Figure 1.
Sphingolipids metabolism in yeast.
Schematic overview of yeast sphingolipid metabolism displaying the metabolic intermediates, genes involved and cellular locations of the enzymatic reactions.
Figure 2.
Acetic acid resistance in S. cerevisiae.
(A) CG379 (wild-type), lac1Δ, lag1Δ, ydc1Δ, ypc1Δ and isc1Δ strains, or (B) CG379 pYES2 (empty vector), lag1Δ transformed with pYES2 or pLAG1, and isc1Δ transformed with pYES2 or pISC1. Cells were exposed to 180 mM acetic acid, pH 3.0, for 200 min. Cell viability was determined by standard dilution plate counts and expressed as a percentage of c.f.u on YPD plates. Values are mean ± SD of at least three independent experiments. Values significantly different from CG379 strain: *** P<0.001, One-way ANOVA and Tukey Test.
Figure 3.
Levels of mitochondrial ROS (A) and superoxide anion (B) in the indicated S. cerevisiae strains exposed to 180 mM acetic acid, pH 3.0, for 0 min (white columns) and 200 min (black columns), using MitoTracker Red CM-H2XRos (A) and DHE (B), respectively. Values are mean+SD of at least three independent experiments. Values significantly different from CG379: * P<0.05, ** P<0.01 and *** P<0.001, One-way ANOVA and Tukey Test.
Figure 4.
Mitochondrial morphology and degradation.
(A) Analysis of mitochondrial morphology in S. cerevisiae strains CG379, lag1Δ and isc1Δ expressing mitochondrial GFP (pGAL-CLbGFP) before (control) and after exposure to 180 mM acetic acid, pH 3.0, for 200 min. Cells were observed by fluorescence microscopy. A representative experiment is shown. Bar, 5 µm. (B) Mitochondrial degradation was assessed in these strains by measuring the loss of mtGFP fluorescence during exposure to 180 mM acetic acid, pH 3.0, up to 240 min. (C) Pep4p activity was detected in extracts of the same strains, by measuring the release of tyrosine-containing acid- soluble peptides from acid-denatured haemoglobin. Values are mean ± SD of at least three independent experiments. Values significantly different from CG379: * P<0.05, ** P<0.01, One-way ANOVA and Tukey Test.
Figure 5.
Western blot analysis of cytochrome c in S. cerevisiae strains CG379, isc1Δ and lag1Δ before (−) and after (+) exposure to 180 mM acetic acid, pH 3.0, for 200 min, in both mitochondrial and cytosolic fractions. Cytosolic phosphoglycerate kinase (Pgk1p) and mitochondrial porin (Por1p) levels were used as loading control of cytosolic and mitochondrial fractions, respectively. A representative experiment is shown of at least two independent experiments with similar results.
Figure 6.
Changes in the levels of sphingolipid species during acetic acid-induced apoptosis.
Relative levels of dihydrosphingosine (DHS), dihydrosphingosine-1-phosphate (DHS-1-P), phytosphingosine (PHS), phytosphingosine-1-phosphate (PHS-1-P), phytoceramides (Phyto-C16-Cer and Phyto-C18-Cer) and α-hydroxylated phytoceramide (α-OH-Phyto-C20-Cer) in S. cerevisiae CG379 (wild-type), lag1Δ and isc1Δ strains after exposure to 180 mM acetic acid, pH 3.0, for 200 min are shown. Values are mean ± SD of three independent experiments. * P<0.05, ** P<0.01, One-way ANOVA and Tukey Test. Levels of α-OH-Phyto-C20-Cer in CG379 reproducibly displayed a high increase after exposure to acetic acid in all experiments, though relative values varied considerably.
Figure 7.
Effect of exogenous C2-phytoceramide.
Survival of S. cerevisiae strain CG379 (A), lag1Δ (B) and isc1Δ (C) exposed to DMSO, 15 µM of C2-phytoceramide, 180 mM acetic acid or 15 µM of C2-phytoceramide and 180 mM acetic acid, for up to 200 min. Cell viability was determined by standard dilution plate counts and expressed as a percentage of c.f.u on YPD plates. Values are mean ± SD of at least three independent experiments. Values significantly different: *** P<0.001, One-way ANOVA and Tukey Test.
Table 1.
List of S. cerevisiae strains used in this study.
Table 2.
List of oligonucleotides used in this study.
Table 3.
List of plasmids used in this study.