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• W1985202015 abstract "Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast,Saccharomyces cerevisiae. Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase. However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs. It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB. Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB. Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane. Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p. These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP. Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast,Saccharomyces cerevisiae. Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase. However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs. It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB. Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB. Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane. Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p. These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP. sphingosine 1-phosphate aureobasidin A dihydrosphingosine dihydroceramide dihydrosphingosine 1-phosphate endoplasmic reticulum fumonisin B1 glycerolphospholipid hemagglutinin inositolphosphorylceramide long chain sphingoid base LCB phosphate mannosyl-diinositolphosphorylceramide phytosphingosine sphingosine kinase types 1 and 2 antibody monoclonal antibody phosphatidylinositol phosphatidylethanolamine phosphatidylcholine diacylglycerol pyrophosphate hexacosanoic acid Sphingolipid metabolites, including ceramide, sphingosine, and sphingosine 1-phosphate (S1P)1 function as important second messengers in mammalian cells, regulating diverse biological processes such as cell growth, differentiation, apoptosis, stress responses, calcium homeostasis, and cell migration (1Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (438) Google Scholar, 2Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 3Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 4Spiegel S. English D. Milstien S. Trends Cell Biol. 2002; 12: 236-242Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Several lines of evidence strongly suggest that the dynamic balance between intracellular ceramide/sphingosine and S1P is an important factor that determines their cellular processes (3Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 5Olivera A. Spiegel S. Prostaglandins. 2001; 64: 123-134Crossref PubMed Scopus (145) Google Scholar). However, the mechanisms by which cells regulate intracellular levels of these lipids as well as their localization and mechanisms of action are largely unknown. The level of S1P is regulated by the metabolic enzymes responsible for its formation, which is catalyzed by sphingosine kinase (6Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 7Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar), and its degradation, which is catalyzed by an endoplasmic reticulum (ER)-bound S1P lyase (8Zhou J. Saba J.D. Biochem. Biophys. Res. Commun. 1998; 242: 502-507Crossref PubMed Scopus (152) Google Scholar, 9Van Veldhoven P.P. Gijsbers S. Mannaerts G.P. Vermeesch J.R. Brys V. Biochim. Biophys. Acta. 2000; 1487: 128-134Crossref PubMed Scopus (97) Google Scholar) and a specific phosphatase (10Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (172) Google Scholar,11Le Stunff H. Peterson C. Thornton R. Milstien S. Mandala S.M. Spiegel S. J. Biol. Chem. 2002; 277: 8920-8927Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In mammalian cells, two sphingosine kinase isoforms have been cloned and characterized (6Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 7Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Although sphingosine kinase type 1 (SPHK1) and type 2 (SPHK2) have a high degree of homology, they have differential tissue expression, temporal developmental expression, and properties, suggesting that they have distinct cellular functions and may regulate levels of S1P differently. Furthermore, SPHK1 is a cytoplasmic enzyme, whereas SPHK2 has several predicted transmembrane regions, suggesting that it is a membrane protein (3Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 25851-25854Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). However, both kinase activities are present in the cytosol and in membranes (6Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 7Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). Another study suggested the presence of additional sphingosine kinases in mammalian tissues: one cytosolic and two membrane-bound activities that are associated with the ER and with plasma membrane (12Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar). In the yeast, Saccharomyces cerevisiae, two genes that encode related sphingosine kinases, named LCB4 andLCB5, have been identified (13Nagiec M.M. Skrzypek M. Nagiec E.E. Lester R.L. Dickson R.C. J. Biol. Chem. 1998; 273: 19437-19442Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). They display 53% amino acid identity. The gene products, referred to as LCB (long chain sphingoid base) kinases, phosphorylate several LCBs, including dihydrosphingosine (DHS), phytosphingosine (PHS), and sphingosine. These predicted cytosolic kinases appear to have similar substrate specificities. Like mammalian kinases, two-thirds of the Lcb4p and one-third of the Lcb5p kinase activity are also found in the membrane fraction, although neither protein contains a membrane localization signal (13Nagiec M.M. Skrzypek M. Nagiec E.E. Lester R.L. Dickson R.C. J. Biol. Chem. 1998; 273: 19437-19442Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Yeast also expresses a conserved S1P lyase encoded by the DPL1 gene (14Saba J.D. Nara F. Bielawska A. Garrett S. Hannun Y.A. J. Biol. Chem. 1997; 272: 26087-26090Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Additionally, LCB3and YSR3 encode LCB phosphate phosphatases (15Qie L. Nagiec M.M. Baltisberger J.A. Lester R.L. Dickson R.C. J. Biol. Chem. 1997; 272: 16110-16117Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 16Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (233) Google Scholar). Both phosphatases are localized to the ER but appear to be functionally distinct (18Mao C. Saba J.D. Obeid L.M. Biochem. J. 1999; 342: 667-675Crossref PubMed Scopus (105) Google Scholar). Although the functions of LCB kinases in yeast are largely unknown, a study using yeast strains with deletions of LCB kinase genes showed that LCB kinases are important regulators of heat-induced cell cycle arrest, and that Lcb4p and Lcb5p function redundantly in this process (19Jenkins G.M. Hannun Y.A. J. Biol. Chem. 2001; 276: 8574-8581Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, recent evidence suggests specific roles for the two LCB kinases. Lcb4p has a function in growth inhibition and cell death (20Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar,21Zhang X. Skrzypek M.S. Lester R.L. Dickson R.C. Curr. Genet. 2001; 40: 221-233Crossref PubMed Scopus (50) Google Scholar) and Lcb5p plays a role in heat-stress resistance during induced thermotolerance (22Ferguson-Yankey S.R. Skrzypek M.S. Lester R.L. Dickson R.C. Yeast. 2002; 19: 573-586Crossref PubMed Scopus (45) Google Scholar). This difference implicates a complexity in the mechanisms in which the two LCB kinases control diverse cellular processes. Earlier studies showed that deletion of LCB3 leads to failure of incorporation of exogenous DHS into sphingolipids, suggesting a possible role for LCB phosphorylation and dephosphorylation in sphingolipid synthesis (16Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (233) Google Scholar). Here, we present evidence that incorporation of exogenous DHS into ceramide requires both Lcb4p and Lcb3p. Using an in vitro ceramide synthesis assay, we showed a specific role for membrane-associated Lcb4p in ceramide synthesis. Although membranes lacking LCB kinases had normal LCBP phosphatase activity, they could not support ceramide synthesis from DHS-1P, suggesting that the actions of Lcb4p and Lcb3p must be concerted on the ER membrane. Our observations provide new insights into the mechanisms by which the cell might functionally separate biosynthetic and signaling pools of LCBP. The yeast strains used in this study are listed in Table I. For the deletion strains, the entire open reading frames were deleted and replaced with the designated genes. Deletions were confirmed by PCR. Three copies of the hemagglutinin (HA) epitope were introduced at the COOH terminus of Lcb4p and Lcb5p using a histidine selection as described previously (23Levine T.P. Wiggins C.A. Munro S. Mol. Biol. Cell. 2000; 11: 2267-2281Crossref PubMed Scopus (133) Google Scholar). RH4950 and RH5257 strains were obtained from RH4946 and RH4953, respectively, by plating onto SD plates without histidine. Expression of Lcb3p tagged with FLAG peptide was performed with the same construct (pYSR2) as described previously (16Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Strains containing pYSR2 were grown overnight in SD (synthetic minimal, 6.7 g/liter yeast nitrogen base without amino acids, 20 g/liter glucose, supplemented with the appropriate amino acids)-uracil (SD-ura) medium containing 2% glucose and resuspended in SD-ura medium containing 2% galactose. Expression of the tagged Lcb3p was then induced by incubating cells with SD-ura medium containing 2% galactose and detected by Western analysis using a mouse mAb M2 against FLAG (Sigma).Table IYeast strains used in this studyStrainGenotypeRH4402MATαlcb4Δ∷URA3 lcb5Δ∷LEU2 pep4–3 ura3 leu2 bar1–1RH4423MATα pep4–3 ura3 leu2 bar1–1RH4427MATαlcb3Δ∷KanMx pep4–3 ura3 bar1–1RH4464MATα lcb3Δ∷KanMx lcb4Δ∷URA3 lcb5Δ∷LEU2 pep4–3 ura3 bar1–1RH4835MATαysr3Δ∷URA3 pep4–3 ura3 leu2 bar1–1RH4836MATα lcb3Δ∷KanMx ysr3Δ∷URA3 pep4–3 ura3 leu2 bar1–1RH4946MATα lcb5Δ∷LEU2 pep4–3 ura3 leu2 his3 trp1 bar1–1RH4948MATαpep4–3 ura3 leu2 his3 trp1 bar1–1RH4950MATα lcb4∷LCB4 3xHA HIS5Sp lcb5Δ∷LEU2 pep4–3 ura3 leu2 his3 trp1 bar1–1RH4951MATαlcb4Δ∷TRP1 lcb5Δ∷LEU2 pep4–3 ura3 leu2 his3 trp1 bar1–1RH4953MATαlcb4Δ∷TRP1 pep4–3 ura3 leu2 his3 trp1 bar1–1RH4978MAT a dpp1Δ∷HIS3 lpp1Δ∷TRP1 pep4–3 ura3 leu2 his3 trp1 lys2 bar1–1RH4981MAT a dpp1Δ∷HIS3 lpp1Δ∷TRP1 lcb3Δ∷KanMx ysr3Δ∷URA3 pep4–3 ura3 leu2 his3 trp1 bar1–1RH5243MATαdpp1Δ∷HIS3 lpp1Δ∷TRP1 lcb4Δ∷URA3 lcb5Δ∷LEU2 pep4–3 ura3 leu2 his3 trp1 bar1–1RH5244MATαdpp1Δ∷HIS3 lpp1Δ∷TRP1 pep4–3 ura3 leu2 his3 trp1 lys2 bar1–1RH5257MATαlcb5∷LCB5 3xHA HIS5Sp lcb4Δ∷TRP1 pep4–3 ura3 leu2 his3 trp1 bar1–1 Open table in a new tab Cell cultures, labeling of lipids with [3H]DHS (American Radiolabeled Chemical Inc., St Louis, MO), or [3H]DHS-1P prepared as below, lipid extraction, and treatment by mild alkaline hydrolysis with NaOH were performed as previously described (24Zanolari B. Friant S. Funato K. Sutterlin C. Stevenson B.J. Riezman H. EMBO J. 2000; 19: 2824-2833Crossref PubMed Scopus (206) Google Scholar). The labeled lipids were separated on thin-layer chromatography (TLC) plates (20 × 20, Merck, Darmstadt, Germany), which were developed in solvent system I (chloroform/methanol/4.2 n NH4OH = 9/7/2, v/v). Radiolabeled lipids were visualized and quantified on a Cyclone Storage phosphor system using a tritium-sensitive screen (Packard, Meriden, CT). Wild-type and mutant cytosol were prepared essentially as described previously (25Salama N.R. Yeung T. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). The preparation of total membranes was performed as described previously (26Funato K. Riezman H. J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (151) Google Scholar). In brief, spheroplasts (from 0.5 A 600units of cells in log phase) were broken in lysis buffer (0.1m sorbitol, 20 mm HEPES, pH 7.4, 150 mm potassium acetate, 2 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml protease inhibitor mixture (pepstatin, leupeptin, and antipain)). Subsequently, unbroken cells and cell debris were removed by centrifugation at 3,000 × g for 10 min at 4 °C, and the resulting supernatants were centrifuged at 100,000 × g for 60 min at 4 °C to collect the membrane fraction. The pellet was washed twice and resuspended in B88 (20 mm HEPES-KOH, pH 6.8, 150 mm potassium acetate, 5 mm magnesium acetate, 250 mmsorbitol). Aliquots were frozen in liquid nitrogen and stored at −80 °C. Protein concentration was determined using the Bio-Rad protein assay kit. [3H]DHS-1P was synthesized enzymatically from [3H]DHS by incubation with wild-type yeast cytosol in the presence of an ATP-regenerating system (1 mm ATP, 40 mm phosphocreatine, 0.2 mg/ml creatine phosphokinase). The synthesized [3H]DHS-1P was separated on TLC plates, which were developed in solvent system I, and then isolated from the TLC plates by scraping, and eluted with chloroform/methanol/water (10/10/3, v/v). The isolated [3H]DHS-1P was dried under nitrogen and partitioned between n-butanol and water as described (24Zanolari B. Friant S. Funato K. Sutterlin C. Stevenson B.J. Riezman H. EMBO J. 2000; 19: 2824-2833Crossref PubMed Scopus (206) Google Scholar). The butanol phase was dried under nitrogen, and lipids were dissolved in ethanol. In vitro ceramide synthase activity was determined as described previously (26Funato K. Riezman H. J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (151) Google Scholar). Membranes (200 μg), cytosol (100 μg), an ATP-regenerating system (1 mmATP, 40 mm phosphocreatine, 0.2 mg/ml creatine phosphokinase), GDP-mannose (50 μm), and either a mix of [3H]DHS and unlabeled DHS (10 and 40 pmol, respectively, 0.5 μCi) or [3H]DHS-1P (0.25 μCi) were first incubated for 15 min at 10 °C. Subsequently, CoA (50 μm) and a mix of liposomes containing hexacosanoic acid (C26) and phosphatidylinositol (PI) (50 μm/250 μm, respectively) were added, and the mixture was incubated for 2 h at 24 °C in a final total volume of 50 μl of B88. The reaction was stopped by addition of 333 μl of chloroform/methanol (1/1, v/v). The organic phase was collected after centrifugation at 13,000 × g for 5 min, and the pellet was re-extracted with 250 μl of chloroform/methanol/water. The extracted lipids were submitted to a mild alkaline treatment with NaOH and then partitioned between n-butanol and water and analyzed by TLC with solvent system II (chloroform/acetic acid = 9/1, v/v). LCBP phosphatase activity was measured by adding [3H]DHS-1P (0.25 μCi) to the membranes (200 μg) in 50 μl of B88 containing apyrase (10 units/ml) and incubating at 24 °C for 60 min. The lipids treated with NaOH were analyzed by TLC with solvent system I as described above. To characterize the nature of membrane association of proteins, cells were grown in SD-ura medium, washed, and resuspended with TNE buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml protease inhibitor mixture). The cells were disrupted with glass beads, and the cell debris and glass beads were removed by centrifugation at 720 × gfor 5 min at 4 °C. One volume of TNE buffer containing 2m NaCl, 5 m urea, 2% Triton X-100, 2% SDS, or 200 mm Na2CO3 (pH 11.5) was added to the supernatants (27Nishikawa S. Nakano A. Biochim. Biophys. Acta. 1991; 1093: 135-143Crossref PubMed Scopus (57) Google Scholar). The mixture was incubated on ice for 30 min and centrifuged at 100,000 × g for 60 min at 4 °C. The resulting pellet and supernatant fractions were subjected to SDS-PAGE and then analyzed by Western blotting using a rat mAb 3F10 against HA (Roche Molecular Biochemicals, Basel, Switzerland), a mouse mAb M2 against FLAG, or a rabbit polyclonal antibody against End3p. For determination of the subcellular localization of proteins, spheroplasts were broken in lysis buffer as described above, and unbroken cells and cell debris were removed by centrifugation at 500 × g for 5 min at 4 °C. After centrifuging again at 500 × g for 5 min at 4 °C, the resulting supernatants (1 ml) were layered onto 1-ml steps of 22, 26, 30, 34, 38, 42, 46, 50, 54, and 60% (w/v) sucrose in 10 mm HEPES, pH 7.4, 1 mm MgCl2(28Schroder S. Schimmoller F. Singer-Kruger B. Riezman H. J. Cell Biol. 1995; 131: 895-912Crossref PubMed Scopus (157) Google Scholar). The gradients were centrifuged at 200,000 × gfor 140 min at 4 °C in a TST41.14 rotor. Eleven fractions of 1 ml were collected from the top of the gradient. Aliquots from each fraction were diluted with 10 mm HEPES, pH 7.4, 1 mm MgCl2 and centrifuged at 100,000 ×g for 60 min at 4 °C. Membrane pellets were then resuspended in B88. For analysis by immunoblotting, samples were solubilized in SDS sample buffer by vortexing and subjected to SDS-PAGE, followed by Western blotting using a rat mAb 3F10 against HA, a rabbit polyclonal Ab against Wbp1p or Emp47p, or a mouse mAb against the yeast vacuolar H+-ATPase 100-kDa subunit (Molecular Probes Europe BV, Leiden, Netherlands). The amount of protein in the fractions was quantified by densitometric scanning on a computing densitometer (Molecular Dynamics, Sunnyvale, CA). Indirect immunofluorescence on whole fixed yeast cells was performed as described by Beck et al. (29Beck T. Schmidt A. Hall M.N. J. Cell Biol. 1999; 146: 1227-1237Crossref PubMed Scopus (249) Google Scholar). For double labeling, a mouse mAb 12CA5 against HA (Roche Molecular Biochemicals, Basel, Switzerland) followed by an indocarbocyanine, Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and a rabbit polyclonal Ab against Kar2p (kindly provided by R. Schekman) followed by a fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) were used. Cells were visualized with a Zeiss Axiophot microscope (100× objective). LCB3 and its homologue, YSR3, encode LCBP phosphatases in yeast (15Qie L. Nagiec M.M. Baltisberger J.A. Lester R.L. Dickson R.C. J. Biol. Chem. 1997; 272: 16110-16117Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). It has been demonstrated that lcb3 deletion mutant cells fail to incorporate exogenously added DHS efficiently into ceramide and subsequently into sphingolipids, whereas endogenously synthesized LCBs are still converted to sphingolipids (16Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (233) Google Scholar, 18Mao C. Saba J.D. Obeid L.M. Biochem. J. 1999; 342: 667-675Crossref PubMed Scopus (105) Google Scholar). These results suggest a possible role for a cycle of phosphorylation and dephosphorylation of exogenous LCB in ceramide synthesis. However, this has not been tested directly. To test whether LCB kinase is required for incorporation of exogenous DHS into sphingolipids, we constructed multiple deletion strains for the two LCB kinases encoded by LCB4 and LCB5 (13Nagiec M.M. Skrzypek M. Nagiec E.E. Lester R.L. Dickson R.C. J. Biol. Chem. 1998; 273: 19437-19442Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and measured sphingolipid synthesis by labeling with [3H]DHS at 24 °C. The doublelcb4/5 deletion mutant cells could not synthesize DHS-1P (Fig. 1, A andB). This phenotype indicates that Lcb4p and Lcb5p are the only LCB kinases in yeast, as described previously (13Nagiec M.M. Skrzypek M. Nagiec E.E. Lester R.L. Dickson R.C. J. Biol. Chem. 1998; 273: 19437-19442Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). As expected,lcb3 mutant cells accumulated DHS-1P. The lcb3mutant cells showed an 8-fold increase in DHS-1P synthesis after a short time labeling (labeling for 30 min) and a 5-fold increase after a long time labeling (labeling for 120 min) when compared with wild-type cells. Quantitative analysis also revealed that incorporation of [3H]DHS into glycerolphospholipids (GPLs: PE, PC, and PI) in the lcb3 mutant was increased 2- to 6-fold compared with wild-type cells throughout the 120-min labeling time. The decrease in DHS-1P in wild-type and lcb3 mutant cells as GPL levels increase is consistent with a previous study, which suggested that DHS is incorporated into GPLs through phosphorylation of DHS and subsequent cleavage by the LCBP lyase, Dpl1p (16Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This idea is further supported by our finding that the lcb4/5 mutant, which fails to produce DHS-1P, could not synthesize GPLs from exogenously added DHS. Consequently, the lcb4/5 mutant cells accumulated more PHS than lcb3 mutant or wild-type cells (Fig. 1 A). Importantly, when the lcb4/5 mutant cells were labeled with [3H]DHS for 30 min, the incorporation of [3H]DHS into sphingolipids (IPC-C and M(IP)2C) was hardly detectable (Fig. 1 B) or was obviously reduced compared with wild-type cells (Fig. 1 C). After longer labeling times, 120 min (Fig. 1 B) and 90 min (Fig. 1 C), the lcb4/5 mutants showed 40–50% of the amount of sphingolipid synthesis found in wild-type cells. In contrast, when lcb4/5 mutant cells were labeled with [3H]myo-inositol for 15 or 30 min at 24 °C, almost normal levels of all sphingolipids were observed (data not shown). This is consistent with a previous finding thatde novo synthesis of sphingolipids is only slightly, if at all affected by deletion of both LCB3 and YSR3genes (18Mao C. Saba J.D. Obeid L.M. Biochem. J. 1999; 342: 667-675Crossref PubMed Scopus (105) Google Scholar). These results suggest that exogenous DHS must undergo a cycle of phosphorylation and dephosphorylation to be efficiently incorporated into sphingolipids. We observed that the lcb4 mutant cells could not make any detectable DHS-1P, as well as GPLs from exogenously added [3H]DHS, but the lcb5 mutant cells could (data not shown). Therefore, we tested whether Lcb4p is the major kinase responsible for sphingolipid synthesis from exogenous DHS. To test this, we quantified the amount of synthesized sphingolipid in the single lcb4 and lcb5 mutant cells. Thelcb4 mutant cells showed 20–30% of the amount of sphingolipid synthesis found in wild-type cells, whereas thelcb5 mutant cells showed only a slight reduction during a long time period of labeling (Fig. 1 C). These results suggest that Lcb4p but not Lcb5p is required for an efficient sphingolipid synthesis from exogenous DHS. During a long labeling period, lcb4/5 mutant cells can synthesize detectable amounts of sphingolipids from exogenous DHS. In contrast, lcb3 mutant cells synthesize no detectable sphingolipid during this time (Fig. 1, A and B). These results raised the question of whether Lcb3p is only required if exogenous DHS become phosphorylated. Indeed, a mutant lacking both the LCB kinases and the major phosphatase (lcb3/4/5) synthesized similar amounts of sphingolipids as wild-type cells in a long term labeling experiment (Fig. 1, A and B). After a shorter labeling time, the lcb3/4/5 mutant resembled thelcb3 or lcb4/5 mutant in the incorporation of exogenous DHS into sphingolipids. Phenotypes in DHS-1P, GPLs, and PHS synthesis observed for thelcb3/4/5 mutant are identical to thelcb4/5 mutant. These results suggest that, if exogenous LCB does not get phosphorylated, it can bypass the normal route for incorporation into sphingolipid, which is dependent upon Lcb3p. The above data suggest that Lcb4p is required for an efficient ceramide synthesis from exogenous DHS. Therefore, we decided to measure ceramide synthase activity directly in an in vitro system that we recently developed (26Funato K. Riezman H. J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (151) Google Scholar, 30Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (218) Google Scholar).In vitro ceramide synthase activity with [3H]DHS requires a membrane fraction, ATP, and cytosol (26Funato K. Riezman H. J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (151) Google Scholar). In addition, the reaction is temperature- and CoA-dependent (26Funato K. Riezman H. J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (151) Google Scholar, 30Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (218) Google Scholar). When wild-type membranes were incubated with wild-type cytosol, an ATP-regenerating system, GDP-mannose, CoA, and a mix of liposomes containing C26 and PI, dihydroceramide (DH-Cer) was preferentially synthesized (Fig.2 A). Under the same conditions, very little phytoceramide was synthesized (Ref. 26Funato K. Riezman H. J. Cell Biol. 2001; 155: 949-959Crossref PubMed Scopus (151) Google Scholar and data not shown). This DH-Cer synthesis was completely inhibited by fumonisin B1 (FuB), a specific inhibitor of ceramide synthase (31Merrill Jr., A.H. van Echten G. Wang E. Sandhoff K. J. Biol. Chem. 1993; 268: 27299-27306Abstract Full Text PDF PubMed Google Scholar), but not by aureobasidin A (AbA), a specific inhibitor of IPC synthase (32Zhong W. Murphy D.J. Georgopapadakou N.H. FEBS Lett. 1999; 463: 241-244Crossref PubMed Scopus (32) Google Scholar). Membrane and cytosol derived from the doublelcb3/ysr3 mutant cells failed to synthesize DH-Cer. The single lcb3 mutant showed a strong reduction of the incorporation of DHS into DH-Cer when compared with wild-type components, whereas ysr3 mutant components showed normal synthesis (Fig. 2 A). These results suggest that Lcb3p and Ysr3p do not have redundant functions in ceramide synthesis. This is consistent with previous studies (18Mao C. Saba J.D. Obeid L.M. Biochem. J. 1999; 342: 667-675Crossref PubMed Scopus (105) Google Scholar). Membrane and cytosol from either the lcb4/5 mutant or thelcb3/4/5 mutant had no detectable ceramide synthase activity. The complete lack of in vitroceramide synthesis (Fig. 2 A) and in vitrosphingolipid synthesis (data not shown) withlcb3/ysr3, lcb4/5, andlcb3/4/5 mutants suggests that thein vitro assay measures only the efficient pathway for ceramide and sphingolipid synthesis that is mediated by LCB phosphorylation and dephosphorylation. Because the incorporation of [3H]DHS into DH" @default.
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• W1985202015 title "Lcb4p Is a Key Regulator of Ceramide Synthesis from Exogenous Long Chain Sphingoid Base in Saccharomyces cerevisiae" @default.
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