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

ABSTRACT 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.

Download full-text


Available from: Kaushlendra Tripathi, Dec 16, 2014
59 Reads
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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.
    Biochimica et Biophysica Acta 10/2013; 1841(5). DOI:10.1016/j.bbalip.2013.10.014 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Science Signaling 10/2013; 6(299):rs14. DOI:10.1126/scisignal.2004515 · 6.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Saccharomyces cerevisiae protein kinase Sch9 has been demonstrated to be an in vitro and in vivo effector of sphingolipid signaling. In this study, the link between Sch9 and sphingolipid metabolism in S. cerevisiae was examined in vivo based on the observation that the sch9Δ mutant displays altered sensitivities to various inhibitors of sphingolipid metabolism, i.e. myriocin and aureobasidin A. Sphingolipid profiling indicated that sch9Δ cells have increased levels of long-chain bases and long-chain base-1 phosphates, decreased levels of several species of (phyto)ceramides, and altered ratios of complex sphingolipids. We show that the TORC1 - Sch9 signaling pathway functions to repress the expression of the ceramidase genes YDC1 and YPC1, thereby unveiling, for the first time in yeast, a nutrient-dependent transcriptional mechanism involved in the regulation of sphingolipid metabolism. Additionally, we established that Sch9 affects the activity of the inositol phosphosphingolipid phospholipase C, Isc1, which is required for ceramide production by hydrolysis of complex sphingolipids. As sphingolipid metabolites play a crucial role in the regulation of stress tolerance and longevity of yeast cells, our data provide a model in which Sch9 regulates the latter phenotypes by acting not only as an effector but also as a regulator of sphingolipid metabolism.
    Molecular biology of the cell 11/2013; 25(1). DOI:10.1091/mbc.E13-06-0340 · 4.47 Impact Factor
Show more