Stress-Activated Degradation of Sphingolipids Regulates Mitochondrial Function and Cell Death in Yeast

Figure 2

Sphingolipid degradation modulates mitochondrial activity in yeast. (a) Schematic overview of the enzymatic conversions of sphingolipid degradation. Downstream enzymatic activities of dihydrosphingosine-1-phosphate are depicted. Only the conversions of dihydrosphingosine and not of other sphingosine species such as phytosphingosine are shown. (b) Oxygen consumption rates of mutants affected in the sphingolipid degradation pathway. Cells were grown in synthetic galactose medium. The fzo1Δ mutant was included as a negative control. The O₂ consumption rate of the wild type was arbitrarily set to 1. (c) Reactive oxygen species (ROS) production in mutants affected in hexadecenal production (dpl1Δ) or degradation (hfd1Δ). 2′,7′-dichlorodihydrofluorescein diacetate assay in the indicated yeast strains before or after salt shock (1 M NaCl, 2 h). ROS levels upon normal growth conditions were set to 1 for each strain background. (d) Intracellular localization of Hfd1p. Cells expressing constitutive Hfd1-GFP and Om14-dsRed fusion proteins were grown in synthetic glucose- or galactose-containing medium. (e, f) Genetic manipulation of the sphingolipid degradation pathway affects cell viability and ROS production. The hexadecenal-producing Dpl1p enzyme was overexpressed under control of the GAL1 promoter in yeast wild type or the hfd1Δ mutant. (e) Growth efficiency was assessed on synthetic agar medium-containing glucose (SD) or galactose (SGal) supplemented or not with 4 μM valinomycin. Alternatively, colony formation was quantified in the same strains (lower panel). Cells from fresh overnight cultures in synthetic glucose medium were diluted in the indicated media to an OD₆₀₀ of 0.1, and growth was allowed for an additional 24 h. Colony-forming units were determined by plating the cells onto YPD agar medium. The colony number obtained for the wt upon the different growth conditions was set to 100. (f) Quantification of ROS production in the same strains grown in synthetic glucose or galactose medium by the 2′,7′-dichlorodihydrofluorescein diacetate assay. (g) Overexpression of Dpl1p causes mitochondrial fragmentation in hfd1Δ mutants. MitoTracker-stained mitochondria were visualized in the indicated yeast cells containing the empty vector or the galactose-inducible DPL1 expression on synthetic galactose medium. Data information: in (b, c, e, and f), data are presented as mean ± SD. Three biological replicates were analyzed. Significant changes with respect to the wild type are marked. , (Student’s t-test).

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