Old Yellow Enzymes, Highly Homologous FMN Oxidoreductases with Modulating Roles in Oxidative Stress and Programmed Cell Death in Yeast
2007; Elsevier BV; Volume: 282; Issue: 49 Linguagem: Inglês
10.1074/jbc.m704058200
ISSN1083-351X
AutoresOsama Odat, Samer Matta, Hadi Khalil, Sotirios C. Kampranis, Raymond Pfau, Philip N. Tsichlis, Antonios M. Makris,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoIn a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H2O2-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, Δoye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H2O2-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H2O2-induced PCD in wild type cells, but accelerate PCD in Δoye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (Δoye2 oye3) is highly resistant to H2O2-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H2O2-induced cell death: in Δoye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast. In a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H2O2-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, Δoye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H2O2-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H2O2-induced PCD in wild type cells, but accelerate PCD in Δoye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (Δoye2 oye3) is highly resistant to H2O2-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H2O2-induced cell death: in Δoye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast. Cell suicide responses regulated through programmed cell death (PCD) 3The abbreviations used are: PCD, programmed cell death; ROS, reactive oxygen species; OYE, Old Yellow Enzyme; GSH, γ-glutamylcystinylglycine; GSSG, glutathione disulfide; CM, complete medium; t-BOOH, tert-butyl hydroperoxide; CHP, cumene hydroperoxide; HE, hydroethidium; DKO, double knock-out; EMS, ethane methyl sulfonate; mit-GFP, mitochondria targeted green fluorescent protein; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; YFP, yellow fluorescent protein. have been documented not only in higher organisms but also in bacteria and yeast. In nature, unicellular organisms exist as populations in an environment with limited resources (1Costerton J.W. Stewart P.S. Greenberg E.P. Science. 1999; 284: 1318-1322Crossref PubMed Scopus (8948) Google Scholar); thus a conserved suicide program in which older or damaged cells sacrifice themselves and release nutrients to the remaining cells promotes continued group survival (2Cornillon S. Foa C. Davoust J. Buonavista N. Gross J.D. Golstein P. J. Cell Sci. 1994; 107: 2691-2704Crossref PubMed Google Scholar, 3Vachova L. Palkova Z. J. Cell Biol. 2005; 169: 711-717Crossref PubMed Scopus (153) Google Scholar, 4Sinclair D.A. Mills K. Guarente L. Trends Biochem. Sci. 1998; 23: 131-134Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Cell death with apoptotic features has been reported in yeast treated with low concentrations of acetic acid or hydrogen peroxide (5Ludovico P. Sousa M.J. Silva M.T. Leao C. Corte-Real M. Microbiology. 2001; 147: 2409-2415Crossref PubMed Scopus (412) Google Scholar, 6Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (868) Google Scholar), with DNA damage induced by UV radiation treatment (7Del Carratore R. Della Croce C. Simili M. Taccini E. Scavuzzo M. Sbrana S. Mutat. Res. 2002; 513: 183-191Crossref PubMed Scopus (89) Google Scholar), after exposure to high levels of mating pheromone (8Severin F.F. Hyman A.A. Curr. Biol. 2002; 12: R233-R235Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), and upon aging (9Herker E. Jungwirth H. Lehmann K.A. Maldener C. Frohlich K.U. Wissing S. Buttner S. Fehr M. Sigrist S. Madeo F. J. Cell Biol. 2004; 164: 501-507Crossref PubMed Scopus (453) Google Scholar). Recently, it has become clear that a core PCD machinery exists in Saccharomyces cerevisiae. For example, yeast with mutations in the CDC48 gene, an AAA family member involved in the fusion of endoplasmic reticulum-derived vesicles (10Latterich M. Frohlich K.U. Schekman R. Cell. 1995; 82: 885-893Abstract Full Text PDF PubMed Scopus (337) Google Scholar), exhibit characteristic hallmarks of apoptotic cell death including DNA fragmentation, chromatin remodeling (11Ahn S.H. Cheung W.L. Hsu J.Y. Diaz R.L. Smith M.M. Allis C.D. Cell. 2005; 120: 25-36Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), and annexin V staining (12Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (680) Google Scholar). Similarly, expression of a mutant form of the mammalian ortholog of CDC48, valosin containing protein, induces mammalian cells to undergo apoptosis (13Shirogane T. Fukada T. Muller J.M. Shima D.T. Hibi M. Hirano T. Immunity. 1999; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Yeast analogs of a number of components of the canonical apoptotic machinery have been described. Yeast homologs for caspase-like proteases, YCA1 (14Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar); for the OMI/HtrA2 protease, NMA111 (YNL123w) (15Fahrenkrog B. Sauder U. Aebi U. J. Cell Sci. 2004; 117: 115-126Crossref PubMed Scopus (177) Google Scholar); for apoptosis inducing factor, YNR074C; and for apoptosis inducing factor-homologous mitochondrion-associated inducer of death (AMID), NDI1, have all been implicated in the regulation of PCD in yeast (16Wissing S. Ludovico P. Herker E. Buttner S. Engelhardt S.M. Decker T. Link A. Proksch A. Rodrigues F. Corte-Real M. Frohlich K.U. Manns J. Cande C. Sigrist S.J. Kroemer G. Madeo F. J. Cell Biol. 2004; 166: 969-974Crossref PubMed Scopus (323) Google Scholar, 17Li W. Sun L. Liang Q. Wang J. Mo W. Zhou B. Mol. Biol. Cell. 2006; 17: 1802-1811Crossref PubMed Scopus (116) Google Scholar). Heterologous expression of mammalian regulators of apoptosis in yeast can influence yeast PCD. Expression of the anti-apoptotic protein Bcl-2 can rescue a superoxide dismutase-deficient yeast strain (18Kane D.J. Sarafian T.A. Anton R. Hahn H. Gralla E.B. Valentine J.S. Ord T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1614) Google Scholar), whereas expression of the pro-apoptotic counterpart Bax or Bak kills yeast in a manner that resembles PCD induced by these proteins in mammalian cells. Upon expression in yeast, Bax localizes mainly in the mitochondria and promotes mitochondrial membrane hyperpolarization (19Gross A. Pilcher K. Blachly-Dyson E. Basso E. Jockel J. Bassik M.C. Korsmeyer S.J. Forte M. Mol. Cell. Biol. 2000; 20: 3125-3136Crossref PubMed Scopus (145) Google Scholar), causing an eventual collapse of ΔΨm and release of reactive oxygen species (ROS) and cytochrome c. This phenotype has been exploited to isolate proteins inhibiting Bax lethality (20Jin C. Reed J.C. Nat. Rev. Mol. Cell. Biol. 2002; 3: 453-459Crossref PubMed Scopus (85) Google Scholar), allowing the identification of BI-1, a highly conserved apoptosis inhibitor (21Chae H.J. Kim H.R. Xu C. Bailly-Maitre B. Krajewska M. Krajewski S. Banares S. Cui J. Digicaylioglu M. Ke N. Kitada S. Monosov E. Thomas M. Kress C.L. Babendure J.R. Tsien R.Y. Lipton S.A. Reed J.C. Mol. Cell. 2004; 15: 355-366Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), enzymes involved in the ROS detoxification such as BI-GST (22Kampranis S.C. Damianova R. Atallah M. Toby G. Kondi G. Tsichlis P.N. Makris A.M. J. Biol. Chem. 2000; 275: 29207-29216Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) and ascorbate peroxidase (23Moon H. Baek D. Lee B. Prasad D.T. Lee S.Y. Cho M.J. Lim C.O. Choi M.S. Bahk J. Kim M.O. Hong J.C. Yun D.J. Biochem. Biophys. Res. Commun. 2002; 290: 457-462Crossref PubMed Scopus (60) Google Scholar), and Ku70, an evolutionarily conserved component of the double-stranded DNA repair machinery (24Sawada M. Sun W. Hayes P. Leskov K. Boothman D.A. Matsuyama S. Nat. Cell Biol. 2003; 5: 320-329Crossref PubMed Scopus (318) Google Scholar). Previous work in our laboratory isolated a series of EMS-mutagenized yeast strains that exhibited resistance to Bax-induced PCD (25Belhocine S. Mbithe C. Dimitrova I. Kampranis S.C. Makris A.M. Cell Death Differ. 2004; 11: 946-948Crossref PubMed Scopus (8) Google Scholar). To further exploit the effect of Bax and obtain insights on the yeast PCD machinery, we have here utilized one such mutant that failed to target Bax to mitochondria, in a reverse genetic screen to identify yeast proteins that restore sensitivity to Bax. This screen identified the C terminus of the conserved flavin mononucleotide (FMN) oxidoreductase OYE2. Suggestively, the highly related OYE3 protein, which is known to heterodimerize with OYE2 (26Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar), had previously been found to modulate Bax-dependent PCD in yeast (27Reekmans R. De Smet K. Chen C. Van Hummelen P. Contreras R. FEMS Yeast Res. 2005; 5: 711-725Crossref PubMed Scopus (20) Google Scholar). In the current study, we show that full-length OYE2 suppressed Bax lethality in wild type yeast, and is a potent antioxidant protein. This activity contrasts with that of OYE3, which antagonized the protective action of OYE2 in H2O2-induced programmed cell death. The effect of OYE3 requires the presence of OYE2, indicating that it is the oye2p-oye3p heterodimer that facilitates PCD. Surprisingly, the absence of both genes rendered cells hyper-resistant to H2O2-induced PCD by increasing their respiratory efficiency. Deletion of OYE2 with other antioxidant genes elevated endogenous ROS and sensitized cells further to H2O2-induced PCD. In the case of Δoye2 glr1 cells, the cellular redox environment with high levels of oxidized glutathione led to gross morphological aberrations, actin cytoskeleton abnormalities, and defects in organelle partition between mother and daughter cells. Together, these results indicate that OYE2 is a connection point between ROS generation, modulation of PCD, and cytoskeletal regulation. Genetic Screen in Yeast—A previously characterized EMS mutagenized yeast strain R13 (his3 ura3 trp1 LexA-operator-LEU2) carrying the pGILDA/Bax plasmid was transformed with a yeast genomic library on the plasmid pJG4-5 (25Belhocine S. Mbithe C. Dimitrova I. Kampranis S.C. Makris A.M. Cell Death Differ. 2004; 11: 946-948Crossref PubMed Scopus (8) Google Scholar). Growing colonies were replica plated on glucose/CM-His,Trp, and galactose-raffinose/CM-His,Trp. Colonies growing on glucose media but not on galactose, where Bax is expressed, were selected for further characterization. The library plasmids were extracted and then reintroduced into fresh cells and were tested for the reproducibility of the Bax resensitization phenotype. The library plasmids capable of restoring Bax lethality were sequenced. Growth Recovery Curves—Fresh overnight cultures of the various yeast strains grown in Glu/CM media or glucose media lacking the amino acid used as auxotrophic marker were washed with dH2O and resuspended in fresh medium at A600 = 0.1. Aliquots were taken at regular intervals and the absorbance was measured. When A600 > 1, aliquots were serially diluted and new measurements were taken. To examine the effect of a transient pulse of H2O2 in cultures overexpressing the OYE proteins, fresh cells were resuspended at A600 = 0.1 in fresh glucose media and 2 h later, at the end of the lag period, the cultures were supplemented with 1.5 or 1.25 mm H2O2 and incubated with shaking at 30 °C. The ability of the cell populations to recover from the H2O2 insult was assessed by measuring growth at A600 at regular intervals. All growth recovery assays were performed independently in triplicate. Plasmid Constructs—The full-lengths of the OYE2 and OYE3 genes were PCR amplified from wild type yeast genomic DNA using primers 5′OYE2(EcoRI) 5′-GAATTCATGCCATTTGTTAAGGACTTTAAGCC-3′ and 3′OYE2(XhoI) 5′-CTCGAGTTAATTTTTGTCCCAACCGAGTTTTAG-3′ for OYE2 and 5′OYE3 5′-CAATTGATGCCATTTGTAAAAGGTTTTGAGCCGATC-3′ and 3′OYE3 5′-CTCGAGTCAGTTCTTGTTCCAACCTAAATCTACTGC-3′ for OYE3. A fusion of the OYE2 with GFP in the C terminus was prepared using a two-step PCR approach with overlapping primers. In the first step, OYE2 was amplified using primers 5′OYE2(EcoRI) and 3′OYE2(GFP), 5′-CTCGCCCTTGCTCACATTTTTGTCCCAACC-3′, and the GFP construct was amplified using the 5′OYE-GFP 5′-GGTTGGGACAAAAATGTGAGCAAGGGCGAC-3′ and the 3′GFP(XhoI) 5′-CTCGAGTTACTTGTACAGCTCGTCCATGCC-3′. The amplified products from the first round were gel extracted and purified. A small aliquot of the two fragments was mixed in a new PCR using the 5′OYE2(EcoRI) and the 3′GFP(XhoI) external primers. All PCR amplifications were made using Platinum Taq polymerase (Invitrogen). The purified fragments were cloned into the pCR2.1 TOPO TA vector according to the manufacturer's instructions. All the cloned inserts were subsequently subcloned into the yeast expression vectors: pJG4-6, expressing the proteins under a galactose promoter; and pYX143 and pYX143-HA (hemagglutinin tagged), which are low copy number vectors (ARS/CEN, LEU2) expressing the genes under the control of the constitutive TPI promoter. Expression of the proteins was verified in PYX143-HA and pJG4-6 in Western blots using antibodies against the HA tag. BY4741 wild type yeast cells were transformed with pYX143-OYE2, pYX143-OYE3, or a control empty plasmid. Protein expression was verified indirectly by parallel cloning of the OYE2 and OYE3 cDNAs into the pYX143-HA vector, which expresses the cDNAs fused to a hemagglutinin tag. However, the untagged vectors were used in all subsequent experiments to eliminate the possibility of any interference of the HA tag. A C-terminal-truncated construct of OYE2 was generated by PCR using the primer 5′OYE2 and 3′OYE2 (1-388) 5′-CTCGAGCTACGTAGGGTAGTCAATGTA-3′. The product was sequenced and subcloned into the pYX143-HA and pYX143 vectors. Mutation of tyrosine 197 to phenylalanine in OYE2 was generated according to the directions of the QuikChange Site-directed Mutagenesis protocol from Stratagene. The complementary primers OYE2(Y197F)5 5′-CCACAGCGCTAACGGTTTCTTGTTGAACCAGTTCTTG-3′ and OYE2(Y197F)3 3′-CAAGAACTGGTTCAACAAGAAACCGTTAGCGCTGTGG-3′ were used in a PCR run for 16 rounds using Pfu Turbo DNA polymerase. Subsequent to DpnI digestion of nonmutated parental DNA, the digest was used to transform bacterial cells. Plasmid DNA from isolated colonies was sequenced to verify the mutation. The Y197F OYE2 cDNA was subcloned into pYX143, pYX143-HA yeast vectors as above. Flow Cytometric Studies—Yeast strains growing in glucose complete media, untill late logarithmic phase, were washed with PBS and stained. Dihydroethidium (HE; D-1168, Molecular Probes) at 4 μm was used as an indicator of endogenous ROS. Yeast cells harboring plasmids were grown in glucose complete media lacking the amino acid used as the auxotrophic marker. To assess cellular responses to pro-oxidants, aliquots of grown cells were treated for 1 h with 1.5 mm hydrogen peroxide (H2O2), 1 mm t-butyl hydroperoxide (t-BOOH), or 0.2 mm cumene hydroperoxide (CHP). Subsequent to treatment, cells were washed extensively with PBS by repeated centrifugations and finally stained with HE as above. Using FACS, 100,000 cells from each sample were measured. Quantification was performed using the Cytomation software (DAKO). Microscopy and Fluorescence Measurements—Mitochondrial import and morphology was visualized using plasmid pVT100U-mitGFP or plasmid pYX142-mitGFP (which expresses GFP fused to a mitochondrial matrix targeting sequence), which were introduced in yeast cells (28Westermann B. Neupert W. Yeast. 2000; 16: 1421-1427Crossref PubMed Scopus (312) Google Scholar). The organelles were observed in a single plane so as to observe the relative diameter of the tubules. Actin filaments were stained by fixing with 4% formaldehyde and staining using rhodamine-phalloidin (R-415, Molecular Probes). Nuclei were stained with Hoechst 33342. To mask fluorescence from mitochondrial DNA a low dose of Mitotracker Red CMXRos (M7512, Molecular Probes) was used as a counterstain. The lumen of yeast vacuoles was stained with 50 μm CMAC (Y-7531, Molecular Probes) for 20 min. At the end of incubations cells were washed twice, resuspended in prewarmed PBS, applied on microscope slides and observed under ×1000 magnification in a fluorescent microscope. Necrotic cells with permeabilized outer membranes were measured by Evans blue staining. In addition to FACS analysis, ROS were also measured using a PerkinElmer LS55 Luminescence spectrometer. Fresh cultures of cells were resuspended in PBS at A600 = 0.5 and 1-ml aliquots of cells were stained with Mitotracker Red CMXRos (M-7512, Molecular Probes) for 15 min in the dark and washed once with PBS, at the end of incubation the cells were resuspended in 2 ml and fluorescence was measured over the optimal emission range. Generation of Double Knock-out Yeast Strains—A yeast strain harboring a deletion of the OYE2 gene was generated by integrating a URA3 cassette originating from the pUG72 plasmid (29Gueldener U. Heinisch J. Koehler G.J. Voss D. Hegemann J.H. Nucleic Acids Res. 2002; 30: e23Crossref PubMed Scopus (752) Google Scholar). The primers 5′OYE2 lox 5′-TCATATTAAGCTAAATATAGACGATAATATAGTATCGATAATGCCACAGCTGAAGCTTCGTACGC-3′ and 3′OYE2 lox 5′-AAATGGTGCTACAAAGTACGGTTAACACTATTAATTTTTGTCCCAACCGCATAGGCCACTAGTGGATCTG-3′, which contain flanking sequences from the 5′ and 3′ of the OYE2 gene, were used to amplify the cassette in a PCR. The amplified DNA fragment was purified and transformed into a Matα strain that originated by repeated backcrosses to a BY4741 background. Proper integration of the URA cassette was verified by PCR using primers 5′GenOYE2, 5′-CACGACAAGATTCTTCTATTGATTATTCACATATGT-3′, and 3′OYE2 lox. To generate double knockouts the Δoye2::URA3 strain was mated to BY4741 deletion strains Δoye3, Δsod1, Δctt1, Δglr1, Δyca1, and Δgsh1 (Research Genetics), which harbor the G418 antibiotic resistance marker on the deleted gene. Diploid cells were induced to sporulate and the regenerating spores were tested in Glu/CM-ura, +G418 plates. The mating type of the double knock-out haploid strains was subsequently assessed. ρ0 strains (lacking functional mitochondria) were generated from the ρ+ Δoye2 oye3 strain by growing cells in YPD broth containing 10 μg/ml ethidium bromide for 3 days (30Braun R.J. Zischka H. Madeo F. Eisenberg T. Wissing S. Buttner S. Engelhardt S.M. Buringer D. Ueffing M. J. Biol. Chem. 2006; 281: 25757-25767Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Dilutions were plated on YPD plates and growing colonies were tested for their capacity to grow on YPGlycerol media. Induction of PCD with H2O2 and Acetic Acid and Colony Viability Assays—Cells growing in logarithmic phase were used to inoculate at very low density in fresh glucose media. A small aliquot was subsequently removed, serially diluted, and plated on rich YPD plates. This represented the 0 time point and viability of 100%. To the diluted cells, H2O2 was added to 1 mm, and cells were incubated for 2 h at 30 °C. Treated cells were enumerated as above and compared with the original count. All experiments were performed independently in triplicate. The presence of necrotic cells was determined by staining with Evans blue and visualization by light microscopy. Acetic acid-induced PCD was performed in accordance to the protocol described by Ludovico et al. (5Ludovico P. Sousa M.J. Silva M.T. Leao C. Corte-Real M. Microbiology. 2001; 147: 2409-2415Crossref PubMed Scopus (412) Google Scholar) using unbuffered Glu/CM media. Chronological Aging Assays—All strains were grown in 10-ml cultures with unbuffered glucose complete media (2% glucose) untill saturation. At the end of the second day of incubation, a small aliquot of cells was removed and the number of live cells was enumerated by serial dilution and plating on YPD plates. This corresponded to the 0 time point. Aliquots of cells were removed regularly and cells were enumerated as above. Determination of Total Glutathione and Glutathione Disulfide—BY4741, Δoye2, Δoye3, Δoye2 oye3, and Δoye2 glr1 cells were grown untill late log phase in Glu/CM. Total glutathione and glutathione disulfide were measured according to a method described by Griffith (31Griffith O.W. Anal. Biochem. 1980; 106: 207-212Crossref PubMed Scopus (4034) Google Scholar) and modified by Kampranis et al. (22Kampranis S.C. Damianova R. Atallah M. Toby G. Kondi G. Tsichlis P.N. Makris A.M. J. Biol. Chem. 2000; 275: 29207-29216Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The FMN Oxidoreductase OYE2 Limits Bax-induced Lethality in Yeast—To identify yeast genes that participate in PCD processes we took advantage of the Bax-induced lethal phenotype in yeast. The Bax-resistant EMS-mutant strain R13, which exhibits defects in mitochondrial protein targeting (25Belhocine S. Mbithe C. Dimitrova I. Kampranis S.C. Makris A.M. Cell Death Differ. 2004; 11: 946-948Crossref PubMed Scopus (8) Google Scholar), was used in a screen to identify yeast proteins that can restore Bax sensitivity. We transformed cells containing a galactose-inducible Bax expression plasmid with a yeast genomic library cloned into the pJG4-5, galactose-inducible, yeast expression vector. Transformed yeast were initially plated on glucose-selective plates at low density to enable us to pick distinct colonies. Approximately 3,000 colonies were replica-plated on glucose and galactose-selective media to induce Bax and library expression from the galactose promoter. One of the library clones that converted Bax-resistant R13 cells back to sensitivity contained the C terminus of OYE2 from amino acid 314 to the end of the gene (amino acids 314-400). The gene is translated from a proximal ATG supplied from the pJG4-5 vector (Fig. 1). Reekmans et al. (27Reekmans R. De Smet K. Chen C. Van Hummelen P. Contreras R. FEMS Yeast Res. 2005; 5: 711-725Crossref PubMed Scopus (20) Google Scholar) have recently shown that deletion of OYE3, an FMN oxidoreductase homologous to OYE2, attenuated Bax-induced growth arrest, cell death, and caused a decrease in NADPH in yeast. Among the EMS mutant yeasts we previously generated, which are resistant to Bax-induced PCD, a significant proportion also showed defects in protein transport to mitochondria, most likely limiting integration of Bax to the outer membrane. Expression of OYE2-(314-400) in R13 cells not only sensitized cells to Bax lethality (Fig. 1A), but also restored the ability of GFP fused to a mitochondrial targeting sequence (mit-GFP) to associate with mitochondria (Fig. 1C), indicating that OYE2 can affect mitochondrial targeting. The size of R13 cells also decreased and resembled wild type appearance. In wild type yeast, Bax causes changes in the morphology of the mitochondria. Whereas in a percentage of cells the organelles become fragmented, the remaining cells exhibit fewer swollen organelles most likely by fusion as a defense response (Fig. 1B). YFP-Bax localized to mitochondria in wild type cells, whereas in the R13 Bax-resistant mutant the YFP fusion clearly fails to target the organelles (Fig. 1B, bottom row). Co-expression of YFP-Bax with the OYE2-(314-400) C terminus reversed the diffused fluorescence, however, only very weak peripheral fluorescence could be detected (data not shown). YFP-Bax is lethal to R13 cells, only when co-expressed with the OYE2-(314-400) despite the difficulty in detecting its fluorescence. Recently TOM22, a component of the complex responsible for initial import of mitochondrial targeted proteins was identified as a Bax receptor (32Bellot G. Cartron P.F. Er E. Oliver L. Juin P. Armstrong L.C. Bornstein P. Mihara K. Manon S. Vallette F.M. Cell Death Differ. 2006; 14: 785-794Crossref PubMed Scopus (132) Google Scholar). This could explain the frequent association between Bax resistance and defects in mitochondrial protein translocation in our mutants (25Belhocine S. Mbithe C. Dimitrova I. Kampranis S.C. Makris A.M. Cell Death Differ. 2004; 11: 946-948Crossref PubMed Scopus (8) Google Scholar). We next overexpressed the full-length open reading frame of OYE2 from the pJG4-4 vector, under the control of a galactose-inducible promoter. In wild type EGY48 cells expressing Bax, overexpression of the full-length OYE2 suppressed Bax lethality, whereas the C terminus OYE2 could not do so (Fig. 2A). OYE2 also reversed to a large extent mitochondrial swelling, as well as excessive mitochondrial fission, both characteristic effects of Bax expression in sensitive yeast strains (Fig. 2B). Full-length OYE2 did not resensitize the R13 mutant cells to Bax (data not shown), suggesting that this action of the OYE2-(314-400) represented a dominant negative effect of the truncated protein. Supporting the OYE2 protective role, Bax expression in Δoye2 was more toxic compared with wild type yeast (data not shown). OYE2 Localizes to Mitochondria—As OYE2 was isolated based on restoring Bax sensitivity to a strain with deficient localization of Bax to its site of action, the mitochondria, we asked if OYE2 might itself associate with mitochondria. A full-length OYE2-GFP fusion was introduced into EGY48 cells under the control of a galactose-inducible promoter. OYE2-GFP localized to distinct domains in the cytoplasm that partially overlapped with mitochondria (Fig. 2C). This indicates that OYE2 localized on the organelle and can thus limit directly the capacity of Bax to insert and oligomerize on the mitochondrial outer membrane. The OYE2-GFP fusion weakly protected wild type cells from Bax lethality and did not act as dominant negative (data not shown). OYE2 and OYE3 Have Opposing Functions in Regulation of Oxidative Stress in Wild Type Yeast—Bax-induced lethality in yeast is strongly linked to enhanced intracellular oxidative stress (33Dimitrova I. Toby G.G. Tili E. Strich R. Kampranis S.C. Makris A.M. FEBS Lett. 2004; 566: 100-104Crossref PubMed Scopus (11) Google Scholar). OYE3 modulates Bax-dependent PCD (27Reekmans R. De Smet K. Chen C. Van Hummelen P. Contreras R. FEMS Yeast Res. 2005; 5: 711-725Crossref PubMed Scopus (20) Google Scholar), and heterodimerizes with OYE2 in vivo and in vitro (26Stott K. Saito K. Thiele D.J. Massey V. J. Biol. Chem. 1993; 268: 6097-6106Abstract Full Text PDF PubMed Google Scholar). Furthermore, OYE2 and OYE3 share 82% identity at the amino acid level, suggesting a related activity. To elucidate the functional relationship of OYE2, and OYE3, we expressed them at moderate levels in yeast and assessed basal and induced levels of oxidative stress. Yeast overexpressing OYE2 or OYE3, or vector-transformed control cells, were treated with 1.5 mm H2O2, with 1 mm of the prooxidant tert-butyl hydroperoxide (t-BOOH), and with 0.2 mm of the prooxidant CHP. After incubation, cells were stained with HE to gauge oxidative stress: Fig. 3A compares FACS-determined HE values for cells overexpressing OYE2 or OYE3 (hatched lines) in reference to cells expressing empty vector (vertical lines) for each condition. In untreated cells, overexpression of OYE2 modestly lowered (-13%), and OYE3 elevated (+24%), endogenous ROS
Referência(s)