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Upstream of Growth and Differentiation Factor 1 (uog1), a Mammalian Homolog of the Yeast Longevity Assurance Gene 1 (LAG1), RegulatesN-Stearoyl-sphinganine (C18-(Dihydro)ceramide) Synthesis in a Fumonisin B1-independent Manner in Mammalian Cells

2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês

10.1074/jbc.m205211200

1083-351X

Krishnan Venkataraman, Christian Riebeling, Jacques Bodennec, Howard Riezman, Jeremy C. Allegood, M. Cameron Sullards, Alfred H. Merrill, Anthony H. Futerman,

Lipid metabolism and biosynthesis

The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B1, an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1,upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3H]sphinganine orl-3-[3H]serine demonstrated thatuog1 conferred fumonisin B1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids ofuog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB1, and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays ofuog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that theN-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis. The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B1, an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1,upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3H]sphinganine orl-3-[3H]serine demonstrated thatuog1 conferred fumonisin B1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids ofuog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB1, and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays ofuog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that theN-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis. Interest in determining the regulatory mechanisms of ceramide metabolism has been stimulated over the past decade by the realization that ceramides formed by turnover of complex sphingolipids, and byde novo synthesis, influence key aspects of cell growth, regulation, differentiation, and death (1Spiegel S. Merrill A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (644) Google Scholar, 2Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (726) Google Scholar, 3Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 4Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (440) Google Scholar, 5Venkataraman K. Futerman A.H. Trends Cell Biol. 2000; 10: 408-412Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 6Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Ceramides are formedde novo by N-acylation of sphinganine to dihydroceramide, which is subsequently desaturated by dihydroceramide desaturase (7Merrill A.H. Wang E. J. Biol. Chem. 1986; 261: 3764-3769Abstract Full Text PDF PubMed Google Scholar, 8Michel C. van Echten-Deckert G. Rother J. Sandhoff K. Wang E. Merrill A.H. J. Biol. Chem. 1997; 272: 22432-22437Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 9Ternes P. Franke S. Zahringer U. Sperling P. Heinz E. J. Biol. Chem. 2002; 277: 25512-25518Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The N-acyltransferase(s), which are referred to herein as (dihydro)ceramide synthase(s), acylate various long chain bases, including sphinganine, sphingosine, and 4-hydroxysphinganine, utilize a wide spectrum of fatty acyl-CoAs, and are inhibited by the mycotoxin, fumonisin B1(FB1) 1The abbreviations used are: FB1, fumonisin B1; ER, endoplasmic reticulum; ESI-MS/MS, electrospray tandem mass spectrometry; GlcCer, glucosylceramide; GSL, glycosphingolipid; HA, hemagglutinin; PDI, protein-disulfide isomerase; SM, sphingomyelin; HEK, human embryonic kidney. 1The abbreviations used are: FB1, fumonisin B1; ER, endoplasmic reticulum; ESI-MS/MS, electrospray tandem mass spectrometry; GlcCer, glucosylceramide; GSL, glycosphingolipid; HA, hemagglutinin; PDI, protein-disulfide isomerase; SM, sphingomyelin; HEK, human embryonic kidney. (10Wang E. Norred W.P. Bacon C.W. Riley R.T. Merrill A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar, 11Wang E. Merrill A.H. Methods Enzymol. 1999; 311: 15-21Crossref Scopus (29) Google Scholar, 12Merrill A.H.J. Sullards C. Allegood J.C. Wang E. Linn S.C. Andras L. Liotta D.C. Hartl M. Humpf H.U. Futerman A.H. Ceramide Signaling. R. G. Landes Co., Austin, TX2002Google Scholar). Kinetic evidence has been obtained for multiple (dihydro)ceramide synthases, but no biochemical or molecular evidence has been obtained to prove their existence. One reason for suggesting that multiple (dihydro)ceramide synthases exist is that FB1 suppresses the synthesis of most, but not all, sphingolipids in vivo, and the residual ceramide formed is preferentially channeled into glycosphingolipids (GSLs) rather than sphingomyelin (SM) (13Merrill Jr., A.H. van Echten G. Wang E. Sandhoff K. J. Biol. Chem. 1993; 268: 27299-27306Abstract Full Text PDF PubMed Google Scholar, 14Harel R. Futerman A.H. J. Biol. Chem. 1993; 268: 14476-14481Abstract Full Text PDF PubMed Google Scholar, 15Meivar-Levy I. Futerman A.H. J. Biol. Chem. 1999; 274: 4607-4612Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Furthermore, ceramide synthesis is thought to occur primarily in the endoplasmic reticulum (ER) (16Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (161) Google Scholar, 17Mandon E.C. Ehses I. Rother J. Van Echten G. Sandhoff K. J. Biol. Chem. 1992; 267: 11144-11148Abstract Full Text PDF PubMed Google Scholar), but recent studies (18Shimeno H. Soeda S. Sakamoto M. Kouchi T. Kowakame T. Kihara T. Lipids. 1998; 33: 601-605Crossref PubMed Scopus (86) Google Scholar, 19Shimeno H. Soeda S. Yasukouchi M. Okamura N. Nagamatsu A. Biol. & Pharm. Bull. 1995; 10: 1335-1339Crossref Scopus (29) Google Scholar) have suggested that ceramide can also be made in a mitochondria-enriched fraction. Until more tools are available to study (dihydro)ceramide synthases, the causes of such differences will remain obscure. This issue is further complicated by the ability to bypass FB1inhibition by formation of ceramide via the reverse activity of an acyl-CoA-independent ceramidase (20El Bawab S. Birbes H. Roddy P. Szulc Z.M. Bielawska A. Hannun Y.A. J. Biol. Chem. 2001; 276: 16758-16766Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Recent studies (21Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (218) Google Scholar) demonstrated that the yeast genes, LAG1(22D'Mello N, P. Childress A.M. Franklin D.S. Kale S.P. Pinswasdi C. Jazwinski S.M. J. Biol. Chem. 1994; 269: 15451-15459Abstract Full Text PDF PubMed Google Scholar) and LAC1, are required for acyl-CoA-dependent ceramide synthesis using very long chain (C26) fatty acids. Yeast strains lacking Lag1p and Lac1p produce greatly reduced levels of ceramides and lack FB1-sensitive ceramide synthase activity (23Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (219) Google Scholar). Moreover, LAG1 regulates glycosylphosphatidylinositol-anchored protein transport from the ER to the Golgi apparatus in yeast (24Barz W.P. Walter P. Mol. Biol. Cell. 1999; 10: 1043-1059Crossref PubMed Scopus (96) Google Scholar), a step that depends on ceramide synthesis (25Horvath A. Sütterlin C. Manning-Krieg U. Movva N.R. Riezman H. EMBO J. 1994; 13: 3687-3695Crossref PubMed Scopus (183) Google Scholar, 26Futerman A.H. Trends Cell Biol. 1995; 5: 377-380Abstract Full Text PDF PubMed Scopus (41) Google Scholar), supporting the idea that the yeast genes either encode for a catalytic subunit of ceramide synthase or are obligatory activators of ceramide synthase (21Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (218) Google Scholar, 23Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (219) Google Scholar). Finally, a tomato gene homolog of LAG1, the Alternaria stem canker locus-1 (ASC1), mediates FB1 resistance in tomato (27Brandwagt B.F. Mesbah L.A. Takken F.L. Laurent P.L. Kneppers T.J. Hille J. Nijkamp H.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4961-4966Crossref PubMed Scopus (177) Google Scholar), implying that ASC1 might encode a FB1-insensitive (dihydro)ceramide synthase in plants (28Brandwagt B.F. Kneppers T.J. Nijkamp H.J. Hille J. Mol. Plant-Microbe Interact. 2002; 15: 35-42Crossref PubMed Scopus (54) Google Scholar). In the current study, we examine the effect of overexpression of a mouse homolog of LAG1, upstream ofgrowth and differentiation factor-1(uog1) (29Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (26) Google Scholar, 30Boyer J. Pascolo S. Richard G.F. Dujon B. Yeast. 1993; 9: 279-287Crossref PubMed Scopus (10) Google Scholar), in mammalian cells. uog1 was originally discovered while screening for transforming growth factor-β family members, and was found to be expressed in various tissues as part of a bicistronic RNA together with growth/differentiation factor-1 (gdf1) (31Lee S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4250-4254Crossref PubMed Scopus (141) Google Scholar). Moreover, it functionally complemented the lethality of a LAG1 andLAC1 double deletion in yeast (29Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (26) Google Scholar). We now show that transfection with uog1 increased, in a FB1-insensitive manner, (dihydro)ceramide synthesis, which was subsequently preferentially channeled into neutral but not acidic GSLs. Unexpectedly, the ceramide and neutral GSLs formed were greatly enriched in C18- (stearoyl) fatty acids. Our data establish that UOG1 is involved in the regulation ofN-stearoyl-sphinganine synthesis in mammalian cells, and we demonstrate for the first time that cells can synthesize ceramides with a high degree of fatty acid selectivity as a result of the increased expression of a single gene product. l-3-[3H]Serine (specific activity of 26 or 31 Ci/mmol) and [14C]methylcholine (specific activity of 56 mCi/mmol) were from Amersham Biosciences. Ceramide, FB1, glucosylceramide (GlcCer), galactosylceramide, SM, palmitic acid, stearic acid, palmitoyl-CoA, Red Taq polymerase, defatted bovine serum albumin, phenylmethylsulfonyl fluoride, leupeptin, antipain, and aprotinin were from Sigma. Stearoyl-CoA was from Laroden Lipid Biochemicals (Malmö, Sweden).d-erythro-Sphinganine,d-erythro-sphingosine,d-erythro-sphinganine 1-phosphate,d-erythro-sphingosine 1-phosphate,d-erythro-stearoyl-sphingosine, a neutral GSL standard mixture, a mono-sialo and di-sialo GSL standard mixture, and (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol were from Matreya (Pleasant Gap, PA). Reverse phase RP-18 columns were from Supelco (Bellefonte, PA). pcDNA 3.0 was from Invitrogen. A pGEMT cloning kit was from Promega Corp. (Madison, WI). Vent polymerase was from New England Biolabs (Beverly, MA). LipofectAMINE was from Invitrogen. Restriction enzymes and DNA-modifying enzymes were either from MBI Fermentas (Vilnius, Lithuania) or from New England Biolabs. A one-step reverse transcriptase-PCR kit was from Qiagen GmbH (Hilden, Germany). A rat anti-HA monoclonal antibody (clone 3F10) was from Roche Molecular Biochemicals; a rabbit anti-protein-disulfide isomerase (PDI) antibody was from Stressgen (Victoria, Canada), and MitoTracker® Deep Red was from Molecular Probes (Eugene, OR). Silica Gel 60 thin layer chromatography (TLC) plates were from Merck. All solvents were of analytical grade and were purchased from Biolab (Jerusalem, Israel). The coding sequences of the human and mousegdf1 genes (GenBankTM accession numbers M62302and M62301) were subject to a Blast search against human and mouse expression sequence tag data bases. Two cDNA clones (GenBankTM accession numbers AU080088 (MNCb-5211) andAU080131 (MNCb-5265)) from mouse brain showed the highest sequence identity with the cDNA sequence of uog1 and were obtained from the National Institute of Infectious Diseases, Japanese Collection of Research Bioresources Gene Bank, Japan. Restriction digestion analysis of both MNCb-5211 and MNCb-5265 demonstrated that the constructs had two distinct open reading frames coding for uog1 and gdf1. The uog1fragment (1.3 kilobases) was released by restriction digestion usingEcoRI and AvrII sites, sub-cloned into a pcDNA 3.0 vector at EcoRI and XbaI sites, and subjected to nucleotide sequencing. Both MNCb-5211 and MNCb-5265 had identical nucleotide sequences, and the derived amino acid sequence from pcDNA-UOG1(5211) and pcDNA-UOG1(5265) demonstrated that there was one amino acid change, from serine to arginine, at position 334 compared with the derived amino acid sequence from mouse UOG1 (31Lee S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4250-4254Crossref PubMed Scopus (141) Google Scholar); because the amino acid sequence was not conserved at this position between human and mouse UOG1, the coding sequence of MNCb-derived clones was not modified. Subsequently, the pcDNA-UOG1(5211) clone derived from MNCb-5211 was used for overexpression studies. Sequence-specific primers with unique restriction sites were designed for the 5′ and 3′ ends of uog1 cDNA, and theuog1 cDNA was amplified by PCR to obtain the 1.08-kb PCR product. The PCR product was sub-cloned into a pGEMT vector, and the positive clones were identified, according to instructions provided in the technical manual for the pGEMT easy vector system. The cDNA insert was also sub-cloned into tagged vectors to create vectors carrying hemagglutinin epitope (HA) tags at the N and C termini of UOG1. The coding sequences were confirmed by nucleotide sequencing. COS-7 cells, grown on glass coverslips, and human embryonic kidney 293T cells (HEK-293T), grown in Nunc tissue culture dishes, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. COS-7 cells were transfected using LipofectAMINE according to the manufacturer's instructions, and HEK-293T cells were transfected by the calcium phosphate method (32). Eighteen hours after transfection, COS-7 cells were incubated with MitoTracker® Deep Red (25 nm) for 15 min and chased for 3 min in fresh medium. The cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline (20 min, 37 °C), and subsequently permeabilized with 0.1% (v/v) Triton X-100 in phosphate-buffered saline, 1% bovine serum albumin for 30 min. Primary antibodies (rabbit anti-PDI, dilution of 1:200; rat anti-HA, 1:500) were applied for 2 h at room temperature. A rhodamine-labeled goat anti-mouse secondary antibody (which cross-reacts with the rat anti-HA primary antibody) was diluted 1:200, and a fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody was diluted 1:500. After a 1-h incubation, slides were mounted in Fluoromount G and viewed by confocal laser scanning microscopy using an Olympus Fluoview FV500 imaging system. Fluorescein isothiocyanate, rhodamine fluorescence, and MitoTracker® Deep Red were viewed using an argon and two helium-neon laser with excitation wavelengths of 488, 543, and 633 nm, respectively. Images were acquired in sequential mode and analyzed using Fluoview 3 imaging software. 24 h after transfection, HEK-293T cells were incubated with or without 20 μm FB1. Subsequently, cells were metabolically labeled either with [4,5-3H]sphinganine or withl-3-[3H]serine and [14C]methylcholine. 9 μCi of [4,5-3H]sphinganine, synthesized by reduction ofd-erythro-sphingosine with NaB[3]H4 (11 Ci/mmol) (16Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (161) Google Scholar), was added to the culture medium 45 h after transfection. Three hours later, cells were washed with phosphate-buffered saline and removed from the dishes by scraping with a rubber policeman. After homogenization by sonication, protein concentration was determined (33Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar), and lipids were extracted (34Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42382) Google Scholar) and dried under N2. Lipids were subjected to alkaline hydrolysis in chloroform, 0.6 n NaOH in methanol (1:1) (37 °C, 2 h) to degrade glycerolipids (35Bodennec J. Koul O. Aguado I. Brichon G. Zwingelstein G. Portoukalian J. J. Lipid Res. 2000; 41: 1524-1531Abstract Full Text Full Text PDF PubMed Google Scholar). Samples were subsequently washed three times with water to remove the NaOH, and the [4/5-3H]sphingolipids 2[4,5-3H]Sphinganine is metabolized to [4,5-3H]dihydroceramide and subsequently to [3H]ceramide, which cannot be differentiated by the TLC solvent used in this experiment. [3H]Ceramide consists of a mixture of [4-3H]ceramide and [5-3H]ceramide (16Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (161) Google Scholar, 42Hirschberg K. Zisling R. van Echten-Deckert G. Futerman A.H. J. Biol. Chem. 1996; 271: 14876-14882Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The resulting lipid is therefore referred to as [4/5-3H](dihydro)ceramide. obtained from the chloroform phase were separated by TLC using chloroform, methanol, 9.8 mm CaCl2 (60:35:8, v/v) as the developing solvent. Cells were incubated withl-3-[3H]serine (30 μCi per dish) and [14C]methylcholine (3 μCi per dish) for 24 h. Lipids were then extracted (36Folch J. Lees M. Stanley G.H.S. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and dissolved in benzene/methanol (1:1, v/v). 3H- and 14C-labeled phospholipids were resolved by two-dimensional TLC using tetrahydrofurane/acetone/methanol/water (50:20:40:6, v/v) as the first developing solvent, and chloroform/acetone/methanol/acetic acid/water (50:20:10:15:5, v/v) as the second developing solvent (35Bodennec J. Koul O. Aguado I. Brichon G. Zwingelstein G. Portoukalian J. J. Lipid Res. 2000; 41: 1524-1531Abstract Full Text Full Text PDF PubMed Google Scholar). Phospholipids were visualized using iodine and recovered from TLC plates by scraping the silica directly into scintillation vials. One ml of methanol and 5 ml of Optima Gold scintillation fluid (Packard Instrument Co.) were added to each vial for scintillation counting, and radioactivity was determined in a Packard 2100 beta radiospectrometer equipped with the Transformed Spectral Index of the External Standard/Automatic Efficiency Control (tSIE/AEC) program for quench correction and counting of double radiolabeled samples. Another aliquot of the lipid extract was used for analysis of [3H]sphingolipids. After alkaline hydrolysis (see above), the lipids were obtained from the chloroform phase, and after drying were reconstituted in 1:1 benzene/methanol (1:1, v/v). [3H]Sphingolipids were separated by TLC using the following developing solvents. (i) [3H](Dihydro)ceramide was separated using chloroform:methanol (50:3.5, v/v). (ii) [3H]GlcCer and [3H]galactosylceramide were separated on a sodium borate-coated TLC plate using chloroform/methanol/water (24:7:1, v/v). (iii) Neutral [3H]GSLs were separated on two-dimensional TLC using tetrahydrofurane/acetone/methanol/water (50:20:40:6, v/v) and chloroform/acetone/methanol/acetic acid/water (50:20:10:15:5, v/v). For analysis of acidic [3H]GSLs, the aqueous phase of the lipid extract (36Folch J. Lees M. Stanley G.H.S. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) was loaded onto Supelco RP-18 disposable cartridges (37Williams M.A. McCluer R.H. J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar) and washed with water. Lipids were eluted using chloroform/methanol (1:1, v/v), dried under N2, mixed with authentic ganglioside standards, and separated by TLC using chloroform/methanol/9.8 mm CaCl2 (60:35:8, v/v). HEK-293T cells were homogenized in 20 mm Hepes, pH 7.4, 25 mm KCl, 250 mm sucrose and 2 mm MgCl2, containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml antipain, 1 μg/ml leupeptin, and 100 kIU/ml aprotinin). Homogenates or microsomal fractions (16Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (161) Google Scholar, 38Venkataraman K. Futerman A.H. Biochim. Biophys. Acta. 2001; 1530: 219-226Crossref PubMed Scopus (32) Google Scholar) (both 200 μg of protein) were incubated with 1.48 μCi of [4,5-3H]sphinganine, 15 μm sphinganine, 20 μm defatted bovine serum albumin (16Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (161) Google Scholar, 38Venkataraman K. Futerman A.H. Biochim. Biophys. Acta. 2001; 1530: 219-226Crossref PubMed Scopus (32) Google Scholar). The reaction was initiated by addition of either 10 μm free fatty acid (palmitate or stearate) or 50 μm palmitoyl-CoA or stearoyl-CoA. Lipids were extracted (34Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42382) Google Scholar) and [4/5-3H](dihydro)ceramide synthesis was analyzed by TLC using chloroform, methanol, 2 m ammonium chloride (40:10:1, v/v). HEK-293T cells were grown in 60-mm plastic culture dishes and transfected by the calcium phosphate method. After 24 h, cells were incubated with or without 20 μm FB1 for a further 24 h. Cells were harvested by trypsinization and collected by centrifugation. The cell pellet was washed once with ice-cold phosphate-buffered saline and lyophilized. The cell pellets were extracted and analyzed for sphingolipids using essentially the conditions described (39Sullards M.C. Methods Enzymol. 2001; 312: 32-45Crossref Google Scholar, 40Sullards, M. C., and Merrill, A. H., Jr. (2001)Science's STKE,//stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/67/pl1;2001, PL1.Google Scholar), which utilize normal phase high pressure liquid chromatography to separate the sphingolipid classes followed by electrospray tandem mass spectrometry (ESI-MS/MS) on a PE-Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ionspray source. Dry N2was used as the nebulizing gas at a flow rate of 6 liters/min. The ionspray needle was held at 5500 V, and the orifice and ring voltages were kept low (40 and 220 V, respectively) to prevent collisional decomposition of molecular ions prior to entry into the first quadrupole, and the orifice temperature was set to 500 °C. N2 was used to collisionally induce dissociations in Q2, which was offset from Q1 by 40–50 V. Q3 was then set to pass molecularly distinctive product ions (N ions) of m/z264.2. Multiple reaction monitoring scans were acquired by setting Q1 and Q3 to pass the precursor and product ions of the most abundant sphingolipid molecular species. For example, for the ceramides, these transitions occur at m/z 538.7/264.4, 566.5/264.4, 622.7/264.4, 648.7/264.4, 650.7/264.4, which corresponds to ceramides with a d18:1 sphingoid base (sphingosine) and C16:0, C18:0, C22:0, C24:1, and C24:0 fatty acids, respectively. The dwell time was 25 ms for each transition. Quantitation was achieved by spiking the samples prior to extraction with the C12 fatty acid homologs of ceramide and SM along with the C8 fatty acid homolog of GlcCer. uog1 was sub-cloned into a pcDNA vector or into a pcDNA-HA vector to give constructs expressing UOG1 with an HA tag at the N (HA-UOG1) or C terminus (UOG1-HA). When the HA tag was at the N terminus of UOG-1, UOG1 was localized in reticular-like structures that were confirmed to correspond to the ER by almost complete co-localization with the ER marker, PDI, in both HEK-293T cells (not shown) and in COS cells (Fig.1); due to their size and morphology, much better resolution of intracellular organelles was obtained with COS cells. In contrast, when the tag was at the C terminus, the Golgi apparatus was strongly labeled and there was less co-localization with PDI. Because UOG1 has a C-terminal K(X)KXX motif (41Nilsson T. Jackson M. Peterson P. Cell. 1989; 58: 707-718Abstract Full Text PDF PubMed Scopus (367) Google Scholar), this suggests that the presence of a tag at the C terminus blocks the recycling and retrieval of UOG1 from the Golgi apparatus to the ER. Neither HA-UOG1 nor UOG1-HA showed any co-localization with the mitochondria-specific dye MitoTracker® (Fig. 1) or with an anti-cytochrome c oxidase IV antibody (not shown). Similar results were obtained with green fluorescent protein-tagged UOG1 constructs. To determine whether UOG1 is involved in regulating (dihydro)ceramide synthesis in mammalian cells, uog1-transfected HEK-293T cells were metabolically labeled with [4,5-3H]sphinganine (15Meivar-Levy I. Futerman A.H. J. Biol. Chem. 1999; 274: 4607-4612Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 42Hirschberg K. Zisling R. van Echten-Deckert G. Futerman A.H. J. Biol. Chem. 1996; 271: 14876-14882Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) or withl-3-[3H]serine, a substrate for the first enzyme in the pathway of sphingolipid biosynthesis, serine palmitoyltransferase (7Merrill A.H. Wang E. J. Biol. Chem. 1986; 261: 3764-3769Abstract Full Text PDF PubMed Google Scholar). There was a 2.8-fold increase in [4/5-3H](dihydro)ceramide synthesis from [4,5-3H]sphinganine (Fig.2 A) and a small increase inl-3-[3H]serine incorporation into [3H]ceramide (Fig. 2 B) inuog1-transfected cells compared with mock-transfected cells. As expected, FB1 inhibited (dihydro)ceramide synthesis in mock-transfected cells using both labeling protocols. However, FB1 only inhibited [4/5-3H](dihydro)ceramide synthesis from [4,5-3H]sphinganine by ∼35% inuog1-transfected cells (Fig. 2 A) and, surprisingly, increased levels of [3H]ceramide synthesis from [3H]serine (Fig. 2 B). Thus, by using both [4,5-3H]sphinganine andl-3-[3H]serine, UOG1 overexpression elevates levels of (dihydro)ceramide synthesis and confers FB1resistance. 3The reason for the difference in the extent of FB1 resistance or of FB1-induced elevation of (dihydro)ceramide synthesis using the two different metabolic labeling protocols (i.e. [4,5-3H]sphinganine and l-3-[3H]serine) is not known but may be due to the different times of labeling (3 versus 24 h), the different amounts of label added, or the different sites of incorporation of l-3-[3H]serine and [4,5-3H]sphinganine into the sphingolipid metabolic pathway. The fate of the (dihydro)ceramide synthesized inuog1-transfected HEK-293T cells was determined by metabolic labeling with l-3-[3H]serine (TableI). In uog1-transfected cells a significant increase in the synthesis of the neutral GSLs, GlcCer, ceramide dihexosides, ceramide trihexosides, and globosides was observed compared with mock-transfected cells (Table I), but there was no elevation of ganglioside synthesis. In FB1-treateduog1-transfected cells, neutral GSL synthesis was elevated to an even greater extent, with ceramide trihexoside and globoside synthesis elevated 10–11-fold; similar to uog1-transfected cells that were