Title: Yeast 1,3-β-Glucan Synthase Activity Is Inhibited by Phytosphingosine Localized to the Endoplasmic Reticulum
Abstract: 1,3-β-d-Glucan, a major filamentous component of the cell wall in the budding yeastSaccharomyces cerevisiae, is synthesized by 1,3-β-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Δgns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Δgns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Δsur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Δgns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo. 1,3-β-d-Glucan, a major filamentous component of the cell wall in the budding yeastSaccharomyces cerevisiae, is synthesized by 1,3-β-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Δgns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Δgns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Δsur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Δgns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo. 1,3-β-glucan synthase dihydrosphingosine endoplasmic reticulum 1,3-β-d-glucan guanosine 5′-(γ-thio)triphosphate inositol phosphoceramide mannosylinositol phosphoceramide mannosyl diinositolphosphorylceramide phytosphingosine polymerase chain reaction 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid In plant and fungi, remodeling of the cell wall is one of the essential processes for cell shape determination. Among cell wall components in the budding yeast Saccharomyces cerevisiae, 1,3-β-d-glucan (glucan) is the main structural component responsible for the rigidity of the cell wall (1Cid V.J. Duran A. Rey F. Snyder M.P. Nombela C. Sanchez M. Microbiol. Rev. 1995; 59: 345-386Crossref PubMed Google Scholar). Glucan is synthesized by a specific biosynthetic enzyme, 1,3-β-glucan synthase (GS)1 (EC 2.4.1.34) localized to the plasma membrane. Yeast GS has been extensively studied both genetically and biochemically, revealing spatial and temporal regulation of cell wall synthesis (1Cid V.J. Duran A. Rey F. Snyder M.P. Nombela C. Sanchez M. Microbiol. Rev. 1995; 59: 345-386Crossref PubMed Google Scholar, 2Smits G.J. Kapteyn J.C. van den Ende H. Klis F.M. Curr. Opin. Microbiol. 1999; 2: 348-352Crossref PubMed Scopus (185) Google Scholar). Recent studies of yeast GS revealed that it is composed of at least two subunits: a putative catalytic subunit encoded by two related genes, FKS1 andFKS2, and predicted to be an intrinsic membrane protein with 16-membrane spanning domains (3Inoue S.B. Takewaki N. Takasuka T. Mio T. Adachi M. Fujii Y. Miyamoto C. Arisawa M. Furuichi Y. Watanabe T. Eur. J. Biochem. 1995; 231: 845-854Crossref PubMed Scopus (169) Google Scholar, 4Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandala S.M. Frommer B.R. Kurtz M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar, 5Mazur P. Morin N. Baginsky W. El-Sherbeini M. Clemas J.A. Nielsen J.B. Foor F. Mol. Cell. Biol. 1995; 15: 5671-5681Crossref PubMed Google Scholar) and a regulatory subunit, a peripheral membrane protein encoded by RHO1 (6Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (392) Google Scholar, 7Drgonova J. Drgon T. Tanaka K. Kollar R. Chen G.C. Ford R.A. Chan C.S.M. Takai Y. Cabib E. Science. 1996; 272: 277-279Crossref PubMed Scopus (301) Google Scholar, 8Mazur P. Baginsky W. J. Biol. Chem. 1996; 271: 14604-14609Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Since GTP-bound Rho1p is required not only for cell wall synthesis but also for intracellular actin organization (9Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar), signal transduction leading to Rho1p plays a key role in cell morphogenesis. Another gene,GNS1, was originally isolated as a positive component required for GS activity in vitro (10El-Sherbeini M. Clemas J.A. J. Bacteriol. 1995; 177: 3227-3234Crossref PubMed Google Scholar). GS activity is severely reduced in the membrane fraction of a Δgns1mutant (10El-Sherbeini M. Clemas J.A. J. Bacteriol. 1995; 177: 3227-3234Crossref PubMed Google Scholar). GNS1 interacts genetically withFKS1: a Δgns1 Δfks1 double mutant grows more slowly and exhibits more reduced GS activity in the membrane fraction than single mutants (10El-Sherbeini M. Clemas J.A. J. Bacteriol. 1995; 177: 3227-3234Crossref PubMed Google Scholar). Although these results suggest thatGNS1 is somehow involved in GS activity, the physiological function of GNS1 remained unsolved since the Δgns1 mutant has a normal glucan content (10El-Sherbeini M. Clemas J.A. J. Bacteriol. 1995; 177: 3227-3234Crossref PubMed Google Scholar). Other lines of evidence revealed that GNS1 is allelic toELO2, which is involved in fatty acid elongation and sphingolipid synthesis (11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 12David D. Sundarababu S. Gerst J.E. J. Cell Biol. 1998; 143: 1167-1182Crossref PubMed Scopus (114) Google Scholar). A Δelo2(Δgns1) mutant is defective primarily in elongation of very long chain fatty acids. Since yeast sphingolipids are structural components of very long chain fatty acids, inability of Δelo2 cells to synthesize very long chain fatty acids results in alteration in the amounts of intermediates in sphingolipid metabolism (11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). In this study, we further investigated the reduced GS activity in the membrane fraction of the Δgns1 mutant. Our results indicate that a PHS accumulation in the Δgns1 mutant causes non-competitive inhibition of GS activity. Media for growth of S. cerevisiae and Escherichia coli are as described previously (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Genetic manipulations and yeast transformations were carried out as described (14Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The E. coli strain SCS1 (Stratagene, San Diego, CA) was used for propagation of plasmids used in this study. The yeast strains used in this work were derivatives of YPH500 (MATα ade2 his3 leu2 lys2 trp1 ura3) (14Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). YOC798 (MATα ade2 his3 leu2 lys2 trp1 ura3 Δgns1::HIS3) was made by transformation of YPH500 with the SphI-SacI fragment of pYO1929. Transformation of YOC798 with pYO1738 resulted in YOC799. YOC2587 (MATα ade2 his3 leu2 lys2 trp1 ura3 Δsur2::cgHIS3) and YOC2588 (MATα ade2 his3 leu2 lys2 trp1 ura3Δipt1::cgHIS3) were made by the PCR method described by Sakumoto et al. (15Sakumoto N., Y. Mukai K. Uchida T. Kouchi J. Kuwajima Nakagawa Y. Sugioka S. Yamamoto E. Furuyama T. Mizubuchi H. Ohsugi N. Sakuno T. Kikuchi K. Matsuoka I. Ogawa N. Kaneko Y. Harashima S. Yeast. 1999; 15: 1669-1679Crossref PubMed Scopus (94) Google Scholar): primers were used to amplify the HIS3 gene of Candida glabratatogether with flanking sequences derived from the upstream and downstream regions of SUR2 and IPT1, respectively. Restriction and modifying enzymes were purchased from TaKaRa (Kyoto, Japan). PHS, erythro-DHS, trypsin, Sephacryl S-1000, protease inhibitors, and reagents for enzyme assays were obtained from Sigma. Reagents for sphingolipids extraction came from Wako (Osaka, Japan). Standard molecular biological techniques were used for the construction of plasmids and PCR (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). pUC119-GNS1 containing the full-length of GNS1was constructed by inserting the 3.0-kilobaseSphI-SalI fragment containing GNS1into pUC119. pYO1929, generated for GNS1 disruption, was made by inserting the BamHI-BamHI fragment ofHIS3 from pJJ215 into the NruI-EcoRV gap of pUC119-GNS1. YEpU-DPL1 was constructed by inserting the 2.6-kilobase XhoI-XhoI fragment amplified by PCR with 5′-CCGCTCGAGCCGACAGTACGACTTAAAAAA-3′ and 5′-CCGCTCGAGTTATTTGTAGAAGGATTGTTT-3′ containing the full-length ofDPL1 into pRS326 (14Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). In order to insert the 3HA epitope at the C terminus of Gns1p, anNheI site was introduced just before the stop codon ofGNS1 as follows. First, pRS314-NcoI was constructed by inserting the NcoI linker at the uniqueBamHI site of pRS314 (14Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Second, pYO1932 was constructed by inserting the SpeI-NcoI fragment containingGNS1 from the DNA clone bank (16Sato K. Nishikawa S. Nakano A. Mol. Biol. Cell. 1995; 6: 1459-1477Crossref PubMed Scopus (91) Google Scholar), theNcoI-NheI fragment amplified by PCR with 5′-TTTGCATACTTATCACCATGG-3′ and 5′-CTAGCTAGCCCTTTTTCTTCTGTGTTG-3′, and the NheI-SalI fragment amplified by PCR with 5′-CTAGCTAGCTAAGTGTAAAATCTTTGA-3′ and 5′-GATTTTCAAATCACAGTCGAC-3′, into pRS314-NcoI. Finally, pYO1738 (GNS1:3HA) was generated by inserting the NheI-NheI fragment containing 3HA from pYT11 (17Takita Y. Ohya Y. Anraku Y. Mol. Gen. Genet. 1995; 246: 269-281Crossref PubMed Scopus (35) Google Scholar) into pYO1932 at the NheI site. Cells were grown at 25 °C in 1 liter of medium in a 2-liter flask rotating in air incubator (Innova 4330) at 150 rpm until theA 600 of the culture reached one. All the following procedures were carried out at 4 °C, unless otherwise stated. The cells were harvested, washed with 1 mm EDTA, and disrupted by vortexing 4 times for 2 min each with 5 ml of glass beads in 20 ml the breaking solution containing 0.5 m NaCl, 1 mm EDTA with 1 mm phenylmethylsulfonyl fluoride. After centrifugation at 1,500 × g for 5 min, the supernatant was collected and transferred to 33 PC tubes (Hitachi). The membrane fraction was collected by centrifugation at 100,000 × g for 30 min in an RP70T rotor (Hitachi) with Himac CP 65β (Hitachi). The resultant pellet was suspended with a membrane buffer containing 50 mm Tris-HCl, pH 7.5, 10 mmEDTA, 1 mm β-mercaptoethanol, and 33% glycerol, homogenized with a Dounce homogenizer, and stored at −80 °C. Purification of GS was carried out by product entrapment as described previously (6Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (392) Google Scholar) with some modifications. GS was solubilized from the membrane fraction by adding 0.2 m NaCl, 20 µm GTPγS, 5 mm dithiothreitol, 0.5% CHAPS, and 0.1% cholesteryl hemisuccinate. This suspension was left on ice for 20 min and centrifuged at 100,000 × g for 30 min. The supernatant was collected (“the detergent fraction”), to which 2.5 mm UDP-Glc and 20 mm potassium fluoride were added, and was subsequently incubated for 45 min at 30 °C. The white polymer of 1,3-β-glucan was collected by a 5-min centrifugation at 1,500 × g at 4 °C. The pellet was washed 3 times with extraction buffer (4 µm GTPγS, 1 mmdithiothreitol, 0.5% CHAPS, and 0.1% cholesteryl hemisuccinate in membrane buffer) containing 5 mm UDP-Glc and was centrifuged at 4,750 × g for 5 min at 4 °C. The resultant pellet was centrifuged again at 421,000 × gfor 10 min at 4 °C. The tight pellet was homogenized in the extraction buffer and the purified GS fraction was recovered in the supernatant after re-centrifugation at 421,000 × g for 10 min. GS activity was measured as formerly reported (4Douglas C.M. Foor F. Marrinan J.A. Morin N. Nielsen J.B. Dahl A.M. Mazur P. Baginsky W. Li W. El-Sherbeini M. Clemas J.A. Mandala S.M. Frommer B.R. Kurtz M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12907-12911Crossref PubMed Scopus (338) Google Scholar). To the detergent fraction that was solubilized with CHAPS and cholesteryl hemisuccinate, 0.1 mg/ml trypsin was added. After a 1-h incubation on ice, trypsin digestion was stopped by addition of 0.2 mg/ml trypsin inhibitor. The samples were then heated to 100 °C for 20 min. Plasma membranes (18Panaretou B. Piper P. Methods Mol. Biol. 1996; 53: 117-121PubMed Google Scholar), microsomes (16Sato K. Nishikawa S. Nakano A. Mol. Biol. Cell. 1995; 6: 1459-1477Crossref PubMed Scopus (91) Google Scholar), and secretory vesicles (19Walworth N.C. Novick P.J. J. Cell Biol. 1987; 105: 163-174Crossref PubMed Scopus (118) Google Scholar) were enriched as described. Nuclei were enriched by sucrose density gradient centrifugation as published before (20Hurt E.C. McDowall A. Schimmang T. Eur. J. Cell Biol. 1988; 46: 554-563PubMed Google Scholar, 21Aris J.P. Blobel G. Methods Enzymol. 1991; 194: 735-749Crossref PubMed Scopus (54) Google Scholar). Vacuoles and lipid particles were isolated by the procedure of Ohsumi and Anraku (22Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar), with the modification that yields the lipid particles fraction (23Leber R. Zinser E. Zellnig G. Paltauf F. Daum G. Yeast. 1994; 10: 1421-1428Crossref PubMed Scopus (209) Google Scholar, 24Hechtberger P. Zinser E. Saf R. Hummel K. Paltauf F. Daum G. Eur. J. Biochem. 1994; 225: 641-649Crossref PubMed Scopus (103) Google Scholar). Mitochondria (25Riezman H. Hase T. van Loon A.P. Grivell L.A. Suda K. Schatz G. EMBO J. 1983; 2: 2161-2168Crossref PubMed Scopus (106) Google Scholar) and Golgi membranes (26Lupashin V.V. Hamamoto S. Schekman R.W. J. Cell Biol. 1996; 132: 277-289Crossref PubMed Scopus (73) Google Scholar) were prepared as described. Cell organelles were fractionated on equilibrium density gradients according to the published procedures (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 17: 659-672Crossref Scopus (128) Google Scholar) with several modifications. Cultures were grown at 25 °C in 4 liters medium until theA 600 of the cultures reached one. Cells lysates made with glass beads were cleared of debris by centrifugation at 1,000 × g for 5 min. The membrane fraction was prepared by centrifugation at 100,000 × g for 2 h. The pellet was resuspended with 5 ml of STE 10 (10% sucrose in breaking solution with protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 2 mg/ml chymostatin, 1 mg/ml leupeptin, 0.5 mg/ml pepstatin, and 0.5 mg/ml aprotinin) and was layered on top of 30 ml of a 20–60% linear sucrose gradient in breaking solution. Samples were centrifuged at 100,000 × g for 20 h at 4 °C in a P28S rotor (Hitachi), and fractions of 3 ml were collected from the top of the gradient. Each fraction was diluted 5-fold by breaking solution and was centrifuged at 100,000 ×g for 1 h at 4 °C in an RP70T rotor (Hitachi). The pellet was suspended with the membrane buffer as described above. Plasma membrane ATPase activity (28Serrano R. Methods Enzymol. 1988; 157: 533-544Crossref PubMed Scopus (233) Google Scholar), Golgi GDPase activity (29Abeijon C. Orlean P. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6935-6939Crossref PubMed Scopus (133) Google Scholar), NADPH-cytochrome c reductase activity (30Schatz G. Klima J. Biochim. Biophys. Acta. 1964; 81: 448-461PubMed Google Scholar), α-d-mannosidase activity (31Yoshihisa T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1988; 263: 5158-5163Abstract Full Text PDF PubMed Google Scholar), kynurenine activity (32Bandlow W. Biochim. Biophys. Acta. 1972; 282: 105-122Crossref PubMed Scopus (47) Google Scholar), and invertase activity (19Walworth N.C. Novick P.J. J. Cell Biol. 1987; 105: 163-174Crossref PubMed Scopus (118) Google Scholar) were assayed in gradient fractions as described previously. Sphingolipids were extracted from [3H]serine-labeled cells as described (11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Radioactive bands were quantified and visualized with FLA-2000 (Fuji Photo Film) using a tritium screen. Sphingolipids were extracted from non-labeled cells as follows. Cultures of 1 liter each were grown at 25 °C until the A 600 of each culture reached one. Cells were washed with 1 mm EDTA, suspended in 15 ml of breaking solution as described above and disrupted with 5 ml of glass beads by vortexing 4 times for 2 min. After addition of 30 ml of methanol and 15 ml of CHCl3, lipids were extracted according to the method of Bligh and Dyer (33Bligh E.G. Dyer W.J. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42352) Google Scholar). After removal of cell debris and glass beads by centrifugation at 1,500 × g for 20 min, 12 ml of H2O, and 15 ml of CHCl3 were added to the supernatant. Protein was removed by centrifuging at 1,500 ×g for 20 min. The lower layer was washed 3 times with 90 ml of CHCl3, methanol, 0.1 m EDTA, 0.1m EGTA, pH 7.0 (2:2:5, v/v), and was dried with a vacuum evaporator. Half of the lipid extract was subjected to mild alkaline methanolysis by incubating with 3 ml of the monomethylamine reagent as prepared by Clarke and Dawson (34Clarke N.G. Dawson R.M. Biochem. J. 1981; 195: 301-306Crossref PubMed Scopus (186) Google Scholar) for 1 h at 52 °C. The resultant alkali-stable lipids were dried under a N2stream, resuspended in 0.1 ml of chloroform/methanol (2:1 by volume), and applied to Whatman Linear K6D silica gel TLC plates. The plates were developed with CHCl3, methanol, 30% NH4OH (9:7:2, v/v) or CHCl3, methanol, 4.2 nNH4OH (9:7:2, v/v). We preferred the 30% NH4OH solvent system since mass spectrometric analysis revealed that the PHS fraction obtained using the 4.2 n NH4OH solvent system contained degraded products of PHS or DHS. The mass spectrometric analysis was performed with Ion Trap LC/MS esquire 3000 (Nippon Bruker Daltonics). The fluorescent intensities of sphingolipids were quantified and visualized with LAS-1000plus (Fuji Photo Film) after spraying with primuline (Pfaltz & Bauer Inc.). Procedures for immunofluorescence microscopy were as described previously (35Pringle J. Adams A. Drubin D. Haarer B. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (600) Google Scholar). Anti-HA (16B12, BAbCO), and anti-Kar2p antibodies were used as primary antibodies. Cells were observed under the Olympus BX-FLA microscope (Olympus, Tokyo). To characterize the reduced GS activity in the membrane fraction ofΔgns1 cells, we determined theK m and V max values of GS activity. A kinetic analysis of GS activity revealed that theΔgns1 mutant has a decreasedV max value, one-fifth that of wild-type cells, while the K m value was not significantly changed (Table I). These results indicate that the reduced GS activity in the Δgns1 mutant is due to the reduced maximum velocity. At least three possibilities are raised based on this observation: (i) the amount of GS diminishes, (ii) an activator of GS decreases, or (iii) an inhibitor of GS accumulates in the membrane fraction of the Δgns1mutant.Table IKm and Vmax values of GS activityStrainK mV maxmmnmol/min/mg proteinWild-type0.37 ± 0.122.92 ± 0.44Δgns10.33 ± 0.060.54 ± 0.01K m and V max values were determined by s/v∼v plot. Average ± S.D. of four independent experiments. Open table in a new tab K m and V max values were determined by s/v∼v plot. Average ± S.D. of four independent experiments. To test the first possibility, we measured the total amounts of Fks1p and Rho1p in Δgns1 cells. Immunoblotting analyses demonstrated that the Fks1p and Rho1p levels in the membrane fraction of Δgns1 cells were indistinguishable from those of wild-type cells (data not shown). InΔgns1 cells, Fks1p and Rho1p exhibited a normal localization pattern (6Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (392) Google Scholar): both are placed at the growing tip of the bud (data not shown). These results suggested that the amount of GS localized to the plasma membrane was not altered inΔgns1 cells. We also examined whether or not Gns1p is a component of the GS complex using a strain expressing Gns1p tagged at its C terminus with 3 repeats of the influenza hemagglutinin (3HA) epitope (Gns1:3HAp). A low-copy plasmid expressing Gns1:3HAp was introduced to theΔgns1 strain. The membrane fraction ofΔgns1 cells expressing Gns1:3HAp exhibited a normal level of GS activity (data not shown), showing that Gns1:3HAp is fully functional. GS was purified by extraction from the membrane fraction followed by product entrapment (see “Experimental Procedures”). In the purified GS fraction with more than 300-fold increase in specific activity (Fig.1 A), the regulatory subunit, Rho1p, was greatly enriched while Gns1:3HAp was apparently lost (Fig.1 B). Examinations of the subcellular localization of Gns1:3HAp by immunofluorescent microscopy revealed that Gns1:3HAp was mainly localized to the ER (Fig. 1 C), exhibiting a localization pattern different from those of Fks1p and Rho1p (6Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (392) Google Scholar). These results strongly suggested that Gns1p is not a tightly bound component of the GS complex. Although we did not rule out the possibility that Gns1p activates GS, we obtained strong evidence suggesting that theΔgns1 mutant contains a GS inhibitor(s). We solubilized the membrane fractions of wild-type andΔgns1 mutant cells with detergents and treated with trypsin and heat to digest proteinous components. It was found that the resultant trypsin digest prepared fromΔgns1 cells had an inhibitory activity to GS, while that from the wild-type cells had little inhibitory effect (Fig.2 A). Dixson plots on GS activity showed linearity at 0.2 and 0.3 mm UDP-glucose and that the two lines intersected on the x axis (Fig.2 B). Taken together, the membrane fraction ofΔgns1 cells likely contains a non-proteinous and non-competitive inhibitor(s) of GS. We analyzed the subcellular distribution of the inhibitor(s) in Δgns1 cells. Because inhibitory activity was mainly recovered from the membrane fraction (data not shown), the membrane fraction of Δgns1 cells was further fractionated on a 20–60% linear sucrose gradient by centrifugation. We first determined the distributions of marker enzymes to check organelle distributions along the gradient. ER membranes were distributed in lower density fractions, while the plasma membrane was found in higher density fractions both in wild-type andΔgns1 fractions (Fig.3 A). Each fraction was solubilized with detergents, followed by treatment with trypsin and heat, and was subjected to the inhibition assay. Fractions around the peak of the plasma membrane exhibited little difference in the inhibitory activity between the wild-type andΔgns1 strains. In contrast, lighter fractions of Δgns1 prominently inhibited GS activity (Fig. 3 C). These results suggested that the inhibitor(s) of the Δgns1 mutant is concentrated not in the plasma membrane, but in lighter fractions containing the peak of ER. The Δgns1mutant is defective in elongation of very long chain fatty acids and synthesis of sphingolipids with very long chain fatty acids (11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Since the inhibitor(s) accumulated in Δgns1 cells is non-proteinous and mainly localized to the membrane fraction, we hypothesized that the inhibitor(s) is a lipid. To test this idea, we examined whether the total lipid extracted fromΔgns1 cells has an inhibitory activity. It was found that the total lipid extracted from theΔgns1 mutant strikingly inhibited GS activity (Fig. 4), while that extracted from wild-type cells had less inhibitory activity. Furthermore, Fig. 4 shows that the sphingolipid prepared from the total lipid of theΔgns1 mutant by mild alkaline had the same inhibitory activity. These results suggested that the inhibitor(s) accumulated in the Δgns1 cells is mainly present in the sphingolipid fraction. In order to identify which sphingolipid(s) inhibits GS activity, we resolved the sphingolipid fraction from [3H]serine-labeled cells by TLC (11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). As previously reported (11Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), the amounts of ceramide, inositol phosphoceramide (IPC), mannosylinositol phosphoceramide (MIPC), and mannosyl diinositolphosphorylceramide (M(IP)2C) decreased in theΔgns1 mutant (Fig.5 A), while both PHS and DHS contents increased 30-fold as compared with those in wild-type cells. Measurements of GS activity in the presence of each sphingolipid demonstrated that the PHS fraction from Δgns1cells strikingly inhibited GS activity (Fig. 5 B). The DHS fraction prepared from Δgns1 cells also inhibited GS activity to some extent. Mutations defective in sphingolipid biosynthesis have been widely studied in S. cerevisiae (Ref. 36Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1438: 305-321Crossref PubMed Scopus (129) Google Scholar; Fig.6 A). We constructedΔsur2 and Δipt1mutants, both of which affect sphingolipid biosynthesis (37Grilley M.M. Stock S.D. Dickson R.C. Lester R.L. Takemoto J.Y. J. Biol. Chem. 1998; 273: 11062-11068Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 38Haak D. Gable K. Beeler T. Dunn T. J. Biol. Chem. 1997; 272: 29704-29710Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 39Dickson R.C. Nagiec E.E. Wells G.B. Nagiec M.M. Lester R.L. J. Biol. Chem. 1997; 272: 29620-29625Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and measured their sphingolipid compositions and GS activities.Δsur2 cells contained an increased amount of DHS, while Δipt1 cells contained normal levels of PHS and DHS (Fig. 6 B). GS activity was specifically reduced in the membrane fraction isolated fromΔsur2 cells (Fig. 6 C). These results also suggested that DHS has GS inhibitory activity. Since DPL1 encodes a possible long-chain base-phosphate lyase that catabolizes sphingolipids (Ref. 40Saba 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 and Fig. 6 A), it is likely that overexpression of DPL1 results in reduced intracellular levels of PHS and DHS (41Grote E. Vlacich G. Pypaert M. Novick P.J. Mol. Biol. Cell. 2000; 11: 4051-4065Crossref PubMed Scopus (36) Google Scholar). In order to test this possibility, we introduced multiple copies of DPL1 to theΔgns1 strain. We found that DPL1overexpression in Δgns1 cells caused a 50% decrease in the amounts of PHS and DHS (Fig.7 A) as well as a reduction in GS inhibition (Fig. 7 B). This result further supported the idea that an accumulation of PHS or DHS or both causes reduced GS activity in the membrane fraction. To examine whether PHS and/or DHS inhibit GS activity, we isolated sphingolipids from wild-type cells, and directly measured their effects on the activity of purified GS. Ceramide, IPC, MIPC, and M(IP)2C did not affect GS activity in the concentration ranges examined. In contrast, PHS and DHS highly inhibited GS activity. The IC50 of PHS was about 0.5 mg/ml (Fig.8 A), which is approximately in the same range as the physiological concentration in theΔgns1 mutant but notably higher than that in wild-type strain (data not shown). Fig. 8 B shows that PHS inhibited GS activity in a non-competitive fashion. DHS also inhibited GS activity non-competitively (Fig. 8 A and data not shown), but the intracellular level of DHS was much lower than that of PHS in wild-type cells (Figs. 5 A and 7 A), suggesting that in vivo PHS is the primary GS inhibitor. Taken together, PHS, the intermediate of sphingolipids that accumulated in the Δgns1 mutant, was judged to be a potent intrinsic inhibitor to GS. In order to investigate the localization of PHS, we measured the PHS contents in purified organelle membranes. The quality of the organelle preparation was monitored by marker enzyme distributions (see “Experimental Procedures”). It was found that each organelle preparation contained only small amounts of other organelles (Table II). 250 µg of sphingolipids extracted from each organelle membrane was subjected to TLC (Fig. 9 A). As previously reported (24Hechtberger P. Zinser E. Saf R. Hummel K. Paltauf F. Daum G. Eur. J. Biochem. 1994; 225: 641-649Crossref PubMed Scopus (103) Google Scholar, 42Patton J.L. Lester R. J. Bacteriol. 1991; 173: 3101-3108Crossref PubMed Google Scholar), IPC was mainly localized to the Golgi membrane, while MIPC and M(IP)2C were concentrated in the plasma membrane. Lipid particles contained neither MIPC nor M(IP)2C. Measurements of the fluorescent intensity of the PHS fraction demonstrated that this sphingolipid was largely localized to the microsomal membrane fraction (Fig. 9 B). The localization pattern of PHS in wild-type cells is the same as that of Gns1p (Ref. 12David D. Sundarababu S. Gerst J.E. J. Cell Biol. 1998; 143: 1167-1182Crossref PubMed Scopus (114) Google Scholar and Fig. 1 C) and was consistent with the distribution of the inhibitor accumulated inΔgns1 mutant cells.Table IICharacterization of yeast subcellular fractions by specific activities of marker enzymesMarker enzymeOrganelle fractionsNucMSGolgiMitVacLPSVPMNADPH:cytochrome creductase1.14.41.01.71.41.50.61.4GDPase14135151862712Kynurenine hydroxylase0.010.010.000.170.010.000.000.00α-d-Mannosidase6556352151Invertase0.00.00.10.00.00.23.20.0PM ATPase0.10.80.10.20.60.62.55.0NADPH: cytochrome c reductase, GDPase, kynurenine hydroxylase, α-d-mannosidase, invertase, and PM ATPase were used as marker enzymes of the ER, the Golgi, mitochondria, vacuoles, secretory vesicles, and the plasma membrane, respectively.The following abbreviations are used in the table: Nuc, nucleus; MS, microsomes; Mit, mitochondria; Vac, vacuoles; LP, lipid particles; SV, secretory vesicles; PM, plasma membrane. Open table in a new tab NADPH: cytochrome c reductase, GDPase, kynurenine hydroxylase, α-d-mannosidase, invertase, and PM ATPase were used as marker enzymes of the ER, the Golgi, mitochondria, vacuoles, secretory vesicles, and the plasma membrane, respectively. The following abbreviations are used in the table: Nuc, nucleus; MS, microsomes; Mit, mitochondria; Vac, vacuoles; LP, lipid particles; SV, secretory vesicles; PM, plasma membrane. It was previously found that the membrane fraction ofΔgns1 cells exhibits reduced GS activity (10El-Sherbeini M. Clemas J.A. J. Bacteriol. 1995; 177: 3227-3234Crossref PubMed Google Scholar). In this study, several lines of evidence indicated that an accumulation of PHS in the Δgns1 mutant causes non-competitive inhibition of GS activity. First,Δgns1 cells accumulated a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Second, theΔgns1 mutant accumulated PHS, which was discovered to inhibit GS non-competitively. Among the six sphingolipids examined, PHS was clearly the most potent GS inhibitor. Third, the localization of PHS to the ER was identical to that of the inhibitor accumulated in Δgns1 cells. Fourth,DPL1 overexpression partially lowered the level of PHS accumulated in the Δgns1 cells, resulting in reduced inhibition of GS. Thus, our results are consistent with the idea that PHS negatively regulates GS activity. PHS inhibits GS activity non-competitively in several possible ways. First, PHS may repress the interaction between Fks1p and Rho1p. For instance, PHS may form a microdomain around Fks1p to inhibit the interaction with the prenylated form of Rho1p required for GS activity (43Inoue S.B. Qadota H. Arisawa M. Watanabe T. Ohya Y. J. Biol. Chem. 1999; 274: 38119-38124Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). It has been reported that the particular lipid fraction isolated from mammalian cells contains glycosylphosphatidylinositol-anchored proteins (44Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 45Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8047) Google Scholar, 46Pralle A. Keller P. Florin E.-L. Simons K. Horber J.K.H. J. Cell Biol. 2000; 148: 997-1008Crossref PubMed Scopus (841) Google Scholar), but does not include prenylated proteins (47Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Likewise, a particular lipid microdomain containing PHS possibly excludes prenylated Rho1p, preventing its interaction with Fks1p. Alternatively, PHS may alter the environment of lipid bilayer, causing inactivation of GS. The physicochemical state of lipid bilayer plays an important role in the activities of a number of enzymes (48Gennis R.B. Biomembranes: Molecular Structure and Function. Springer-Verlag, New York1989: 199-234Crossref Google Scholar). Since wild-type cells contain PHS on the ER membrane, it is likely that PHS inhibits GS activity on the ER. Many membrane-bound proteins localized to the plasma membrane are synthesized on the ER and transported through the secretory pathway (49Govindan B. Novick P. J. Exp. Zool. 1995; 273: 401-424Crossref PubMed Scopus (16) Google Scholar). Fks1p, a putative catalytic subunit of GS, accumulated in intracellular organelles when vesicular transport was blocked by secmutations, 2M. Abe, H. Oadota, and Y. Ohya, unpublished results. suggesting that Fks1p is transported along this pathway to the plasma membrane after its synthesis on the ER. Furthermore, the GS activity was reduced in the membrane fraction of sec12-1, sec16-2, andsec21-1 cells,2 all of which are defective in transport from the ER to the Golgi at the restrictive temperature (50Novick P. Ferro S. Schekman R. Cell. 1981; 25: 461-469Abstract Full Text PDF PubMed Scopus (528) Google Scholar,51Kaiser C.A. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (539) Google Scholar). Furthermore, the reduction in GS activity in the secmutants is diminished by overexpression ofDPL1.2 These results raise a possibility that a basal level of PHS residing in the ER functions to prevent nascent GS from being activated in the ER. If this is the case, it is the first example suggesting that a sphingolipid is involved in the inactivation of an enzyme at a specific organelle. In the case of many yeast enzymes that remain inactive until transported to their specific organelles, activation of enzymes is brought about by a modification or cleavage of their precursors. N-Glycosylation is required for activation of invertase (52Chu F.K. Watorek W. Maley F. Arch. Biochem. Biophys. 1983; 233: 543-555Crossref Scopus (59) Google Scholar, 53Esmon P, C. Esmon B.E. Schauer I.E. Taylor A. Schekman R. J. Biol. Chem. 1987; 263: 8832-8837Google Scholar) and exo-β-1,3-glucanase (54Basco R.D. Hernandez L.M. Munox M.D. Olivero I. Andaluz E. Del Rey F. Larriba G. Biochem. J. 1994; 304: 917-922Crossref PubMed Scopus (9) Google Scholar), both of which are then transported to the plasma membrane. The N-linked oligosaccharide has been shown to affect the activities of these enzymes by stabilizing protein-protein interactions or by altering the affinity to their substrates. Peptide cleavage is required for activation of vacuolar enzymes such as proteinase A (55Wolff A.M. Din N. Petersen J.G. Yeast. 1996; 12: 823-832Crossref PubMed Scopus (16) Google Scholar) and carboxypeptidase Y (56Stevens T. Esmon B. Schekman R. Cell. 1982; 30: 439-448Abstract Full Text PDF PubMed Scopus (371) Google Scholar, 57Mechler B. Muller H. Wolf D.H. EMBO J. 1987; 6: 2157-2163Crossref PubMed Scopus (52) Google Scholar). Further study will be necessary to test whether sphingolipid in fact affects activities of nascent GS transported through the ER. We thank Dr. Kunio Kitada for the C. glabrata genome, Osamu Kondoh for technical advice on measurements of GS activity, Akiko Enju for technical advice on lipid analysis, Drs. Ken Sato and Yumiko Saito for technical advice on cell fractionation, and Nippon Bruker Daltonics K. K. for mass spectrometric analysis. We also thank Dr. Keiichi Homma for reading the manuscript and a member of laboratory of Signal transduction for helpful discussions.