Cungui Mao

Involvement of Yeast Sphingolipids in the Heat Stress Response of Saccharomyces cerevisiae

A role for sphingolipids in the yeast heat stress response has been suggested by the isolation of suppressors of mutants lacking these lipids, which are unable to grow at elevated temperatures. The current study examines the possible role of sphingolipids in the heat adaptation of yeast cells as monitored by growth and viability studies. The suppressor of long chain base auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was corrected by transformation with serine palmitoyltransferase. Thus, the deficiency in sphingolipids and not the suppressor mutation was the cause of the heat-sensitive phenotype of the SLC strain 7R4. The ability of sphingolipids to rescue the heat-sensitive phenotype was examined, and two endogenous yeast sphingoid backbones, phytosphingosine and dihydrosphingosine, were found to be most potent in this effect. Next, the effect of heat stress on the levels of the three major classes of sphingolipids was determined. The inositol phosphoceramides showed no change over a 1.5-h time course. However, the four detected species of sphingoid bases increased after 15 min of heat stress from 1.4- to 10.8-fold. The largest increases were seen in two sphingoid bases, C20 phytosphingosine and C20 dihydrosphingosine, which increased 6.4- and 10.8-fold over baseline, respectively. At 60 min of heat stress two species of yeast ceramide increased by 9.2- and 10.6-fold over baseline. The increase seen in the ceramides was partially decreased by Fumonisin B1, a ceramide synthase inhibitor. Therefore, heat stress induces accumulation of sphingoid bases and of ceramides, probably throughde novo synthesis. Taken together, these results demonstrate that sphingolipids are involved in the yeast heat stress adaptation.

Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate

Ceramidases catalyze hydrolysis of ceramides to generate sphingosine (SPH), which is phosphorylated to form sphingosine-1-phosphate (S1P). Ceramide, SPH, and S1P are bioactive lipids that mediate cell proliferation, differentiation, apoptosis, adhesion, and migration. Presently, 5 human ceramidases encoded by 5 distinct genes have been cloned: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). Each human ceramidase has a mouse counterpart. AC, NC, and ACER1-3 have maximal activities in acidic, neutral, and alkaline environments, respectively. ACER1-3 have similar protein sequences but no homology to AC and NC. AC and NC also have distinct protein sequences. The human AC (hAC) was implicated in Farber disease, and hAC may be important for cell survival. The mouse AC (mAC) is needed for early embryo survival. NC is protective against inflammatory cytokines, and the mouse NC (mNC) is required for the catabolism of ceramides in the digestive tract. ACER1 is critical in mediating cell differentiation by controlling the generation of SPH and S1P and that ACER2s role in cell proliferation and survival depends on its expression or the cell type in which it is found. Here, we discuss the role of each ceramidase in regulating cellular responses mediated by ceramides, SPH, and S1P.

Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity.

Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screeningSaccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast geneYPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed byin vitro studies using an Escherichia coliexpression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1was cloned as a fumonisin B1-resistant gene.

Cloning and Characterization of a Saccharomyces cerevisiae Alkaline Ceramidase with Specificity for Dihydroceramide

In a previous study, we reported that theSaccharomyces cerevisiae gene YPC1 encodes an alkaline ceramidase with a dual activity, catalyzing both hydrolysis and synthesis of yeast ceramide (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876–6884). In this study, we have identified a YPC1homologue in S. cerevisiae that also encodes an alkaline ceramidase. We show that these two ceramidases have different substrate specificity, such that YPC1p preferentially hydrolyzes phytoceramide, whereas the new ceramidase YDC1p hydrolyzes dihydroceramide preferentially and phytoceramide only slightly. Neither enzyme hydrolyzes unsaturated mammalian-type ceramide. In contrast to YPC1p, YDC1p had only minor in vitro reverse activity of catalyzing dihydroceramide formation from a free fatty acid and dihydrosphingosine and no activity with phytosphingosine. Overexpression of YDC1p had no reverse activity in non-stressed yeast cells, but like YPC1p suppressed the inhibition of growth by fumonisin B1 albeit more modestly. Deletion of YDC1 andYPC1 or both did not apparently affect growth, suggesting neither gene is essential. However, the Δydc1 deletion mutant but not the Δypc1 deletion mutant was sensitive to heat stress, indicating a role for dihydroceramide but not phytoceramide in heat stress responses, and suggesting that the two enzymes have distinct physiological functions.

Identification and Characterization of Saccharomyces cerevisiae Dihydrosphingosine-1-phosphate Phosphatase

We have identified the yeast sphingosine resistance gene (YSR2) of Saccharomyces cerevisiae as encoding a protein that specifically dephosphorylates dihydrosphingosine 1-phosphate (DHS-1-P), and we refer to this protein as dihydrosphingosine-1-phosphate phosphatase. Overexpression of YSR2 conferred sphingosine resistance to the dihydrosphingosine-1-P lyase-defective mutant (JS16) of S. cerevisiae, which is hypersensitive to sphingosine. Theysr2Δ deletion mutant of S. cerevisiaeaccumulated DHS-1-P compared with its wild type strain upon labeling withd-erythro-[4,5-3H]dihydrosphingosine, whereas overexpression of YSR2 increased dephosphorylation of DHS-1-P. An epitope-tagged fusion protein (YSR2-Flag) was partially purified and found to specifically dephosphorylate DHS-1-P to yield dihydrosphingosine. YSR2 failed to dephosphorylate ceramide 1-phosphate or phosphatidic acid. Functionally, the mutant bearing theysr2Δ deletion decreased labeling of sphingolipids and increased labeling of glycerolipids dramatically following in vivo labeling withd-erythro-[3H]dihydrosphingosine, but it slightly affected labeling of sphingolipids with inositol. Taken together, these results identify YSR2 as dihydrosphingosine-1-phosphate phosphatase. They also raise the intriguing possibility that phosphorylation followed by dephosphorylation is required for incorporation of exogenous long chain sphingoid bases into sphingolipids.

Role of human sphingosine-1-phosphate phosphatase 1 in the regulation of intra- and extracellular sphingosine-1-phosphate levels and cell viability

Sphingosine-1-phosphate (S1P) is a highly bioactive lipid that exerts numerous biological effects both intracellularly as a second messenger and extracellularly by binding to its G-protein-coupled receptors of the endothelial differentiation gene family (S1P receptors-(1–5)). Intracellularly, at least two enzymes, sphingosine kinase and S1P phosphatase, regulate the activity of S1P by governing the phosphorylation status of S1P. To study the regulation of S1P levels, we cloned the human isoform of S1P phosphatase 1 (hSPPase1). The hSPPase1 has 78% homology to the mouse SPPase at the amino acid level with 6–8 possible transmembrane domains. Confocal microscopy revealed green fluorescent protein-tagged hSPPase1, expressed in either MCF7 or HEK293 cells, co-localized to endoplasmic reticulum with calreticulin. According to Northern blot analysis, hSPPase1 is expressed in most tissues, with the strongest levels found in the highly vascular tissues of placenta and kidney. Transient overexpression of hSPPase1 exhibited a 2-fold increase in phosphatase activity against S1P and dihydro-S1P, indicating that the expressed protein was functional. Small interfering RNA (siRNA) knockdown of endogenous hSPPase1 drastically reduced hSPPase1 mRNA levels, as confirmed by reverse transcription PCR, and resulted in an overall 25% reduction of in vitro phosphatase activity in the membrane fractions. Sphingolipid mass measurements in hSPPase1 siRNA knockdown cells revealed a 2-fold increase of S1P levels and concomitant decrease in sphingosine. In vivo labeling of hSPPase1 siRNA-treated cells showed accumulation of S1P within cells, as well as significantly increased secretion of S1P into the media, indicating that hSPPase1 regulates secreted S1P. In addition, siRNA-induced knockdown of hSPPase1 endowed resistance to tumor necrosis factor-α and the chemotherapeutic agent daunorubicin. Collectively, these data suggest that regulation of hSPPase1 with the resultant changes in cellular and secreted S1P could have important implications to cell proliferation, angiogenesis, and apoptosis.

Identification of ISC1 (YER019w) as Inositol Phosphosphingolipid Phospholipase C inSaccharomyces cerevisiae

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show thatISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression ofISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates ofISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [3H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.

Cystic Fibrosis Transmembrane Regulator Regulates Uptake of Sphingoid Base Phosphates and Lysophosphatidic Acid MODULATION OF CELLULAR ACTIVITY OF SPHINGOSINE 1-PHOSPHATE

Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. Sphingosine 1-phosphate (SPP) has recently emerged as an important lipid messenger and a ligand for the endothelial differentiation gene receptor family of proteins through which it mediates its biologic effects. Recent studies in Saccharomyces cerevisiae in our laboratory implicated the yeast oligomycin resistance gene (YOR1), a member of the ATP binding cassette family of proteins, in the transport of SPP. The cystic fibrosis transmembrane regulator is a unique member of the ATP binding cassette transporter family and has high homology with YOR1. We therefore set out to investigate if this member of the family can regulate SPP transport. We demonstrate that C127/cystic fibrosis transmembrane regulator (CFTR) cells, expressing wild type CFTR, exhibited significantly higher uptake of sphingosine 1-phosphate than either cells expressing a mutant CFTR C127/ΔF508 or C127/mock-transfected cells. This effect was specific, dose-dependent, and competed off by dihydrosphingosine 1-phosphate and lysophosphatidic acid. There was no difference in uptake of sphingosine, C16-ceramide, sphingomyelin, lysophingomyelin, phosphatidylcholine, lysophosphatidylcholine, or phosphatidic acid among the different cell lines. Pretreatment with forskolin or isobutylmethylxanthine to stimulate cAMP did not affect the uptake in any of the cell lines. Moreover, we found that mitogen-activated protein kinase activation by SPP was less responsive in C127/CFTR as compared with C127/mock-transfected cells, suggesting that uptake of SPP by CFTR may divert it from interacting with its cell surface receptors and attenuate signaling functions. Taken together, these data implicate CFTR in uptake of SPP and the related phosphorylated lipids dihydrosphingosine 1-phosphate and lysophosphatidic acid. This uptake influences the availability of SPP to modulate biologic activity via endothelial differentiation gene receptors. These studies may have important implications to cystic fibrosis.

Yeast sphingolipids: metabolism and biology.

Sphingolipids have recently emerged as important bioactive molecules in addition to being critical structural components of cellular membranes. These molecules have been implicated in regulating cell growth, differentiation, angiogenesis, apoptosis, and senescene. To study sphingolipid mediated biology, it is necessary to investigate sphingolipid metabolism and its regulation. The yeast Saccharomyces cerevisiae has allowed such studies to take place as the sphingolipid metabolic and regulatory pathways appear conserved across species. Using yeast genetic approaches most enzymes of sphingolipid metabolism have been identified and cloned which has led to identification of their mammalian homologues. Many of the yeast enzymes are targets of fungal toxins thus underscoring the importance of this pathway in yeast cell regulation. This review focuses on the yeast sphingolipid metabolic pathway and its role in regulation of yeast biology. Implication of the insights gained from yeast to mammalian cell regulation are discussed.

Golgi alkaline ceramidase regulates cell proliferation and survival by controlling levels of sphingosine and S1P

Sphingosine‐1‐phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum‐free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) ‐mediated inhibition of S1P1, a G‐protein‐coupled receptor for S1P. The RNAi‐mediated down‐regulation of haCER2 enhanced the serum deprivation‐induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up‐regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up‐regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P‐mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine.—Xu, R., Jin, J., Hu, W., Sun, W., Bielawski, J., Szulc, Z., Taha, T., Obeid, L. M., Mao, C. Golgi alkaline ceramidase regulates cell proliferation and survival by controlling levels of sphingosine and S1P FASEB J. 20, 1813–1825 (2006)