Identification of C18:1-phytoceramide as the candidate lipid mediator for hydroxyurea resistance in yeast

Recent studies showed that deletion of ISC1, the yeast homologue of the mammalian neutral sphingomyelinase, resulted in an increased sensitivity to hydroxyurea (HU). This raised an intriguing question as to whether sphingolipids are involved in pathways initiated by HU. In this study, we show that HU treatment led to a significant increase in Isc1 activity. Analysis of sphingolipid deletion mutants and pharmacological analysis pointed to a role for ceramide in mediating HU resistance. Lipid analysis revealed that HU induced increases in phytoceramides in WT cells but not in isc1Δ cells. To probe functions of specific ceramides, we developed an approach to supplement the medium with fatty acids. Oleate (C18:1) was the only fatty acid protecting isc1Δ cells from HU toxicity in a ceramide-dependent manner. Because phytoceramide activates protein phosphatases in yeast, we evaluated the role of CDC55, the regulatory subunit of ceramide-activated protein phosphatase PP2A. Overexpression of CDC55 overcame the sensitivity to HU in isc1Δ cells. However, addition of oleate did not protect the isc1Δ,cdc55Δ double mutant from HU toxicity. These results demonstrate that HU launches a lipid pathway mediated by a specific sphingolipid, C18:1-phytoceramide, produced by Isc1, which provides protection from HU by modulating Swe1 levels through the PP2A subunit Cdc55.