Figure 8.

Ceramide homeostasis and enhanced catabolism of inositol SLs. (A) Inositol-SLs are labile in sec14-1^ts tlg2Δ double mutants. Wild-type, sec14-1^ts, sec14-1^ts tlg2Δ, and sec14-1^ts tlg2Δ kes1Δ yeast were radiolabeled to steady state with ³[H]inositol (20 μCi/ml) at 30°C. Subsequently, cells were washed and resuspended in glucose-SD media and incubated for 3 h at 37°C in the presence of unlabeled inositol (12 μM). Lipids were extracted from cells and subject to alkaline methanolysis. Inositol-SLs were quantified by liquid scintillation counting and relative inositol-SL stability for each strain is expressed as the ratio of [³H] after chase:[³H] before chase. Values represent averages (and standard deviations) from three independent experiments. (B) Inositol SL profiles are altered in tlg2Δ mutants. Wild-type, sec14-1^ts, tlg2Δ, sec14-1^ts tlg2Δ, and sec14-1^ts tlg2Δ kes1Δ yeast were labeled to steady state with [³H]inositol (20 μCi/ml). After a 2-h shift to the restrictive temperature for the sec14-1^ts allele (37°C), lipids were extracted from 5 OD_{600 nm} of cells and subject to alkaline methanolysis. TLC profiles for the inositol-SLs for yeast strains of indicated genotype are shown and each lipid assignment (indicated at left) was determined by R_f. (C) Quantitative inositol SLomics. Complex SLs were extracted from 25 OD₆₀₀ of yeast and analyzed by mass spectrometry. Lipid mass was calculated by comparison with internal standards. Each SL species is represented as the log₁₀ of the mutant:wild-type ion intensity ratio. Species shown in red report decreased mass in mutant relative to wild-type, whereas green indicates increase. (D) Incorporation of a ppn1Δ allele into the sec14-1^ts tlg2Δ genetic background restores UPR activity. UPR activity was monitored via the UPRE::LACZ reporter in wild-type, sec14-1^ts tlg2Δ, sec14-1^ts tlg2Δ isc1Δ, and sec14-1^ts tlg2Δ ppn1Δ yeast strains as indicated. Yeast were cultured at the permissive temperature (30°C) to mid-logarithmic growth phase and then shifted to the restrictive temperature (37°C) for 2 h before analysis. As control for UPR activity, wild-type yeast were challenged with DTT (final concentration, 5 mM) for 60 min before assay.