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 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.

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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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    ABSTRACT: Humans and yeast possess alkaline ceramidases located in the early secretory pathway. Single deletions of the highly homologous yeast alkaline ceramidases YPC1 and YDC1 have very little genetic interactions or phenotypes. Here we performed chemical-genetic screens to find deletions/conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities in sphingolipid biosynthesis detected by metabolic labeling do not alter the mass spectrometric lipid profile of ypc1∆ydc1∆ cells. Ceramides of ypc1∆ydc1∆ remained normal even in presence of aureobasidin A, an inhibitor of inositolphosphorylceramide synthase. Moreover, in caloric restriction conditions Ypc1p reduces chronological life span. A novel finding is that, when working backwards as a ceramide synthase in vivo, Ypc1p prefers C24 and C26 fatty acids as substrates, whereas it prefers C16:0, when solubilized in detergent and working in vitro. Therefore, its physiological activity may not only concern the minor ceramides containing C14 and C16. Intriguingly, so far the sole discernable benefit of conserving YPC1 for yeast resides with its ability to convey relative resistance towards H2 O2 . This article is protected by copyright. All rights reserved.
    Full-text · Article · May 2014 · FEMS Yeast Research
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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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    ABSTRACT: Sphingolipids play a key role in cells as structural components of membrane lipid bilayers and signaling molecules implicated in important physiological and pathological processes. Their metabolism is tightly regulated. Mechanisms controlling sphingolipid metabolism are far from being completely understood. However, they already reveal the integration of sphingolipids in the whole metabolic network as signaling devices that coordinate different metabolic pathways. A picture of sphingolipids integrated into metabolic networks might help to understand sphingolipid homeostasis. This review describes recent advances in the regulation of de novo sphingolipid synthesis with a focus on the bridges that exist with other metabolic pathways and the importance of this crosstalk in the control of sphingolipid homeostasis. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
    Full-text · Article · Oct 2013 · Biochimica et Biophysica Acta
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    ABSTRACT: Ceramide, the central molecule of sphingolipid metabolism, is an important bioactive molecule that participates in various cellular regulatory events and that has been implicated in disease. Deciphering ceramide signaling is challenging because multiple ceramide species exist, and many of them may have distinct functions. We applied systems biology and molecular approaches to perturb ceramide metabolism in the yeast Saccharomyces cerevisiae and inferred causal relationships between ceramide species and their potential targets by combining lipidomic, genomic, and transcriptomic analyses. We found that during heat stress, distinct metabolic mechanisms controlled the abundance of different groups of ceramide species and provided experimental support for the importance of the dihydroceramidase Ydc1 in mediating the decrease in dihydroceramides during heat stress. Additionally, distinct groups of ceramide species, with different N-acyl chains and hydroxylations, regulated different sets of functionally related genes, indicating that the structural complexity of these lipids produces functional diversity. The transcriptional modules that we identified provide a resource to begin to dissect the specific functions of ceramides.
    Full-text · Article · Oct 2013 · Science Signaling
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