The role of ceramides in the mitochondrial apoptotic pathway induced by acetic acid

Abstract

The yeast Saccharomyces cerevisiae undergoes a mitochondrial-dependent regulated cell death in response to different stimuli, exhibiting typical markers of mammalian apoptosis. However, the upstream signalling 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. We have previously characterized the contribution of enzymes involved in ceramide metabolism to apoptotic cell death induced by acetic acid in yeast. We showed 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 ROS production, reduced mitochondrial alterations and less translocation of cytochrome c into the cytosol in response to acetic acid. In this work, the interaction between these sphingolipid metabolism enzymes, as well as ceramide, in the modulation of acetic acid-induced regulated cell death was assessed. We found that deletion of both LAG1 and ISC1 is not more advantageous, since deletion of both genes does not increase the survival of yeast cells to acetic acid. This suggests that blocking both metabolic pathways precludes cells from counteracting the effects of acetic acid treatment via production of a pool of sphingolipids with a pro-survival role. Consistently, we also observed that these single mutant displayed a decrease in some phytoceramide species and increased of LCB-1Ps. Additionally, combined exposure to C2- phytoceramide and acetic acid resulted in significantly greater sensitivity than individual exposure to each agent in both wild-type and isc1Δ and lag1Δ mutant cells, suggesting that phytoceramide is sufficient to induce cell death and sensitize cells to acetic acid. Pkh1p, Ypk1p, Sch9p, Sit4p, Tor1p and Hog1p have been shown to mediate the effects of sphingolipids and/or regulate sphingolipid levels, mitochondria function and oxidative stress in response to several stimuli. The role of these signalling proteins in acetic acid-induced apoptosis, possibly acting through Isc1p, was therefore assessed. Our results suggest that Pkh1p, Ypk1p, Sch9p and Sit4p, but not Tor1p and Hog1p, regulate acetic acid-induced cell death. Isc1p and Sit4p seem to act independently in acetic acid-induced cell death. However an involvement of Sit4p in the regulation of mitochondrial function and resistance of the isc1Δ mutant cannot be totally discarded. We also found that acetic acid exposure leads to Pkh1p dependent-phosphorylation of both Sch9p and Ypk1p and that Isc1p acts in the same pathway as Sch9p. Both sch9Δ and isc1Δ single and double mutants lacking Isc1p and Sch9p have the same resistant phenotype, and we observed that SCH9 deletion impairs the translocation of Isc1p to mitochondria upon acetic acid-induced cell death. Unexpectedly, these resistant mutants exhibited higher levels of mitochondria ceramides than the wild-type cells. However, we found that the higher resistance of all mutants correlates with higher levels of endogenous LCB-1-P, which suggest that changing the sphingolipid balance in favour of the LCBPs in mitochondria or whole cells results in increased survival to acetic acid. The role of the Pkh1p-Ypk1p pathway in the resistance of isc1Δ was also addressed. We observed that Pkh1p is necessary for isc1Δ resistance to acetic acid-induced apoptosis. Indeed, double deletion of ISC1 and PKH1 had a drastic effect on cell survival, which was associated with increased ROS accumulation and release of cytochrome c, abnormal cell morphology and accumulation of large amounts of chitin, suggesting an involvement of the CWI pathway. Accordingly, we found the CWI pathway signals acetic acid-induced cell death, as blocking signal transduction through this pathway or over-activation of the CWI renders cells more resistant or more sensitive to acetic acid. In conclusion, the overall results suggest that Pkh1p-Ypk1p and Pkh1p- Sch9p pathways play a different role in acetic acid-induced cell death, through the regulation and activation of Isc1p and Lag1p, which contribute to production of sphingolipids that lead to cell death.