Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de Novo synthesis

Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the several-fold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C₁₈ and C₁₀ phytosphingosines. Since the most studied mechanism for ceramide generation in Animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [³H]phytosphingosine and [³H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol- P-ceramide, and mannose(inositol-P)₂-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [³H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or beth.