We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast. 1. Introduction In Saccharomyces cerevisiae the highly homologous genes RAS1 and RAS2 encode small G-proteins that are activated by the guanine nucleotide exchange factors (GEFs), Cdc25 and Sdc25 [1, 2] and inactivated by the GTPase-activating proteins (GAPs), Ira1 and Ira2 [3]. GEFs and GAPs control the switch of the two small monomeric proteins between the active GTP-bound and the inactive GDP-bound state. The Ras proteins and the GPCR system [4–6] constitute two branches that modulate the activity of adenylate cyclase (Cyr1), according to the glucose availability in the environment. In turn Cyr1 [7] activates cAMP-dependent protein kinase (PKA) through cAMP. The amount of this second messenger is also regulated at the level of degradation by the two phosphodiesterases, Pde1 and Pde2. PKA plays a major role in the modulation of metabolism, stress resistance, cell growth, proliferation, morphogenesis, and aging [8]. Recently, our group expressed a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3), which binds Ras-GTP with a much higher affinity than Ras-GDP, to investigate the localization of active Ras in wild-type and in mutant strains in the cAMP/PKA pathway [9]. Our results showed that in W303-1A wild-type cells the probe
References
[1]
S. Jones, M.-L. Vignais, and J. R. Broach, “The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to Ras,” Molecular and Cellular Biology, vol. 11, no. 5, pp. 2641–2646, 1991.
[2]
S. Rudoni, S. Colombo, P. Coccetti, and E. Martegani, “Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae,” Biochimica et Biophysica Acta, vol. 1538, no. 2-3, pp. 181–189, 2001.
[3]
K. Tanaka, M. Nakafuku, T. Satoh et al., “S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein,” Cell, vol. 60, no. 5, pp. 803–807, 1990.
[4]
S. Zaman, S. I. Lippman, X. Zhao, and J. R. Broach, “How Saccharomyces responds to nutrients,” Annual Review of Genetics, vol. 42, pp. 27–81, 2008.
[5]
J. M. Thevelein and J. H. De Winde, “Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae,” Molecular Microbiology, vol. 33, no. 5, pp. 904–918, 1999.
[6]
F. Rolland, J. Winderickx, and J. M. Thevelein, “Glucose-sensing and -signalling mechanisms in yeast,” FEMS Yeast Research, vol. 2, no. 2, pp. 183–201, 2002.
[7]
G. F. Casperson, N. Walker, and H. R. Bourne, “Isolation of the gene encoding adenylate cyclase in Saccharomyces cerevisiae,” Proceedings of the National Academy of Sciences of the United States of America, vol. 82, no. 15, pp. 5060–5063, 1985.
[8]
B. Smets, R. Ghillebert, P. De Snijder et al., “Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae,” Current Genetics, vol. 56, no. 1, pp. 1–32, 2010.
[9]
S. Broggi, E. Martegani, and S. Colombo, “Live-cell imaging of endogenous Ras-GTP shows predominant Ras activation at the plasma membrane and in the nucleus in Saccharomyces cerevisiae,” The International Journal of Biochemistry & Cell Biology, vol. 45, no. 2, pp. 384–394, 2013.
[10]
J. E. Leadsham, K. Miller, K. R. Ayscough et al., “Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast,” Journal of Cell Science, vol. 122, no. 5, pp. 706–715, 2009.
[11]
Y. Hu, W. Lu, G. Chen et al., “K-rasG12V transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis,” Cell Research, vol. 22, no. 2, pp. 399–412, 2012.
[12]
S. Büttner, T. Eisenberg, D. Carmona-Gutierrez et al., “Endonuclease G regulates budding yeast life and death,” Molecular Cell, vol. 25, no. 2, pp. 233–246, 2007.
[13]
F. Madeo, E. Fr？hlich, and K.-U. Fr？hlich, “A yeast mutant showing diagnostic markers of early and late apoptosis,” Journal of Cell Biology, vol. 139, no. 3, pp. 729–734, 1997.
[14]
F. Madeo, E. Herker, S. Wissing, H. Jungwirth, T. Eisenberg, and K.-U. Fr？hlich, “Apoptosis in yeast,” Current Opinion in Microbiology, vol. 7, no. 6, pp. 655–660, 2004.
[15]
F. Madeo, E. Herker, C. Maldener et al., “A caspase-related protease regulates apoptosis in yeast,” Molecular Cell, vol. 9, no. 4, pp. 911–917, 2002.
[16]
N. Guaragnella, C. Pereira, M. J. Sousa et al., “YCA1 participates in the acetic acid induced yeast programmed cell death also in a manner unrelated to its caspase-like activity,” FEBS Letters, vol. 580, no. 30, pp. 6880–6884, 2006.
[17]
W. Li, L. Sun, Q. Liang, J. Wang, W. Mo, and B. Zhou, “Yeast AMID homologue Ndi1p displays respiration-restricted apoptotic activity and is involved in chronological aging,” Molecular Biology of the Cell, vol. 17, no. 4, pp. 1802–1811, 2006.
[18]
S. Wissing, P. Ludovico, E. Herker et al., “An AIF orthologue regulates apoptosis in yeast,” Journal of Cell Biology, vol. 166, no. 7, pp. 969–974, 2004.
[19]
B. Fahrenkrog, U. Sauder, and U. Aebi, “The S. cerevisiae HtrA-like protein Nma111p is a nuclear serine protease that mediates yeast apoptosis,” Journal of Cell Science, vol. 117, no. 1, pp. 115–126, 2004.
[20]
J. Qiu, J.-H. Yoon, and B. Shen, “Search for apoptotic nucleases in yeast: role of Tat-D nuclease in apoptotic DNA degradation,” The Journal of Biological Chemistry, vol. 280, no. 15, pp. 15370–15379, 2005.
[21]
I. Orlandi, M. Bettiga, L. Alberghina, and M. Vai, “Transcriptional profiling of ubp10 null mutant reveals altered subtelomeric gene expression and insurgence of oxidative stress response,” The Journal of Biological Chemistry, vol. 279, no. 8, pp. 6414–6425, 2004.
[22]
M. Ligr, F. Madeo, E. Fr？hlich, W. Hilt, K.-U. Fr？hlich, and D. H. Wolf, “Mammalian Bax triggers apoptotic changes in yeast,” FEBS Letters, vol. 438, no. 1-2, pp. 61–65, 1998.
[23]
F. Madeo, E. Fr？hlich, M. Ligr et al., “Oxygen stress: a regulator of apoptosis in yeast,” Journal of Cell Biology, vol. 145, no. 4, pp. 757–767, 1999.
[24]
L. Du, Y. Su, D. Sun et al., “Formic acid induces Yca1p-independent apoptosis-like cell death in the yeast Saccharomyces cerevisiae,” FEMS Yeast Research, vol. 8, no. 4, pp. 531–539, 2008.
[25]
P. Ludovico, M. J. Sousa, M. T. Silva, C. Le？o, and M. C？rte-Real, “Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid,” Microbiology, vol. 147, no. 9, pp. 2409–2415, 2001.
[26]
P. Ludovico, F. Rodrigues, A. Almeida, M. T. Silva, A. Barrientos, and M. C？rte-Real, “Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae,” Molecular Biology of the Cell, vol. 13, no. 8, pp. 2598–2606, 2002.
[27]
N. Guaragnella, L. Antonacci, S. Passarella, E. Marra, and S. Giannattasio, “Hydrogen peroxide and superoxide anion production during acetic acid-induced yeast programmed cell death,” Folia Microbiologica, vol. 52, no. 3, pp. 237–240, 2007.
[28]
S. Giannattasio, A. Atlante, L. Antonacci et al., “Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger,” FEBS Letters, vol. 582, no. 10, pp. 1519–1525, 2008.
[29]
N. Guaragnella, L. Antonacci, S. Giannattasio, E. Marra, and S. Passarella, “Catalase T and Cu, Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae,” FEBS Letters, vol. 582, no. 2, pp. 210–214, 2008.
[30]
N. Guaragnella, A. Bobba, S. Passarella, E. Marra, and S. Giannattasio, “Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1,” FEBS Letters, vol. 584, no. 1, pp. 224–228, 2010.
[31]
F. Madeo, D. Carmona-Gutierrez, J. Ring, S. Büttner, T. Eisenberg, and G. Kroemer, “Caspase-dependent and caspase-independent cell death pathways in yeast,” Biochemical and Biophysical Research Communications, vol. 382, no. 2, pp. 227–231, 2009.
[32]
D. Ahuatzi, A. Riera, R. Peláez, P. Herrero, and F. Moreno, “Hxk2 regulates the phosphorylation state of Mig1 and therefore its nucleocytoplasmic distribution,” The Journal of Biological Chemistry, vol. 282, no. 7, pp. 4485–4493, 2007.
[33]
F. Moreno, D. Ahuatzi, A. Riera, C. A. Palomino, and P. Herrero, “Glucose sensing through the Hxk2-dependent signalling pathway,” Biochemical Society Transactions, vol. 33, no. 1, pp. 265–268, 2005.
[34]
B. J. Thomas and R. Rothstein, “Elevated recombination rates in transcriptionally active DNA,” Cell, vol. 56, no. 4, pp. 619–630, 1989.
[35]
L. C. Robinson, J. B. Gibbs, M. S. Marshall, I. S. Sigal, and K. Tatchell, “CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae,” Science, vol. 235, no. 4793, pp. 1218–1221, 1987.
[36]
R. J. Rothstein, “One-step gene disruption in yeast,” Methods in Enzymology, vol. 101, pp. 202–211, 1983.
[37]
M. Bettiga, L. Calzari, I. Orlandi, L. Alberghina, and M. Vai, “Involvement of the yeast metacaspase Yca1 in ubp10Δ-programmed cell death,” FEMS Yeast Research, vol. 5, no. 2, pp. 141–147, 2004.
[38]
N. Casatta, A. Porro, I. Orlandi, L. Brambilla, and M. Vai, “Lack of Sir2 increases acetate consumption and decreases extracellular pro-aging factors,” Biochimica et Biophysica Acta, vol. 1833, no. 3, pp. 593–601, 2013.
[39]
S. Colombo, D. Ronchetti, J. M. Thevelein, J. Winderickx, and E. Martegani, “Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae,” The Journal of Biological Chemistry, vol. 279, no. 45, pp. 46715–46722, 2004.
[40]
P. Fabrizio and V. D. Longo, “Chronological aging-induced apoptosis in yeast,” Biochimica et Biophysica Acta, vol. 1783, no. 7, pp. 1280–1285, 2008.
[41]
M. A. S. Khan, P. B. Chock, and E. R. Stadtman, “Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 48, pp. 17326–17331, 2005.
[42]
J. C. Wolfman, S. M. Planchon, J. Liao, and A. Wolfman, “Structural and functional consequences of c-N-Ras constitutively associated with intact mitochondria,” Biochimica et Biophysica Acta, vol. 1763, no. 10, pp. 1108–1124, 2006.
[43]
T. G. Bivona, S. E. Quatela, B. O. Bodemann et al., “PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis,” Molecular Cell, vol. 21, no. 4, pp. 481–493, 2006.
[44]
A. Rebollo, D. Pérez-Sala, and C. Martínez-A, “Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis,” Oncogene, vol. 18, no. 35, pp. 4930–4939, 1999.
