d-ribo-phytosphingosine-1-phosphate and dihydrosphingosine-1-phosphate

Our knowledge of sphingolipid metabolism and function in Saccharomyces cerevisiae is growing rapidly. Here we discuss the current status of sphingolipid metabolism including recent evidence suggesting that exogenous sphingoid long-chain bases must first be phosphorylated and then dephosphorylated before incorporation into ceramide. Phenotypes of strains defective in sphingolipid metabolism are discussed because they provide hints about the undiscovered functions of sphingolipids and are one of the major reasons for studying this model eukaryote. The long-chain base phosphates, dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate, have been hypothesized to play roles in heat stress resistance, perhaps acting as signaling molecules. We evaluate the data supporting this hypothesis and suggest future experiments needed to verify it. Finally, we discuss recent clues that may help to reveal how sphingolipid synthesis and total cellular sphingolipid content are regulated.

Topics: Ceramides; Hot Temperature; Phenotype; Saccharomyces cerevisiae; Sphingolipids; Sphingosine

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) β-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice.

Topics: Glucosylceramidase; Glucosylceramides; Plant Proteins; Plants; Seeds; Sphingosine

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.

Topics: Arabidopsis; Botrytis; Cell Death; Cyclopentanes; Isoleucine; Mutation; Oxylipins; Plant Diseases; Plant Growth Regulators; Pseudomonas syringae; Reactive Oxygen Species; Salicylic Acid; Signal Transduction; Sphingolipids; Sphingosine

An efficient enrichment method using immobilized metal affinity chromatography (IMAC) was developed for selective extraction of bioactive sphingoid base-1-phosphates (SB1Ps) from adventitious roots of Hypericum perforatum cultured in bioreactor. The phosphate-selective IMAC enrichment coupled with LC-MS/MS enabled sensitive analysis of low-abundance SB1Ps present in the root biomass, which would not be feasible otherwise due to severe interferences from complex biological matrices. The time-dependent growth rate and production of SB1Ps from adventitious roots were investigated. The level of phytosphingosine-1-phosphate, which was found to be the major SB1Ps, reached a maximum amount of 635.6pmolpergram of dry weight after 3weeks of culture and decreased between 3 and 5weeks of culture subsequently. On the other hand, sphingosine-1-phosphate and sphinganine-1-phosphate were present at levels of 18.91 and 73.15pmolpergram of dry weight, respectively, after a week of culture and their level decreased thereafter.

Topics: Bioreactors; Biotechnology; Chloroform; Chromatography, Affinity; Chromatography, High Pressure Liquid; Hypericum; Lysophospholipids; Mass Spectrometry; Metals; Methanol; Plant Roots; Solvents; Sphingosine; Time Factors

Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P4 G-protein-coupled receptor. Similarly to reports on the S1P1 receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P1 receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp186 at position 4.64 in the fourth transmembrane domain interacts by a cation-pi interaction with the ammonium group of S1P. Lys204 at position 5.38 forms an ion pair with the S1P. The S1P4 and S1P1 receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure-activity relationships. In particular, the binding pocket of mS1P4 (mouse S1P4) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P1 (human S1P4) places the ammonium recognition site 2 A (1 A=0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Cell Membrane; Cell Movement; Cricetinae; Endothelial Cells; Humans; Ligands; Lysophospholipids; Mice; Molecular Structure; Multigene Family; Mutation; Receptors, Lysosphingolipid; Sequence Homology, Amino Acid; Sphingosine; Substrate Specificity

Topics: Aldehyde-Lyases; Pyridoxal Phosphate; Sphingosine

Topics: Acetyltransferases; Acyltransferases; Chromatography, Thin Layer; Fungal Proteins; Growth Inhibitors; Membrane Proteins; Mixed Function Oxygenases; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine C-Palmitoyltransferase; Signal Transduction; Sphingolipids; Sphingosine

Sphingolipid metabolites in mammals can function as signaling molecules with cell-specific functions. In Saccharomyces cerevisiae, phosphorylated long chain bases, such as dihydrosphingosine 1-phosphate and phytosphingosine 1-phosphate, have also been implicated in stress responses. To further explore the biological roles of these molecules, we created disruption mutants for LCB4, LCB5, DPL1, YSR2, YSR3, and SUR2. LCB4 and LCB5 encode kinases that phosphorylate long chain bases. DPL1 and YSR2/YSR3 are involved in degradation of the phosphorylated long chain bases. SUR2 catalyzes conversion of dihydrosphingosine to phytosphingosine. We adapted an HPLC method to measure intracellular concentrations of the phosphorylated long chain bases. Double mutants of dpl1 and ysr2 were inviable, whereas dpl1 ysr2 lcb4 triple mutants were viable. Further, growth inhibition associated with accumulated phosphorylated long chain bases was observed in the triple mutant dpl1 ysr2 lcb4 overexpressing LCB4 or LCB5. These results indicate that phosphorylated long chain bases can inhibit cell growth. Mutants defective in both YSR2 and SUR2, which accumulated dihydrosphingosine 1-phosphate only, grew poorly. The phenotypes of the ysr2 sur2 mutants were suppressed by overexpression of DPL1. Our results clearly show that elevated levels of phosphorylated long chain bases have an antiproliferative effect in yeast.

Topics: Fungal Proteins; Gene Expression Regulation, Fungal; Genes, Fungal; Phosphorylation; Phosphotransferases; Saccharomyces cerevisiae; Sphingosine

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25 degreesC, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37 degreesC causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44 degreesC heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.

Topics: Heat Stress Disorders; Models, Biological; Mutation; Phospholipids; Saccharomyces cerevisiae; Sphingolipids; Sphingosine