Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys47) was essential for Tsa1 activity, while Tsa1C170S lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or tsa1CCS lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The tsa1CCS rad51– or tsa1 rad51–induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.
References
[1]
Rhee SG, Chae HZ, Kim K (2005) Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 38: 1543–1552.
[2]
Wood ZA, Schroder E, Robin Harris J, Poole LB (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28: 32–40.
[3]
Park SG, Cha MK, Jeong W, Kim IH (2000) Distinct physiological functions of thiol peroxidase isoenzymes in Saccharomyces cerevisiae. J Biol Chem 275: 5723–5732.
[4]
Poole LB (2005) Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases. Arch Biochem Biophys 433: 240–254.
[5]
Biteau B, Labarre J, Toledano MB (2003) ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 425: 980–984.
[6]
Chang TS, Jeong W, Woo HA, Lee SM, Park S, et al. (2004) Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine. J Biol Chem 279: 50994–51001.
Tanaka T, Izawa S, Inoue Y (2005) GPX2, encoding a phospholipid hydroperoxide glutathione peroxidase homologue, codes for an atypical 2-Cys peroxiredoxin in Saccharomyces cerevisiae. J Biol Chem 280: 42078–42087.
[12]
Maiorino M, Ursini F, Bosello V, Toppo S, Tosatto SC, et al. (2007) The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases. J Mol Biol 365: 1033–1046.
[13]
Wong CM, Zhou Y, Ng RW, Kung Hf HF, Jin DY (2002) Cooperation of yeast peroxiredoxins Tsa1p and Tsa2p in the cellular defense against oxidative and nitrosative stress. J Biol Chem 277: 5385–5394.
[14]
Jang HH, Lee KO, Chi YH, Jung BG, Park SK, et al. (2004) Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function. Cell 117: 625–635.
[15]
Monje-Casas F, Michan C, Pueyo C (2004) Absolute transcript levels of thioredoxin- and glutathione-dependent redox systems in Saccharomyces cerevisiae: response to stress and modulation with growth. Biochem J 383: 139–147.
[16]
Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, et al. (2003) Global analysis of protein expression in yeast. Nature 425: 737–741.
[17]
Munhoz DC, Netto LE (2004) Cytosolic thioredoxin peroxidase I and II are important defenses of yeast against organic hydroperoxide insult: catalases and peroxiredoxins cooperate in the decomposition of H2O2 by yeast. J Biol Chem 279: 35219–35227.
[18]
Huang ME, Rio AG, Nicolas A, Kolodner RD (2003) A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations. Proc Natl Acad Sci U S A 100: 11529–11534.
[19]
Wong CM, Siu KL, Jin DY (2004) Peroxiredoxin-null yeast cells are hypersensitive to oxidative stress and are genomically unstable. J Biol Chem 279: 23207–23213.
[20]
Huang ME, Kolodner RD (2005) A biological network in Saccharomyces cerevisiae prevents the deleterious effects of endogenous oxidative DNA damage. Mol Cell 17: 709–720.
[21]
Cha MK, Choi YS, Hong SK, Kim WC, No KT, et al. (2003) Nuclear thiol peroxidase as a functional alkyl-hydroperoxide reductase necessary for stationary phase growth of Saccharomyces cerevisiae. J Biol Chem 278: 24636–24643.
[22]
Pedrajas JR, Miranda-Vizuete A, Javanmardy N, Gustafsson JA, Spyrou G (2000) Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity. J Biol Chem 275: 16296–16301.
[23]
Veal EA, Findlay VJ, Day AM, Bozonet SM, Evans JM, et al. (2004) A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase. Mol Cell 15: 129–139.
[24]
Vivancos AP, Castillo EA, Biteau B, Nicot C, Ayte J, et al. (2005) A cysteine-sulfinic acid in peroxiredoxin regulates H2O2-sensing by the antioxidant Pap1 pathway. Proc Natl Acad Sci U S A 102: 8875–8880.
[25]
Jara M, Vivancos AP, Calvo IA, Moldon A, Sanso M, et al. (2007) The peroxiredoxin Tpx1 is essential as a H2O2 scavenger during aerobic growth in fission yeast. Mol Biol Cell 18: 2288–2295.
[26]
Ragu S, Faye G, Iraqui I, Masurel-Heneman A, Kolodner RD, et al. (2007) Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death. Proc Natl Acad Sci U S A 104: 9747–9752.
[27]
Chae HZ, Uhm TB, Rhee SG (1994) Dimerization of thiol-specific antioxidant and the essential role of cysteine 47. Proc Natl Acad Sci U S A 91: 7022–7026.
[28]
Bossy-Wetzel E, Green DR (2000) Detection of apoptosis by annexin V labeling. Methods Enzymol 322: 15–18.
[29]
Frohlich KU, Fussi H, Ruckenstuhl C (2007) Yeast apoptosis—from genes to pathways. Semin Cancer Biol 17: 112–121.
[30]
Madeo F, Frohlich E, Ligr M, Grey M, Sigrist SJ, et al. (1999) Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145: 757–767.
[31]
Madeo F, Herker E, Maldener C, Wissing S, Lachelt S, et al. (2002) A caspase-related protease regulates apoptosis in yeast. Mol Cell 9: 911–917.
[32]
Ahn SH, Cheung WL, Hsu JY, Diaz RL, Smith MM, et al. (2005) Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae. Cell 120: 25–36.
[33]
D'Autreaux B, Toledano MB (2007) ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 8: 813–824.
[34]
Fourquet S, Huang ME, D'Autreaux B, Toledano MB (2008) The dual functions of thiol-based peroxidases in H2O2 scavenging and signaling. Antioxid Redox Signal 10: 1565–1576.
[35]
Neumann CA, Krause DS, Carman CV, Das S, Dubey DP, et al. (2003) Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression. Nature 424: 561–565.
[36]
Lee TH, Kim SU, Yu SL, Kim SH, Park DS, et al. (2003) Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice. Blood 101: 5033–5038.
[37]
Iraqui I, Faye G, Ragu S, Masurel-Heneman A, Kolodner RD, et al. (2008) Human peroxiredoxin PrxI is an orthologue of yeast Tsa1, capable of suppressing genome instability in Saccharomyces cerevisiae. Cancer Res 68: 1055–1063.
[38]
Jeong JS, Kwon SJ, Kang SW, Rhee SG, Kim K (1999) Purification and characterization of a second type thioredoxin peroxidase (type II TPx) from Saccharomyces cerevisiae. Biochemistry 38: 776–783.
[39]
Kouzminova EA, Rotman E, Macomber L, Zhang J, Kuzminov A (2004) RecA-dependent mutants in Escherichia coli reveal strategies to avoid chromosomal fragmentation. Proc Natl Acad Sci U S A 101: 16262–16267.
[40]
Buttner S, Bitto A, Ring J, Augsten M, Zabrocki P, et al. (2008) Functional mitochondria are required for alpha-synuclein toxicity in aging yeast. J Biol Chem 283: 7554–7560.
[41]
Guscetti F, Nath N, Denko N (2005) Functional characterization of human proapoptotic molecules in yeast S. cerevisiae. FASEB J 19: 464–466.
[42]
Cheung WL, Ajiro K, Samejima K, Kloc M, Cheung P, et al. (2003) Apoptotic phosphorylation of histone H2B is mediated by mammalian sterile twenty kinase. Cell 113: 507–517.
[43]
Weinberger M, Ramachandran L, Feng L, Sharma K, Sun X, et al. (2005) Apoptosis in budding yeast caused by defects in initiation of DNA replication. J Cell Sci 118: 3543–3553.
[44]
Qi H, Li TK, Kuo D, Nur EKA, Liu LF (2003) Inactivation of Cdc13p triggers MEC1-dependent apoptotic signals in yeast. J Biol Chem 278: 15136–15141.
[45]
Mazzoni C, Mancini P, Verdone L, Madeo F, Serafini A, et al. (2003) A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast. Mol Biol Cell 14: 721–729.
[46]
Schmidt KH, Pennaneach V, Putnam CD, Kolodner RD (2006) Analysis of gross-chromosomal rearrangements in Saccharomyces cerevisiae. Methods Enzymol 409: 462–476.
[47]
Chen C, Kolodner RD (1999) Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Nat Genet 23: 81–85.
[48]
Amberg DC, Burke DJ, Strathern JN (2005) Methods in yeast genetics. Cold spring harbor laboratory press.
[49]
Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27.
[50]
Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A, et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.
[51]
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, et al. (2006) Current protocols in molecular biology. New York, N.Y.: John Wiley & Sons, Inc.
[52]
Foiani M, Marini F, Gamba D, Lucchini G, Plevani P (1994) The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication. Mol Cell Biol 14: 923–933.
[53]
Lea DE, Coulson CA (1948) The distribution of the numbers of mutants in bacterial populations. J Genet 49: 264–285.
