Dr. Amin Bredan for writing assistance, Dr. Wouter Laroy for critical reading of the manuscript, André Reekmans for helpful discussions concerning growth curve fittings, and Sylviane Dewaele, Liesbeth Desmyter and Lieselot Defrancq for expert technical assistance. The authors acknowledge support from the Fund for Scientific Research-Flanders and the Bijzonder Onderzoeksfonds.

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