The yeast Saccharomyces cerevisiae undergoes a mitochondrial-dependent programmed cell death in response to different stimuli, such as acetic acid, with features similar to those of mammalian apoptosis. However, the upstream signaling events in this process, including those leading to mitochondrial membrane permeabilization, are still poorly characterized. Changes in sphingolipid metabolism have been linked to modulation of apoptosis in both yeast and mammalian cells, and ceramides have been detected in mitochondria upon apoptotic stimuli. In this study, we aimed to characterize the contribution of enzymes involved in ceramide metabolism to apoptotic cell death induced by acetic acid. We show that isc1Δ and lag1Δ mutants, lacking inositol phosphosphingolipid phospholipase C and ceramide synthase, respectively, exhibited a higher resistance to acetic acid that was associated with lower levels of some phytoceramide species. Consistently, these mutant cells displayed lower levels of ROS production and reduced mitochondrial alterations, such as mitochondrial fragmentation and degradation, and decreased translocation of cytochrome c into the cytosol in response to acetic acid. These results suggest that ceramide production contributes to cell death induced by acetic acid, especially through hydrolysis of complex sphingolipids catalyzed by Isc1p and de novo synthesis catalyzed by Lag1p, and provide the first in vivo indication of its involvement in mitochondrial outer membrane permeabilization in yeast.
References
[1]
Buttner S, Eisenberg T, Herker E, Carmona-Gutierrez D, Kroemer G, et al. (2006) Why yeast cells can undergo apoptosis: death in times of peace, love, and war. J Cell Biol 175: 521–525.
[2]
Ludovico P, Sousa MJ, Silva MT, Leao C, Corte-Real M (2001) Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 147: 2409–2415.
[3]
Ludovico P, Rodrigues F, Almeida A, Silva MT, Barrientos A, et al. (2002) Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol Biol Cell 13: 2598–2606.
[4]
Wissing S, Ludovico P, Herker E, Buttner S, Engelhardt SM, et al. (2004) An AIF orthologue regulates apoptosis in yeast. J Cell Biol 166: 969–974.
[5]
Fannjiang Y, Cheng WC, Lee SJ, Qi B, Pevsner J, et al. (2004) Mitochondrial fission proteins regulate programmed cell death in yeast. Genes Dev 18: 2785–2797.
[6]
Pereira C, Chaves S, Alves S, Salin B, Camougrand N, et al. (2010) Mitochondrial degradation in acetic acid-induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier. Mol Microbiol 76: 1398–1410.
[7]
Pereira C, Camougrand N, Manon S, Sousa MJ, Corte-Real M (2007) ADP/ATP carrier is required for mitochondrial outer membrane permeabilization and cytochrome c release in yeast apoptosis. Mol Microbiol 66: 571–582.
[8]
Bartke N, Hannun YA (2009) Bioactive sphingolipids: metabolism and function. J Lipid Res 50Suppl: S91–96.
[9]
Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9: 139–150.
[10]
Maceyka M, Payne SG, Milstien S, Spiegel S (2002) Sphingosine kinase, sphingosine-1-phosphate, and apoptosis. Biochim Biophys Acta 1585: 193–201.
[11]
Taha TA, Mullen TD, Obeid LM (2006) A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death. Biochim Biophys Acta 1758: 2027–2036.
[12]
Morales A, Lee H, Goni FM, Kolesnick R, Fernandez-Checa JC (2007) Sphingolipids and cell death. Apoptosis 12: 923–939.
[13]
Gudz TI, Tserng KY, Hoppel CL (1997) Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem 272: 24154–24158.
[14]
Siskind LJ, Fluss S, Bui M, Colombini M (2005) Sphingosine forms channels in membranes that differ greatly from those formed by ceramide. J Bioenerg Biomembr 37: 227–236.
[15]
Chipuk JE, McStay GP, Bharti A, Kuwana T, Clarke CJ, et al. (2012) Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis. Cell 148: 988–1000.
[16]
Jenkins GM, Richards A, Wahl T, Mao C, Obeid L, et al. (1997) Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae. J Biol Chem 272: 32566–32572.
[17]
Wells GB, Dickson RC, Lester RL (1998) Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis. J Biol Chem 273: 7235–7243.
Sawai H, Okamoto Y, Luberto C, Mao C, Bielawska A, et al. (2000) Identification of ISC1 (YER019w) as inositol phosphosphingolipid phospholipase C in Saccharomyces cerevisiae. J Biol Chem 275: 39793–39798.
[20]
Vaena de Avalos S, Okamoto Y, Hannun YA (2004) Activation and localization of inositol phosphosphingolipid phospholipase C, Isc1p, to the mitochondria during growth of Saccharomyces cerevisiae. J Biol Chem 279: 11537–11545.
[21]
Kitagaki H, Cowart LA, Matmati N, Vaena de Avalos S, Novgorodov SA, et al. (2007) Isc1 regulates sphingolipid metabolism in yeast mitochondria. Biochim Biophys Acta 1768: 2849–2861.
[22]
Kitagaki H, Cowart LA, Matmati N, Montefusco D, Gandy J, et al. (2009) ISC1-dependent metabolic adaptation reveals an indispensable role for mitochondria in induction of nuclear genes during the diauxic shift in Saccharomyces cerevisiae. J Biol Chem 284: 10818–10830.
[23]
Betz C, Zajonc D, Moll M, Schweizer E (2002) ISC1-encoded inositol phosphosphingolipid phospholipase C is involved in Na+/Li+ halotolerance of Saccharomyces cerevisiae. Eur J Biochem 269: 4033–4039.
[24]
Cowart LA, Okamoto Y, Lu X, Hannun YA (2006) Distinct roles for de novo versus hydrolytic pathways of sphingolipid biosynthesis in Saccharomyces cerevisiae. Biochem J 393: 733–740.
[25]
Matmati N, Kitagaki H, Montefusco D, Mohanty BK, Hannun YA (2009) Hydroxyurea sensitivity reveals a role for ISC1 in the regulation of G2/M. J Biol Chem 284: 8241–8246.
[26]
Almeida T, Marques M, Mojzita D, Amorim MA, Silva RD, et al. (2008) Isc1p plays a key role in hydrogen peroxide resistance and chronological lifespan through modulation of iron levels and apoptosis. Mol Biol Cell 19: 865–876.
[27]
Barbosa AD, Osorio H, Sims KJ, Almeida T, Alves M, et al. (2011) Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells. Mol Microbiol 81: 515–527.
[28]
Barbosa AD, Graca J, Mendes V, Chaves SR, Amorim MA, et al. (2012) Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging. Mech Ageing Dev 133: 317–330.
[29]
D'Mello NP, Childress AM, Franklin DS, Kale SP, Pinswasdi C, et al. (1994) Cloning and characterization of LAG1, a longevity-assurance gene in yeast. J Biol Chem 269: 15451–15459.
[30]
Jiang JC, Kirchman PA, Allen M, Jazwinski SM (2004) Suppressor analysis points to the subtle role of the LAG1 ceramide synthase gene in determining yeast longevity. Exp Gerontol 39: 999–1009.
[31]
Siskind LJ, Feinstein L, Yu T, Davis JS, Jones D, et al. (2008) Anti-apoptotic Bcl-2 family proteins disassemble ceramide channels. J Biol Chem 283: 6622–6630.
[32]
Aerts AM, Zabrocki P, Francois IE, Carmona-Gutierrez D, Govaert G, et al. (2008) Ydc1p ceramidase triggers organelle fragmentation, apoptosis and accelerated ageing in yeast. Cell Mol Life Sci 65: 1933–1942.
[33]
Carmona-Gutierrez D, Reisenbichler A, Heimbucher P, Bauer MA, Braun RJ, et al. (2011) Ceramide triggers metacaspase-independent mitochondrial cell death in yeast. Cell Cycle 10: 3973–3978.
[34]
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.
[35]
Perrone GG, Tan SX, Dawes IW (2008) Reactive oxygen species and yeast apoptosis. Biochim Biophys Acta 1783: 1354–1368.
[36]
Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408: 239–247.
[37]
Cabiscol E, Piulats E, Echave P, Herrero E, Ros J (2000) Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J Biol Chem 275: 27393–27398.
[38]
Costa VM, Amorim MA, Quintanilha A, Moradas-Ferreira P (2002) Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7. Free Radic Biol Med 33: 1507–1515.
[39]
Valenti D, Vacca RA, Guaragnella N, Passarella S, Marra E, et al. (2008) A transient proteasome activation is needed for acetic acid-induced programmed cell death to occur in Saccharomyces cerevisiae. FEMS Yeast Res 8: 400–404.
[40]
Karbowski M, Youle RJ (2003) Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ 10: 870–880.
[41]
Heinrich M, Wickel M, Schneider-Brachert W, Sandberg C, Gahr J, et al. (1999) Cathepsin D targeted by acid sphingomyelinase-derived ceramide. EMBO J 18: 5252–5263.
[42]
Burtner CR, Murakami CJ, Kennedy BK, Kaeberlein M (2009) A molecular mechanism of chronological aging in yeast. Cell Cycle 8: 1256–1270.
Hwang O, Kim G, Jang YJ, Kim SW, Choi G, et al. (2001) Synthetic phytoceramides induce apoptosis with higher potency than ceramides. Mol Pharmacol 59: 1249–1255.
[45]
Grant CM, MacIver FH, Dawes IW (1997) Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. FEBS Lett 410: 219–222.
[46]
Mullen TD, Obeid LM (2011) Ceramide and apoptosis: Exploring the enigmatic connections between sphingolipid metabolism and programmed cell death. Anticancer Agents Med Chem
[47]
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.
[48]
Guaragnella N, Antonacci L, Giannattasio S, Marra E, Passarella S (2008) Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae. FEBS Lett 582: 210–214.
[49]
Siskind LJ, Colombini M (2000) The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis. J Biol Chem 275: 38640–38644.
[50]
Perera MN, Ganesan V, Siskind LJ, Szulc ZM, Bielawski J, et al. (2012) Ceramide channels: Influence of molecular structure on channel formation in membranes. Biochim Biophys Acta 1818: 1291–1301.
[51]
Ganesan V, Perera MN, Colombini D, Datskovskiy D, Chadha K, et al. (2010) Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane. Apoptosis 15: 553–562.
[52]
Stiban J, Caputo L, Colombini M (2008) Ceramide synthesis in the endoplasmic reticulum can permeabilize mitochondria to proapoptotic proteins. J Lipid Res 49: 625–634.
[53]
Parra V, Eisner V, Chiong M, Criollo A, Moraga F, et al. (2008) Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis. Cardiovasc Res 77: 387–397.
[54]
Marques M, Mojzita D, Amorim MA, Almeida T, Hohmann S, et al. (2006) The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae. Microbiology 152: 3595–3605.
[55]
Mollapour M, Piper PW (2007) Hog1 mitogen-activated protein kinase phosphorylation targets the yeast Fps1 aquaglyceroporin for endocytosis, thereby rendering cells resistant to acetic acid. Mol Cell Biol 27: 6446–6456.
[56]
Barbosa AD, Gra？a J, Mendes V, Chaves SR, Amorim MA, et al. (2012) Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging. Mech Ageing Dev in press.
[57]
Herrero P, Fernandez R, Moreno F (1985) Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae. Arch Microbiol 143: 216–219.
[58]
Jones EW (1990) Vacuolar proteases in yeast Saccharomyces cerevisiae. Methods Enzymol 185: 372–386.
[59]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
[60]
Bielawski J, Pierce JS, Snider J, Rembiesa B, Szulc ZM, et al. (2010) Sphingolipid analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Adv Exp Med Biol 688: 46–59.
[61]
Montefusco DJ, Newcomb B, Gandy JL, Brice SE, Matmati N, et al. (2012) Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability. J Biol Chem 287: 9280–9289.
[62]
Okamoto K, Perlman PS, Butow RA (1998) The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud. J Cell Biol 142: 613–623.
