Role of C2-phytoceramide in mediating cell death in yeast

Abstract

Ceramides are naturally occurring sphingolipids that cause several biological effects, including induction of cell cycle arrest, cell maturation, terminal cell differentiation, cell senescence, and cell death. It is well established that ceramide induces both apoptosis and autophagy in mammalian cells. However, the molecular mechanisms induced by ceramide in yeast, as well as the signalling systems involved, are poorly understood. It is known that phytoceramides, assumed to be the yeast counterpart of the mammalian ceramides, mediate regulation of cell growth and stress responses in yeast. However, their role has proved difficult to define. In this study, we characterized the effect of exogenous N-Acetyl-phytosphingosine (C2-phytoceramide) on Saccharomyces cerevisiae W303 1A cells. We found that C2-phytoceramide induced death in a dose dependent manner (10-40 µM), but did not trigger authophagy, since cell survival was not rescued in the absence of Atg5p and C2-phytoceramide did not induce Atg8-GFP cleavage. We then tested whether C2-phytoceramide-induced death was apoptotic in nature. We found that exposure to C2-phytoceramide resulted in increased nuclear condensation (15 %), but not in a significant increase in DNA fragmentation. It did not lead to ROS accumulation and, accordingly, loss of cell viability could not be rescued by treating cells with ROS scavengers. Loss of cell viability could also not be rescued by overexpression of the anti-apoptotic protein Bcl-2. Other apoptotic markers are under characterization. Nonetheless, C2-phytoceramide-induced death was only partially accompanied by an increase in cells with loss of membrane integrity, indicating is it is a regulated process. We determined the involvement of several pathways in signalling C2-phytoceramide-induced death. We found that the mutant strain W303 isc1Δ (unable to generate ceramide by degradation of complex lipids) displayed higher sensitivity to C2-phytoceramide. Several mutant strains lacking components of Mitogen Activated Protein Kinase (MAPK) cascades were also tested. We found that the W303 hog1Δ mutant strain displayed increased sensitivity to C2-phytoceramide, while the ste20Δ strain displayed increased resistance. The overall results suggest that C2-phytoceramide leads to cell death in yeast and points to a crosstalk between ceramide and MAPK signalling pathways in this process.