Human Homologues of LAG1 Reconstitute Acyl-CoA-dependent Ceramide Synthesis in Yeast
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m307554200
ISSN1083-351X
AutoresIsabelle Guillas, James C. Jiang, Christine Vionnet, Carole Roubaty, Danièle Uldry, Rachel Chuard, Jinqing Wang, S. Michal Jazwinski, Andreas Conzelmann,
Tópico(s)Lipid metabolism and biosynthesis
ResumoLag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1Δ lac1Δ double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1Δ lac1Δ yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1Δ lac1Δ yeast mutants. Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1Δ lac1Δ double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1Δ lac1Δ yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1Δ lac1Δ yeast mutants. The only structural sphingolipids of Saccharomyces cerevisiae are the inositol phosphorylceramides (IPCs), 1The abbreviations used are: IPC, inositolphosphorylceramide; MIPC, mannosyl-inositolphosphorylceramide; DHS, dihydrosphingosine; PHS, phytosphingosine; PI, phosphatidylinositol; MMA, monomethylamine; BSA, bovine serum albumin; wt, wild type; FOA, fluoroorotic acid; HOX, homeobox; TLC, thin layer chromatography; TLC, TRAM, LAG1, and CLN8. mannosyl-IPCs (MIPCs), and inositol phosphoryl-MIPC, which together represent a significant fraction of membrane lipids, especially in the plasma membrane (1Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1426: 347-357Crossref PubMed Scopus (169) Google Scholar, 2Patton J.L. Lester R.L. J. Bacteriol. 1991; 173: 3101-3108Crossref PubMed Google Scholar, 3Hechtberger P. Zinser E. Saf R. Hummel K. Paltauf F. Daum G. Eur. J. Biochem. 1994; 225: 641-649Crossref PubMed Scopus (104) Google Scholar). Recent progress has resulted in the identification of yeast genes involved in all enzymatic steps that are required for their biosynthesis (4Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 2002; 1583: 13-25Crossref PubMed Scopus (198) Google Scholar). The intermediates in sphingolipid synthesis, dihydrosphingosine (DHS), phytosphingosine (PHS), and their 1-phosphorylated derivatives, as well as free ceramides, have been proposed to act as signal transduction molecules governing heat stress responses, endocytosis, glycosyl phosphatidylinositol protein transport, ubiquitin-dependent degradation of membrane channels, and progression through G1 (for review, see Ref. 4Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 2002; 1583: 13-25Crossref PubMed Scopus (198) Google Scholar). A key role in the sphingolipid pathway is played by ceramide synthase, as it not only catalyzes an essential biosynthetic reaction, but also influences the levels of long chain bases and ceramides, which have signaling function. The simultaneous deletion of LAG1 and its close homologue LAC1 eliminates all detectable acyl-CoA-dependent ceramide biosynthesis in yeast microsomes (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar, 6Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (221) Google Scholar). Moreover, lag1Δ lac1Δ cells have a drastically reduced amount of normal ceramides and IPCs, but exhibit a marked accumulation of free DHS and a compensatory increase of C26:0 fatty acids, which seem to be used for making a new form of phosphatidylinositol (PI) that we call PI′ (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar, 6Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (221) Google Scholar). Whereas the single deletion of LAG1 or LAC1 had no abnormal growth phenotype, the concomitant deletion of LAG1 and LAC1 caused osmotic fragility, calcofluor white hypersensitivity, and a significant decrease of the growth rate in the genetic background of W303 cells, and the same double mutation was lethal in the background of YPK9 cells (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar, 7Barz W.P. Walter P. Mol. Biol. Cell. 1999; 10: 1043-1059Crossref PubMed Scopus (96) Google Scholar, 8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar). Lethality of YPK9 lag1Δlac1Δ (herein named YPK9.2Δ) can be overcome by overexpression of LAG1 homologues from man (LAG1Hs, also named LASS1) or Caenorhabditis elegans (LAG1Ce-1, also named hyl-1), two genes showing 26 and 32% identity to yeast LAG1, respectively (8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar). A recent paper has shown that the murine UOG1, which has 81% identity with LAG1Hs, and 27% identity with yeast LAG1, strongly induces a C18-CoA-specific ceramide synthase activity, when overexpressed in a mammalian cell line (9Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill A.H.J. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Thus, the pivotal role of LAG1 proteins in the ceramide synthase reaction is established in both yeast and mammalian cells, but it presently is not understood whether LAG1 proteins are part of the catalytically active enzyme or are essential regulators of the ceramide synthase. This question has led us to investigate the substrate specificity of human and yeast LAG1 proteins. We found that expression of some human homologues of hitherto unknown function in YPK9.2Δ not only restored viability but also allowed the acyl-CoA-dependent synthesis of ceramides that are not usually made but can be used for IPC biosynthesis. Strains, Growth Conditions, and Materials—S. cerevisiae strains used in this study are listed in Table I. Cells were grown on rich medium (YPD) or minimal media SDaa or SGaa, containing 2% glucose or galactose and uracil (U) and adenine (A) if required (10Benghezal M. Benachour A. Rusconi S. Aebi M. Conzelmann A. EMBO J. 1996; 15: 6575-6583Crossref PubMed Scopus (153) Google Scholar, 11Meyer U. Benghezal M. Imhof I. Conzelmann A. Biochemistry. 2000; 39: 3461-3471Crossref PubMed Scopus (71) Google Scholar) at 30 °C. Chemicals, radiochemicals, and inhibitors were from sources described (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar, 12Flury I. Benachour A. Conzelmann A. J. Biol. Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Plasmid pYES6/CT was from Invitrogen AG (Basel, Switzerland). Acyl-CoAs were synthesized as described (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). Oligonucleotides and DNA sequencing services were obtained at Microsynth (Balgach, Switzerland).Table IYeast strainsStrainsGenotypeReferenceW303-1AMATa can1-100 ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1(29Kirchman P.A. Kim S. Lai C.Y. Jazwinski S.M. Genetics. 1999; 152: 179-190Crossref PubMed Google Scholar)YPK9MATa ade2-101 ochre his3-Δ200 leu2-Δ1 lys2-801 amber trp1-Δ63 ura3-52(29Kirchman P.A. Kim S. Lai C.Y. Jazwinski S.M. Genetics. 1999; 152: 179-190Crossref PubMed Google Scholar)YPK9 lac1ΔSame as YPK9, but lac1Δ::LEU2(8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar)YPK9 lag1ΔSame as YPK9, but lag1Δ::TRP1(8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar)YPK9 lac1Δlag1Δ pBM150:LAG1 apBM150 is a CEN-ARS, URA3 vector possessing the yeast GAL1,10 promoter. (here named YPK9.2Δ.LAG1)Same as YPK9, but lac1Δ::LEU2 lag1Δ::TRP1 and containing pBM150:LAG1 apBM150 is a CEN-ARS, URA3 vector possessing the yeast GAL1,10 promoter.(8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar)YPK9 lac1Δlag1Δ pBM150:LAG1Hs apBM150 is a CEN-ARS, URA3 vector possessing the yeast GAL1,10 promoter. (here named YPK9.2Δ.LAG1Hs)Same as YPK9, but lac1Δ::LEU2 lag1Δ::TRP1 and containing pBM150:LAG1Hs apBM150 is a CEN-ARS, URA3 vector possessing the yeast GAL1,10 promoter.(8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar)YPK9.2Δ.C1As YPK9, but lac1Δ::LEU2 lag1Δ::TRP1 + pYES6/CT with GAL1UAS - Clone 1This studyYPK9.2Δ.C4As YPK9, but lac1Δ::LEU2 lag1Δ::TRP1 + pYES6/CT with GAL1UAS - Clone 4This studyYPK9.2Δ.LASS2Same as YPK9.2Δ.LAG1 but with LASS2 under GAL1 promoter in pYES6/CTThis studyYPK9.2Δ.CLN8Same as YPK9.2Δ.LAG1 but with CLN8 under GAL1 promoter in pYES6/CTThis studyEMY 27 (elo2Δ)MATα ura3 trp1 leu2 elo2::LEU2R. SchneiterEMA 40 (elo3Δ)MATa ura3 trp1 leu2 elo3::URA3R. SchneiterYRXS 12 (mtr7-1)MATa acc1-2150R. Schneitera pBM150 is a CEN-ARS, URA3 vector possessing the yeast GAL1,10 promoter. Open table in a new tab Conditional Expression of Clones 1 and 4, LASS2, and CLN8 — Human cDNA clones were obtained from the Deutsches Ressourcenzentrum für Genomforschung (RZPD; www.rzpd.de/), namely clone 1 (NCBI entry BC010032; RZPD, IRALp962C1927Q2), clone 4 (NCBI entry AK022151; RZPD, IRALp962D1425Q2), and CLN8 (NCBI entry BC007725; RZPD, IMAGp958E241166Q2). LASS2 (NCBI entry XM_041889) was from Origene Technologies Inc. (www.origene.com/home.html). The conditional expression of human cDNAs in yeast was achieved by the insertion of the open reading frames behind the GAL1,10 promoter of the vector pYES6/CT carrying as selective marker a blasticidin resistance gene (Invitrogen AG, Basel, Switzerland). Primers to amplify the cDNA open reading frames between suitable restriction sites are shown in Table II. Reverse primers included a stop codon so that the constructs remained without any tag in pYES6/CT. CLN8 and LASS2 were also inserted as a EagI/EcoRI fragment into a modified version of multicopy vector pRS425 obtained from R. Arkowitz (University of Nice, Nice, France). In this vector, the potent TPI1 promoter had been inserted between SacII and EagI and the LEU2 marker was replaced by ADE2. The sequences of the inserts of all vectors constructed were verified by sequencing.Table IIOligonucleotides usedTarget geneName of primerOligonucleotide sequenceSize of productClone 1 into pYES1FCGGGGTACCAAGCTTAAATAAAATGctccagaccttgtatgattacttctg11401RCCGCTCGAGTCAgtcattcttacgatggttgttattgaClone 4 into pYES4FCGCGGATCCAAAAAATGctgtccagtttcaacgagtgg11824RGCTCTAGACTAtgtggctgttgtgtgcctgtLASS2 into pYES2FCGGGGTACCAAATAAAATGgctgtcactgtggataaac7562RGCTCTAGATCAgtcattcttacgatggttgttatCLN8 into pYES8FCGGGGTACCAAATAAAATGaatcctgcgagcgat8588RGCTCTAGACTAtggcctcttcttccgcYeast LAG1LAG1Ftgttgtaattcgaccattca155LAG1RtcttgaacaaccacaaatcaLASS2 into pRS102.F1GCCGCGGCCGAATAAAatggctgtcactgtggataaa721102.R1GGGAATTCTAGAtcagtcattcttacgatggttgCLN8 into pRS101.F1GCCGCGGCCGAATAAAatgaatcctgcgagcg884101.R1GGGAATTctatggcctcttcttccgca Open table in a new tab Microsomal Ceramide Synthase Assay—Microsomes were prepared and resuspended in lysis buffer as described (13Canivenc-Gansel E. Imhof I. Reggiori F. Burda P. Conzelmann A. Benachour A. Glycobiology. 1998; 8: 761-770Crossref PubMed Scopus (61) Google Scholar). Unless stated otherwise, assays contained the equivalent of 33 μg of protein (3.3 μl of microsomes or 33–50 μl of detergent extract), 6–10 μCi of [4,5-3H]DHS (2–3.3 nmol = 20–33 μm) and 10–13 nmol (100–130 μm) of acyl-CoA or free fatty acids in a final volume of 0.1 ml. Acyl-CoA or free fatty acids were added in 3 μl from a stock in 1% Zwittergent 3-16 so that assays contained 0.77 mm Zwittergent (critical micellar content = 0.01–0.06 mm). Unless stated otherwise, the assay buffer was 100 mm Tris-HCl, pH 7.5, 1 mm ATP, 30 mm creatine phosphate, and 1 mg/ml creatine kinase, and labeling reactions were incubated at 37 °C for 2–4 h. In some assays, the microsomes were first incubated for 15 min in 0.5 mg/ml delipidated BSA on ice, spun through a cushion of 0.8 m sorbitol, 25 mm potassium phosphate, pH 7.4, at 13,000 × g for 30 min, and the following assay was done in the same phosphate buffer instead of Tris-HCl using the protocol of Wang and Merrill (14Wang E. Merrill A.H.J. Methods Enzymol. 2000; 311: 15-21Crossref PubMed Scopus (29) Google Scholar). At the end the lipids were twice extracted with chloroform-methanol (1:1), and extracts were dried and desalted by butanol-water partitioning. Alternatively, ceramides were extracted by addition of 0.6 ml of 0.55 m aqueous NH4OH, followed by vortexing and centrifugation. The lower chloroform phase was washed twice with 0.6 ml of water and dried. Microsomes were lysed in 0.2 or 0.3% digitonin (critical micellar content = 0.075%) in 100 mm Tris-HCl, pH 7.5, for 2 h at 4 °C under shaking. Lysates were ultracentrifuged at 4 °C, 100,000 × g for 1 h. The final digitonin concentration in the assays using solubilized proteins was usually 0.1%. Lipids were analyzed by ascending TLC using solvents 1, 2, or 3 described previously (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). Radioactivity was detected and quantified by one-dimensional radioscanning, and then the TLC plates were sprayed with EN3HANCE and exposed to film at –80 °C. Metabolic Labeling and Lipid Extraction—Cells harboring the human cDNAs were grown over night on SGaaUA, whereas control YPK9.2Δ.LAG1 cells were depleted of Lag1p on SDaaA for 30 h before being labeled. 10 OD600 units of exponentially growing cells were labeled with 60 μCi of myo-[2-3H]inositol, and 30 OD600 units with 100 μCi of [3H]serine for 2 h at 30 °C. Lipid extraction and desalting were done as described (15Reggiori F. Canivenc-Gansel E. Conzelmann A. EMBO J. 1997; 16: 3506-3518Crossref PubMed Scopus (101) Google Scholar). Lipids were deacylated by mild base treatment using monomethylamine (MMA) as described (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). Characterization of the Microsomal Ceramide Synthase Assay—The ceramide synthase assay described previously (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar) contained microsomes, [3H]DHS, divalent cations, an ATP-regenerating system, and C26:0-CoA. As can be seen in Fig. 1A, when the amount of crude microsomes was varied in such an assay, a maximal amount of [3H]ceramide was generated, when the assay contained 33 μg of microsomal protein. At higher protein concentrations, the amount of product formed decreased. This can be explained by surface dilution, i.e. the fact that these substrates partition into the membranes and their effective concentration, expressed as mol % of membrane lipids, decreases when more membranes are added. Similar findings were also obtained by Morell and Radin (16Morell P. Radin N.S. J. Biol. Chem. 1970; 245: 342-350Abstract Full Text PDF PubMed Google Scholar). Fig. 1B shows that the reaction was linear with time for over 2 h, irrespective of the amount of [3H]DHS we added, and that the amount of [3H]ceramide formed was strictly proportional to the amount of radioactivity added. Thus, in our standard conditions (20 μm [3H]DHS, 2 h of labeling), [3H]DHS is limiting, as expected from the finding that the apparent Km value for DHS in detergent is very high (27 mol %) (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). Digitonin was the only detergent that solubilized the activity in good yield. When using solubilized membrane proteins, we obtained ∼2-fold more activity/mg of protein than with crude microsomes. This may be explained by an enrichment of the enzyme when detergentinsoluble proteins are removed by centrifugation. When digitonin-solubilized microsomal proteins were used, the amount of product made increased with the amount of membrane proteins added into the assay up to 100 μg of protein/assay (Fig. 1C). The non-linearity at higher protein concentrations was expected, as most [3H]DHS was transformed into [3H]ceramide and, thus, [3H]DHS was exhausted. Maximal ceramide synthase activity could be observed over a broad pH range extending from pH 6.5 to 9.5 (Fig. 1D). The same broad pH profile was found in membranes from YPK9 lag1Δ and YPK9 lac1Δ single mutants, in which only either Lac1p or Lag1p is operating (data not shown). Thus, the two homologues cannot be distinguished in this respect. Although EGTA and EDTA, at 10 mm, had no effect on the assay, all divalent cations except for Mg2+ were strongly inhibitory. Thus, addition of Mg2+, Ca2+, Mn2+, Cu2+, and Zn2+ at 5 mm reduced the activity to 85, 37, 31, 26, and 7% of the activity found without divalent cations. 2-Mercaptoethanol, a reducing agent, was without any effect at 7.5 mm, caused a 50% inhibition at 30 mm, and completely inhibited the synthase reaction at 60 mm. Our initial microsomal system contained ATP to maintain ionic gradients (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). However, ATP also allows for biosynthesis of acyl-CoA from a free fatty acid and CoA. By consequence, in assays where ATP is present, free CoA, generated by the ceramide synthase itself or through hydrolysis of acyl-CoA, could be used to activate other free fatty acids present in the microsomes and the acyl-CoA that finally is used to make [3H]ceramide may not be the one we add into the assay. Here we find that ATP is not required in the yeast microsomal system and that as much [3H]ceramide is made in its absence as in its presence, although massive amounts of apyrase (20 units/ml) inhibited the enzyme by 50%. (This could be the result of a nonspecific effect, but ATP also may have an unidentified indirect effect.) Microsomes from lcb4 lcb5 cells lacking LCB kinase displayed a normal incorporation of [3H]DHS into ceramides in the presence of C26-CoA, further illustrating that ATP is not required in this system (data not shown). This result is in contrast to the findings of Funato et al. (30Funato K. Lombardi R. Vallee B. Riezman H. J. Biol. Chem. 2003; 278: 7325-7334Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), who found that lcb4 lcb5 microsomes could not incorporate [3H]DHS into ceramide. We have presently no explanation for this discrepancy. Thus, the yeast ceramide synthase is similar to the mammalian synthase in that it does not require a metal cofactor, ATP, or a thiol-protecting agent (16Morell P. Radin N.S. J. Biol. Chem. 1970; 245: 342-350Abstract Full Text PDF PubMed Google Scholar). Substrate Specificity of Lag1p and Lac1p—IPCs found in S. cerevisiae almost exclusively contain PHS or DHS linked to a very long chain fatty acid, mostly C26:0 but also some C24:0, both of which can be hydroxylated (17Smith S.W. Lester R.L. J. Biol. Chem. 1974; 249: 3395-3405Abstract Full Text PDF PubMed Google Scholar, 18Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). To check whether this prevalence of very long chain fatty acids is the result of the specificity of the ceramide synthase, we tested the ability of the ceramide synthase to use acyl-CoAs of different length. It appeared that C26-CoA was the best substrate, but slightly fewer hydrophobic ceramides were also generated in the presence of C24-CoA, C20-CoA, and C18-CoA (Fig. 2A). The quantification of these bands showed that, with C24-CoA, C20-CoA, and C18-CoA, the synthase generated 77, 19, and 4% of the amount it made with C26-CoA. This is in agreement with the finding that elo3Δ cells, making mostly C22 and C20, are viable and make a normal amount of IPC (18Oh C.S. Toke D.A. Mandala S. Martin C.E. J. Biol. Chem. 1997; 272: 17376-17384Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). We further wanted to compare the fatty acid specificity of Lag1p and Lac1p. For this, the fatty acid preferences of microsomes from lag1Δ and lac1Δ single mutants were tested. After having repeated the experiment shown in Fig. 2B, we do not feel that there is any reproducible difference in the fatty acid preference between the two strains. The same specificity profile was also found in microsomes of lag1Δ lac1Δ double mutants overexpressing either Lag1p or Lac1p (data not shown). As shown in Fig. 2B, Lag1p (operative in lac1Δ) seems to have stronger activity than Lac1p (operative in lag1Δ). This is in agreement with the finding that W303 lag1Δ show a higher sensitivity to calcofluor white than W303 lac1Δ (data not shown). To determine the apparent Km values for acyl-CoA, the standard labeling assay with YPK9 wild type (wt) microsomes was realized in presence of increasing amounts of the two preferred substrates C26-CoA and C24-CoA. Optimal rates of synthesis were attained at 0.33 and 0.14 mm, corresponding to 13 and 6 mol %, respectively (Fig. 3A). It appears that the preference for C26-over C24-CoA becomes more pronounced with higher concentrations of acyl-CoA than what is used in standard assays (5 mol %). Additionally, the lower activity of the synthase with C24-CoA could not be overcome by increasing the concentration of C24-CoA, as with higher concentrations of acyl-CoA the activity reached a maximum and even dropped, possibly because of a detergent effect of acyl-CoA. A similar inhibition at higher concentrations of C24-CoA were noted by Morell and Radin (16Morell P. Radin N.S. J. Biol. Chem. 1970; 245: 342-350Abstract Full Text PDF PubMed Google Scholar). In view of this possible inhibition, Km values can only be estimated roughly but the linear increase of activity up to 13 mol % nevertheless argues that ceramide synthase has a Km value for C26-CoA, which is certainly higher than the concentration of 0.1–0.13 mm (4–6 mol %) that is used in our standard assay. According to our former studies (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar), the yeast ceramide synthase displays an apparent Km value for DHS of 27 mol % (when measured with the digitonin-solubilized enzyme). As mentioned above, the relatively high apparent Km values for DHS and acyl-CoA explain why we lost synthase activity in Fig. 1A, when high amounts of microsomes were added.Fig. 3Rate of formation of ceramide as a function of acyl-CoA concentration. 100 μg of microsomal proteins (crude microsomes) of YPK9 wt cells were incubated with 6 μCi of [3H]DHS in the presence of C26-CoA or C24-CoA at concentrations ranging from 0.014 to 1.4 mm (0.6–39 mol %). Zwittergent concentration was the same in all assays (0.89 mm). Lipids were extracted, analyzed by TLC in solvent system 1, and quantified by radioscanning. Results were plotted as percentage of [3H]DHS-C26 formed as compared with total radioactivity in the corresponding lane versus mol % of acyl-CoA of total lipids (for definition see Fig. 1 legend).View Large Image Figure ViewerDownload Hi-res image Download (PPT) LAG1Hs Induces Ceramide Synthase Activity in YPK9 —As expression of LAG1Hs (LASS1) rescues the lethality and restores the replicative life span of YPK9.2Δ cells (8Jiang J.C. Kirchman P.A. Zagulski M. Hunt J. Jazwinski S.M. Genome Res. 1998; 8: 1259-1272Crossref PubMed Scopus (28) Google Scholar), we undertook to examine the lipid profile and ceramide synthase activity of the rescued YPK9.2Δ.LAG1Hs strain. As shown in Fig. 4, LAG1Hs could restore an IPC profile to YPK9.2Δ as quantitatively and qualitatively similar as that for yeast LAG1 (lanes 2 and 5 versus lanes 3 and 6). Moreover, the cells containing LAG1Hs did not make PI′, lipids a and b, or IPC/B, lipids that are typically found in cells lacking ceramide synthase (5Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar). Indeed, YPK9.2Δ.LAG1Hs cells grow at the same rate as YPK9.2Δ.LAG1 (not shown). Thus, it would appear that LAG1Hs can replace the activity of LAG1 in ceramide synthesis, although we cannot ensure that this would also be true, if LAG1Hs were not overexpressed. When analyzing the microsomal ceramide synthase of YPK9.2Δ.LAG1Hs cells, we found that they contained an acyl-CoA-dependent activity that preferentially uses C26-CoA and C24-CoA (Fig. 5). The preference for C26-CoA over C24-CoA was even more pronounced with LAG1Hs than with yeast LAG1. As human cells do not usually contain ceramides with C26 fatty acids, we wondered whether the membrane environment of the yeast cell may have an influence on the acyl specificity of the synthase, e.g. by recruiting acyl-CoAs of a certain chain length. We argued that, if this were the case, detergent solubilization may liberate the enzyme from its lipid environment and may thus change the acyl chain specificity. Therefore, the synthase of YPK9.2Δ.LAG1Hs was tested after solubilization in digitonin. As shown in Fig. 6, Lag1Hsp again showed a very pronounced preference for C26-CoA and this suggested that Lag1Hsp has an intrinsic specificity for very long chain fatty acids. However, YPK9 microsomes showed a relatively higher activity with C18-CoA after detergent solubilization (compare Fig. 6 to Figs. 5 and 8), indicating that detergent solubilization may allow for specificity changes in certain cases.Fig. 5In vitro ceramide synthase specificity in microsomes from cells expressing LAG1Hs. Crude microsomes (100 μg of microsomal proteins/assay) from exponentially growing YPK9.2Δ.LAG1 or YPK9.2Δ.LAG1Hs cells were incubated with 20 μCi [3H]DHS in the presence of the indicated acyl-CoAs (0.1 mm) or free fatty acids (0.1 mm). The radiolabeled lipids were extracted and analyzed by TLC using solvent system 1. This experiment was done 3 times with the same result.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Effect of detergent solubilization on the substrate specificity of ceramide synthase. Fifty μg of digitonin-solubilized microsomal proteins from YPK9 or YPK9.2Δ.LAG1Hs were used in assays with 6 μCi of [3H]DHS in presence of different acyl-CoAs or corresponding free fatty acids and an ATP regenerating system. Controls are: c1 = [3H]DHS; c2 = [3H]DHS with C26-CoA in 0.3% digitonin buffer but without protein; c3 = 50 μg of solubilized microsomal proteins with [3H]DHS but without C26-CoA. All three controls were incubated and extracted as the other samples. The final concentration of digitonin in all assays was 0.3%. The experiment was done twice with the identical result.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8Overexpression of human clones can rescue YPK9 lac1Δlag1Δ. A, YPK9.2Δ.LAG1, YPK9 containing the empty pYES6/CT vector, YPK9.2Δ.CLN8, YPK9.2Δ.LASS2, YPK9.2Δ.C1, and YPK9.2Δ.C4 were plated on SGaaUA or SDaaUA containing blasticidin (50 μg/ml) and FOA at 1 mg/ml. Cells were streaked heavily and plates were photographed after 4 days at 30 °C. B, cells from plates shown in A were streaked onto SGaaA, SGaaUA, or SGaaUA containing FOA. C, colonies of strains either
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