Identification of C18:1-Phytoceramide as the Candidate Lipid Mediator for Hydroxyurea Resistance in Yeast

Stony Brook University, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2013; 288(24). DOI: 10.1074/jbc.M112.444802
Source: PubMed


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 pharmacologic 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. In order to probe functions of specific ceramides, we developed an approach to supplement the medium with fatty acids. Oleate (C-18:1) was the only fatty acid protecting isc1Δ cells from HU toxicity in a ceramide-dependent manner. Since 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 isc1Δ,cdc55Δ double mutant from HU toxicity. These results demonstrate that HU launches a lipid pathway mediated by a specific sphingolipid, C-18:1 phytoceramide, produced by Isc1, which provides protection from HU by modulating Swe1 levels through PP2A subunit Cdc55.

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Available from: Kaushlendra Tripathi, Dec 16, 2014
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    • "Overexpression of YPC1 has been proposed to make cells resistant to high concentrations of PHS in the medium by lowering free PHS levels through reverse activity (Mao et al., 2000b); Ypc1p thus may channel PHS into C26-containing complex sphingolipids. On the other hand, a recent report also shows that a large panel of less abundant phytoceramides containing nonhydroxylated fatty acids with 14–26 C atoms accumulate when cells are growing in presence of hydroxurea, and that their accumulation is significantly reduced by overexpression of YPC1 (Matmati et al., 2013). This strongly suggests that such ceramides are broken down by overexpressed YPC1, and that ceramidase activity in vivo is by no means restricted to ceramides with very long chain fatty acids. "
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    • "Growing evidence supports the importance of substrate specificity of enzymes belonging to the degradation pathway in production of bioactive SLs. Recent observations in S. cerevisiae support the role of a specific ceramide species generated by Isc1p (yeast ceramidase) in the resistance to hydroxyurea [26]. In mammals, bioactive sphingosine is mainly produced by ceramidase activity [21] and in plants, the alkaline ceramidase also presents a defined substrate selectivity [17]. "
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