Genetic, Biochemical, and Transcriptional Responses of Saccharomyces cerevisiae to the Novel Immunomodulator FTY720 Largely Mimic Those of the Natural Sphingolipid Phytosphingosine

Welsch, Carole A.; Roth, Lukas; Goetschy, Jean François; Movva, N. Rao

doi:10.1074/jbc.m406179200

Cited by 26 publications

(33 citation statements)

References 40 publications

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“…One simple way to elicit PHS accumulation is by preventing its catabolism. This can be achieved by adding N,N-DMS, a specific inhibitor of the first step (phosphorylation) of sphingoid base degradation (Welsch et al, 2004). Alternatively, the prevention of ceramide synthesis also results in PHS accumulation.…”

Section: Resultsmentioning

confidence: 99%

Sphingolipids Function as Downstream Effectors of a Fungal PAQR

et al. 2008

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The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as D-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steadystate levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.The PAQR superfamily of proteins is composed of membrane-bound receptors (Tang et al., 2005) and includes two mammalian proteins that function as receptors for the hormone adiponectin, which represents a molecular link between obesity and type II diabetes. Other members of this family sense and respond to progesterone, making them excellent candidates for the long-sought-after receptors that mediate many of the rapid nontranscriptional effects of progesterone. Because of arguable similarity to G-protein-coupled receptors, it has been proposed that PAQRs function as a new class of GPCRs (Thomas et al., 2007). However, this assignment is highly debatable, and, in reality, almost nothing is known about how these proteins transduce signals. What is needed to explore this complicated question is a simple system with which to study the functionality of PAQR receptors.We chose to use the model eukaryote, Saccharomyces cerevisiae, to study these receptors for several reasons. First, the S. cerevisiae genome encodes 4 proteins-Izh1p, Izh2p, Izh3p, and Izh4p-in the PAQR superfamily of receptors (Lyons et al., 2004), and the study of yeast orthologs as models for human proteins has been a remarkably fruitful method for characterizing poorly understood protein families

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Section: Resultsmentioning

confidence: 99%

Sphingolipids Function as Downstream Effectors of a Fungal PAQR

et al. 2008

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“…These membrane receptor proteins are well known to serve as transducers of various signals, but no examples have yet been described in which these proteins possess transporter activity. Rsb1 is localized to the plasma membrane and contains two N-linked glycosylation sites in its N terminus consistent with the topology expected for a 7-TM receptor protein (9).Work from several laboratories has demonstrated that increased function of the high affinity tryptophan transporter Tat2 influences PHS resistance (3,4,10). Because Rsb1 resembles a 7-TM receptor protein, we hypothesized that Rsb1 might * This work was supported, in whole or in part, by National Institutes of Health Grants GM75120 and GM49825 (to W. S. M.-R. 4 The abbreviations used are: LCB, long chain base; 7-TM, seven-transmembrane receptor protein; HePC, hexadecylphosphocholine; PHS, phytosphingosine; eGFP, enhanced GFP; ABC, ATP-binding cassette.…”

mentioning

confidence: 85%

“…Work from several laboratories has demonstrated that increased function of the high affinity tryptophan transporter Tat2 influences PHS resistance (3,4,10). Because Rsb1 resembles a 7-TM receptor protein, we hypothesized that Rsb1 might * This work was supported, in whole or in part, by National Institutes of Health Grants GM75120 and GM49825 (to W. S. M.-R. 4 The abbreviations used are: LCB, long chain base; 7-TM, seven-transmembrane receptor protein; HePC, hexadecylphosphocholine; PHS, phytosphingosine; eGFP, enhanced GFP; ABC, ATP-binding cassette.…”

mentioning

confidence: 99%

Regulation of Yeast Nutrient Permease Endocytosis by ATP-binding Cassette Transporters and a Seven-transmembrane Protein, RSB1

Johnson

Hanson

Manoharlal

et al. 2010

Journal of Biological Chemistry

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Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein.More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1⌬ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5⌬ yor1 strains and reduced in rsb1⌬ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.Sphingolipids represent one of the major components of the lipid fraction of the eukaryotic plasma membrane. Biosynthesis of these lipids proceeds through production of ceramide that is formed from the linkage of a long chain base (LCB) 4 with a very long chain fatty acid. In the yeast Saccharomyces cerevisiae, one of the two LCBs produced in vivo is referred to as phytosphingosine (PHS) (for reviews see Refs.1, 2). PHS is required for sphingolipid production but also has regulatory properties in terms of subcellular localization of proteins. Elevated levels of PHS cause mislocalization of nutrient permeases from the plasma membrane to the vacuole where these proteins are degraded (3, 4). Regulation of PHS levels in the cell is tightly controlled and important to ensure normal metabolism.One of the best described routes of PHS degradation is provided by the LCB-phosphate lyase Dpl1 (5). This enzyme breaks LCB phosphate into an aldehyde and ethanolamine phosphate limiting accumulation of LCBs. Strains lacking Dpl1 are hypersensitive to PHS. This phenotype was exploited to identify an integral membrane protein designated Rsb1 that, when overproduced, suppressed the PHS hypersensitivity of a dpl1⌬ strain (6). Evidence was presented that elevated levels of Rsb1 led to increased LCB efflux. More recent work demonstrated that loss of the multidrug transporters Pdr5 and Yor1 from cells led to a strong increase in PHS tolerance...

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“…Welsch et al (40) showed that Saccharomyces cerevisiae cells respond similarly to FTY720 and phytosphingosine treatment, using single gene deletants and transcriptional profiling. Phytosphingosine is phosphorylated by Sphk2 but not Sphk1, whereas sphingosine and dihydrosphingosine are substrates for both Sphk1 and Sphk2 (2,36).…”

Section: Sbr Mutants Are Resistant To Endogenous Long Chain Bases-mentioning

confidence: 99%

Essential Requirement for Sphingosine Kinase 2 in a Sphingolipid Apoptosis Pathway Activated by FTY720 Analogues

Don

Martínez-Lamenca

Webb

et al. 2007

Journal of Biological Chemistry

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The clinical immunosuppressant FTY720 is a sphingosine analogue that, once phosphorylated by sphingosine kinase 2 (Sphk2), is an agonist of multiple receptor subtypes for sphingosine 1-phosphate. Short exposures to FTY720 afford long term protection in lymphoproliferative and autoimmune disease models, presumably by inducing apoptosis in subsets of cells essential for pathogenesis. Sphingosine itself is pro-apoptotic, and apoptosis induced with FTY720 or sphingosine is thought to proceed independently of their phosphorylation. Following chemical mutagenesis of Jurkat cells we isolated mutants that are selectively resistant to FTY720 analogue AAL(R), as well as natural sphingolipid bases, including sphingosine. Cells lacking functional Sphk2 were resistant to apoptosis induced with AAL(R), indicating that apoptosis proceeds through AAL(R) phosphorylation. Phosphorylation of AAL(R) was also required for induction of lymphocyte apoptosis in mice, as apoptosis was not induced with the non-phosphorylatable chiral analogue, AAL(S). Apoptosis was induced in the spleen but not the thymus of mice administered 1 mg/kg AAL(R), correlating with levels of AAL(R)-phosphate (AFD(R)) in organ extracts. AFD(R) did not induce apoptosis when added to the cell culture medium, indicating that it induces apoptosis through an intracellular target. NBD-labeled AAL(R) localized to the endoplasmic reticulum, and AAL(R) treatment resulted in elevated cytosolic calcium, Bax redistribution from cytosol to mitochondrial and endoplasmic reticulum membranes, and caspase-independent mitochondrial permeabilization in Jurkat cells. We therefore describe an apoptotic pathway triggered by intracellular accumulation of sphingolipid base phosphates and suggest that sphingoid base substrates for Sphk2 acting intracellularly could be useful in the treatment of lymphoproliferative diseases.The sphingolipids are a class of lipids characterized by a serine head group with one or two fatty acyl tails. The common constituent of all sphingolipids is the long chain base (LCB), 3 and the most commonly occurring LCBs in mammals are sphingosine (Fig. 1A) and dihydrosphingosine. Sphingosine may be phosphorylated by sphingosine kinases 1 and 2 (Sphk1 and Sphk2), yielding sphingosine 1-phosphate (S1P), which has mitogenic and pro-survival properties (1, 2). Ceramides are the N-acylated precursors of sphingosine, generated either through the breakdown of the abundant membrane lipid sphingomyelin, or through de novo synthesis of sphingolipids from palmitoyl coenzyme A and serine. Ceramides have been widely implicated in apoptosis, leading to the proposal of a sphingolipid rheostat, with pro-survival S1P at one end and pro-apoptotic ceramide at the other (1). Sphingosine itself is pro-apoptotic when added exogenously to cells, and its levels are elevated in response to a number of pro-apoptotic stimuli, including antiFas, tumor necrosis factor, and dexamethasone (3, 4). S1P is secreted from cells, following phosphorylation of sphingosine on internal membranes, and may...

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Genetic, Biochemical, and Transcriptional Responses of Saccharomyces cerevisiae to the Novel Immunomodulator FTY720 Largely Mimic Those of the Natural Sphingolipid Phytosphingosine

Cited by 26 publications

References 40 publications

Sphingolipids Function as Downstream Effectors of a Fungal PAQR

Sphingolipids Function as Downstream Effectors of a Fungal PAQR

Regulation of Yeast Nutrient Permease Endocytosis by ATP-binding Cassette Transporters and a Seven-transmembrane Protein, RSB1

Essential Requirement for Sphingosine Kinase 2 in a Sphingolipid Apoptosis Pathway Activated by FTY720 Analogues

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