Necessary Role for the Lag1p Motif in (Dihydro)ceramide Synthase Activity

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues. Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues. In recent years, the sphingolipid ceramide has become established as a bioactive lipid regulating cellular senescence, apoptosis, and stress responses (1Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar). It is, therefore, important to understand how enzymes that generate ceramide function work and how they are regulated. The focus of this study is (dihydro)ceramide synthase (sphingosine N-acyltransferase EC 2.3.1.24), the enzyme that utilizes long-chain bases, sphinganine or sphingosine, and fatty acid CoAs with varying chain length to produce dihydroceramide or ceramide (2Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). Depending on the source of the long-chain base substrate, (dihydro)ceramide synthase can be part of de novo synthesis or part of the recycling of ceramides. The initial biochemical characterization of (dihydro)ceramide synthase was performed in liver and brain microsomes and in mitochondria-rich fractions before the gene or genes encoding this enzyme were known (3Sribney M. Biochim. Biophys. Acta. 1966; 125: 542-547Crossref PubMed Scopus (71) Google Scholar, 4Morell P. Radin N.S. J. Biol. Chem. 1970; 245: 342-350Abstract Full Text PDF PubMed Google Scholar, 5Shimeno H. Soeda S. Sakamoto M. Kouchi T. Kowakame T. Kihara T. Lipids. 1998; 33: 601-605Crossref PubMed Scopus (85) Google Scholar). Interestingly, the first (dihydro)ceramide synthesis genes were not initially cloned as genes involved in ceramide synthesis but as UOG1 (upstream of growth and differentiation factor 1) in mammals (6Lee S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4250-4254Crossref PubMed Scopus (140) Google Scholar), Lag1 (longevity assurance gene 1) in yeast (7D'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 Asc1 (Alternaria stem cancer resistance gene 1) in tomato (8Brandwagt 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 (175) Google Scholar). New homologues of the yeast Lag1 have been described in previous studies (9Pan H. Qin W.X. Huo K.K. Wan D.F. Yu Y. Xu Z.G. Hu Q.D. Gu K.T. Zhou X.M. Jiang H.Q. Zhang P.P. Huang Y. Li Y.Y. Gu J.R. Genomics. 2001; 77: 58-64Crossref PubMed Scopus (70) Google Scholar, 10Guillas I. Jiang J.C. Vionnet C. Roubaty C. Uldry D. Chuard R. Wang J. Jazwinski S.M. Conzelmann A. J. Biol. Chem. 2003; 278: 37083-37091Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 11Venkataraman K. Futerman A.H. FEBS Lett. 2002; 528: 3-4Crossref PubMed Scopus (56) Google Scholar, 12Cai X.F. Tao Z. Yan Z.Q. Yang S.L. Gong Y. DNA Seq. 2003; 14: 79-86Crossref PubMed Scopus (13) Google Scholar, 13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar, 14Lahiri S. Futerman A.H. J. Biol. Chem. 2005; 280: 33735-33738Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the products of some of the homologues were found to be involved in ceramide synthesis (15Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (217) Google Scholar, 16Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (218) Google Scholar, 17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 18Spassieva S.D. Markham J.E. Hille J. Plant J. 2002; 32: 561-572Crossref PubMed Scopus (134) Google Scholar). Currently, this large family of membrane proteins comprises homologues in all genetically studied eukaryotes and some prokaryotes such that there are six paralogues in mice, six in humans, three in Arabidopsis, and two in baker's yeast (11Venkataraman K. Futerman A.H. FEBS Lett. 2002; 528: 3-4Crossref PubMed Scopus (56) Google Scholar). According to recently accepted nomenclature, the six paralogues in mammals are referred to as LASS (longevity assurance homologues) (9Pan H. Qin W.X. Huo K.K. Wan D.F. Yu Y. Xu Z.G. Hu Q.D. Gu K.T. Zhou X.M. Jiang H.Q. Zhang P.P. Huang Y. Li Y.Y. Gu J.R. Genomics. 2001; 77: 58-64Crossref PubMed Scopus (70) Google Scholar). Different LASS paralogues show different specificity for the chain length of the fatty acid CoA substrate; e.g. LASS1/UOG1 utilize C18 CoA (17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), and LASS5 prefers C16 CoA (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar, 19Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Two recent publications provide evidence suggesting (dihydro)ceramide synthase might operate differently in different organisms. In yeast, there is an additional subunit necessary for the reaction, Lip1p (Lag1/Lac1 interaction protein) (20Vallee B. Riezman H. EMBO J. 2005; 24: 730-741Crossref PubMed Scopus (116) Google Scholar). Lahiri and Futerman (14Lahiri S. Futerman A.H. J. Biol. Chem. 2005; 280: 33735-33738Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) provide evidence in mammals for the LASS5 homologue as a bona fide (dihydro)ceramide synthase. All members of the LASS/Lag family of proteins share similar transmembrane profiles of four to seven predicted transmembrane domains (11Venkataraman K. Futerman A.H. FEBS Lett. 2002; 528: 3-4Crossref PubMed Scopus (56) Google Scholar). Membrane localization of some of the members, including yeast Lac1p (longevity assurance gene cognate 1) (21Barz W.P. Walter P. Mol. Biol. Cell. 1999; 10: 1043-1059Crossref PubMed Scopus (96) Google Scholar) and mammalian LASS1, LASS4, LASS5, and LASS6 (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar, 17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 19Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), is established to be in the endoplasmic reticulum. The LASS/Lag proteins share similar transmembrane profiles with a larger group of proteins, such as translocating chain-associating membrane (TRAM) 2The abbreviations used are: TRAM, translocating chain-associating membrane; DHS, dihydrosphingosine. 2The abbreviations used are: TRAM, translocating chain-associating membrane; DHS, dihydrosphingosine. protein, and a protein mutated in neuronal ceroid lipofuscinoses, CLN8 (22Winter E. Ponting C.P. Trends Biochem. Sci. 2002; 27: 381-383Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This shared is known as the stretch of 52 amino acids initially described in the work of Jiang J.C. Kirchman P.A. M. J. Jazwinski S.M. 1998; PubMed Scopus Google Scholar) and shared by LASS/Lag homologues This amino acid motif is known as the Lag1p but significance for (dihydro)ceramide synthase is currently unknown. In this work, we to the of this Lag1p motif in (dihydro)ceramide synthase this eight point mutations were in the Lag1p motif of the mouse LASS1 gene and four in the human LASS5 and the proteins were used to study enzyme Six of the mutations the activity of LASS1 in cells and in vitro as as two of the mutations in the human LASS5, suggesting that the Lag1p motif is necessary for (dihydro)ceramide synthase activity of the LASS family and cells were in and with and and The growth and are are is LASS1, the was mouse LASS1 by The for the LASS1 was a a with mouse The was cloned and the the was used to the LASS1 of the conserved amino acids in the Lag1p motif of the mouse LASS1 gene were was with the the The LASS1 was used as a and the used to generate point mutations are described in The mutations were by DNA not LASS5, a human LASS5 was and the of the The of the was The of the human LASS5 was by to a The of LASS5 was with and and used to the of LASS5. mutations in the Lag1p motif of LASS5 were by as described for LASS1 with shown in used in for Lag1p motif mutants of mouse in a used in for Lag1p motif mutants of human in a of HeLa and of cells were with LASS1 or LASS5, Lag1p motif and using to the were and in with an were for to cells and The were for to the were for 1 to were in and protein were using the were on a and to a membrane using J. T. Scholar). proteins were with as a and as a and with the In of microsomes were used as an enzyme source for the in vitro ceramide synthase and or C16 fatty acid CoA in was for The enzyme was by microsomes to the the was for and the was by of with an for were two using of a of and of 1 in in and by of the lipid was with a and of P.P. Biochim. Biophys. Acta. PubMed Scopus (137) Google Scholar) and was used to the of the of and ceramide were performed on a in a using a of the J. A. PubMed Scopus Google Scholar). to cells were with the sphingosine, and with an and in of 1 in in the were on the and the with a to the and were and using the was on the by an with the known of the and an of the The were the The the were to the and with the using a in the Lag1p of LASS1 in HeLa the functional significance of the Lag1p we eight conserved amino acids the motif and eight point mutants in an mouse LASS1 as in The substitutions were to the of the amino acid It has been shown that of LASS1 results in an of C18 ceramide (17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). the Lag1p motif mutants were in HeLa cells and tested for to cellular C18 the cells were and to lipid by and show as with of LASS1 cellular of C18 and by Interestingly, six of eight Lag1p motif in HeLa showed in cellular of C18 and suggesting that mutations the function of However, the two mutants and were to cellular C18 and ceramide to a similar or Moreover, of the ceramide were by LASS1 or by of the eight mutants that LASS1 is a C18 ceramide synthase and that the mutations not fatty acid is that the for LASS1 and all of the mutants was not as by of protein all of the cells in the Lag1p of LASS1 in we the of the C18 and ceramide in the cells LASS1 or Lag1p motif mutants to an of (dihydro)ceramide synthase activity in vitro. used the that were by for cellular ceramide to microsomes and used microsomes as an enzyme source in an in vitro The in vitro (dihydro)ceramide synthase was performed using a and highly sensitive This utilizes DHS, a as a for the (dihydro)ceramide synthase reaction, is to a can be and by of in vitro ceramide synthase activity this with as a long-chain base and C18 fatty acid CoAs as a fatty acid CoA substrate, was that microsomes cells LASS1 and the mutants and with were the an of in vitro (dihydro)ceramide synthase The six mutants that not show ceramide in cells not show a in activity as with the in vitro results with C18 fatty acid CoA as a are in with the the in and the that the Lag1p motif is important for the (dihydro)ceramide synthase activity of CoAs in for LASS1 an in cellular of ceramide as as C18 the in vitro (dihydro)ceramide synthase activity was performed using that microsomes cells LASS1, with microsomes showed of in vitro ceramide synthase Moreover, the mutants and to show an of in vitro (dihydro)ceramide synthase activity with as a not the used to ceramide in cells not an additional CoA was CoA was used as a for the in vitro ceramide synthase activity microsomes cells LASS1 to show an of activity as with the that LASS1 not utilize cis-C18:1 fatty acid CoAs as substrates in vitro. LASS1 a is a known inhibitor of (dihydro)ceramide synthase in vitro K. Sullards M.C. Allegood J. Wang E. M. Merrill Jr., A.H. Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar). However, in human cells LASS1, fumonisin was shown to cellular of C18 suggesting that LASS1 is fumonisin (17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). was to mutations in the Lag1p motif the fumonisin effect on LASS1 in LASS1, the eight and the were in HeLa the cells were for an additional with fumonisin of ceramide in fumonisin and cells are shown in Interestingly, was found that fumonisin not the of in cells LASS1 in ceramide with This that LASS1 is sensitive to fumonisin in HeLa fumonisin the of C16 ceramide the of all ceramide not can be in fumonisin had similar on ceramide for all of the Lag1p motif that of the Lag1p motif not the fumonisin of The Lag1p for of LASS eight amino acids mutated in the Lag1p motif of LASS1 are conserved in all of the LASS proteins, but different LASS proteins have different fatty acid CoA specificity (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar), and therefore, is that they not the Lag1p motif for the Lag1p motif is for the ceramide synthase activity of LASS family proteins, the Lag1p motif of a different LASS homologue was this four point mutations were in the Lag1p motif of the human LASS5 of the and were the same substitutions as in the Lag1p motif of LASS1 and to the effect of mutations on the activity of LASS1 and LASS5. The point is the same amino acid as but is a is that is this amino acid of the Lag1p motif of another LASS/Lag homologue, the tomato HeLa cells were with LASS5, LASS5 Lag1p motif and the cells were and used for the of the microsomes were used as an enzyme source in an in vitro (dihydro)ceramide synthase with as the long-chain base and C16 fatty acid CoA as the fatty acid CoA The C16 fatty acid CoA is of the fatty acid CoA substrates described for LASS5 in vitro (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar, 19Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The were performed the same as the described for LASS1 and that of the LASS5 the in vitro (dihydro)ceramide synthase activity with C16 fatty acid CoA as a Moreover, the and amino acid substitutions the in vitro (dihydro)ceramide synthase activity of human LASS5, the and mutations not the ceramide synthase activity of human LASS5. The and mutations had the same effect on the in vitro activity of human LASS5 as the and mutations had on the in vitro activity of mouse LASS1 and that the Lag1p motif is necessary for the (dihydro)ceramide synthase activity of LASS homologues and suggesting that the Lag1p motif is not a conserved of LASS/Lag proteins but is for The that the showed similar in vitro activity as the LASS5, the was that conserved mutations are this of the Lag1p motif and the of the motif for ceramide synthase In addition, we investigated the effect of the of the LASS5 and Lag1p motif mutants on cellular ceramide in the and of fumonisin HeLa cells were with LASS5, the Lag1p motif point or an of the were with fumonisin The fumonisin the and was performed for another All of the were the and to and in to the effect of LASS5 on the in vitro activity of microsomes the of LASS5 not the of C16 ceramide in cells The same was for the and an in in vitro In addition, ceramide were not by the of the LASS5 or the Lag1p motif fatty acid CoA was shown to be of the substrates for LASS5 in vitro (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar). and the ceramide not were not by the of LASS5 or the Lag1p motif point the of ceramide in cells and the in vitro (dihydro)ceramide synthase activity of the LASS5 or the Lag1p motif mutants in HeLa de novo synthesis of C16 ceramide is to be regulated. Moreover, fumonisin the of all ceramide in LASS5, as as in the Lag1p motif cells in HeLa LASS5 to is fumonisin The study the functional significance of the Lag1p motif of LASS The Lag1p motif has been as a stretch of amino acids that is conserved LASS/Lag homologues J.C. Kirchman P.A. M. J. Jazwinski S.M. 1998; PubMed Scopus Google Scholar), but there were for functional have shown that six of eight mutated conserved amino acids in the Lag1p motif to be essential to the function of LASS1, to loss of enzyme activity in cells and in vitro. The two mutants and not the Moreover, three amino acid point mutations in the Lag1p motif of the human LASS5, the same as the mutations in LASS1, had the same effect on the activity of LASS5 as they had on In addition, our results show that LASS1 a cis-C18:1 fatty acid CoA as a and that LASS1, LASS5, as as the mutants are sensitive to fumonisin in HeLa results show for the first that the Lag1p motif is for (dihydro)ceramide synthase activity of LASS The that the Lag1p motif is important is for all LASS/Lag proteins, they share the same conserved amino acids in the motif. The loss of function of the Lag1p motif mutants in LASS/Lag homologues different this In our mutations of or in the Lag1p motif of mouse LASS1 or human LASS5 had a effect on the enzyme activity of In of our a recent study by and Riezman Riezman H. Biochem. J. PubMed Scopus Google Scholar) that yeast Lag1 with mutated or in Lag1p motif to the of a In the same yeast the Lag1p motif mutations not the membrane of Interestingly, LASS/Lag proteins can in Lag1p showed that mouse LASS1 enzyme activity was to and and Riezman Riezman H. Biochem. J. PubMed Scopus Google Scholar) show that yeast Lag1 is to the the the same is to Moreover, conserved in the Lag1p motif of the human LASS5 was this was mutated to LASS5 on the LASS5 activity is that is not conserved LASS/Lag homologues The homologue has that and the tomato homologue evidence that Lag1p motif is with (dihydro)ceramide synthesis the of the of LASS/Lag proteins and and CLN8 not contain the Lag1p motif and have not been shown to be involved in ceramide synthesis. The function of is is a of the and is for the endoplasmic or in the membrane of or proteins D. E. S. PubMed Scopus Google Scholar, D. Cell. Full Text PDF PubMed Scopus Google Scholar). The function of CLN8 is but in a the mammalian CLN8 was not to a yeast yeast Lag homologues Lag1 and suggesting that CLN8 is not involved in ceramide synthesis I. Jiang J.C. Vionnet C. Roubaty C. Uldry D. Chuard R. Wang J. Jazwinski S.M. Conzelmann A. J. Biol. Chem. 2003; 278: 37083-37091Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). the Lag1p motif is important for the function of all LASS/Lag homologues, is not to be involved in of the fatty acid different LASS/Lag homologues utilize fatty acid CoAs with different chain (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar, 17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 19Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In of this are our showed that of the mutations in the motif of LASS1 the cellular of of the ceramide C18 and suggesting that the Lag1p motif is not involved in the fatty acid CoA specificity of to the that the cellular of the C18 and as a of of the in vitro showed that C18 (but not fatty acid CoAs were substrates for (dihydro)ceramide synthase activity of microsomes cells similar for the in vitro fatty acid CoA specificity of LASS1 was in a recent study of (13Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar). show that CoA is not a for In our in vitro were performed for of the of fatty acid and and was a for we not all of the fatty acid CoA as substrates for LASS1 in vitro. substrates used in the were and the we used not and ceramides. is that a CoA can be a for LASS1, or the ceramide that the of C18 ceramide in cells is a of of the fatty acid the synthesis of This that of the C18 fatty acid is by the of ceramide synthesis. is that of LASS1 and LASS5 in cells had different on C18 and C16 and of LASS1 resulted in an of C18 and ceramide with the the of LASS5 to a of C16 or ceramide In HeLa the of C16 ceramide was the of C18 ceramide and the same of in C16 or C18 ceramide have a different on the of the ceramide It is important to in our the (dihydro)ceramide synthase activity with C16 fatty acid CoA as was and with the that C16 ceramide is of the ceramide in HeLa in HeLa the C16 ceramide is and that the of C16 ceramide not be by synthase but by some of the enzymes of the sphingolipid important point to the that fumonisin the of C18 ceramide in cells in to results (17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) suggesting that LASS1 is a fumonisin (dihydro)ceramide synthase. Moreover, in this the of C16 ceramide of HeLa cells LASS5 and with fumonisin showed with the is in to a previous C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) C16 ceramide as a of LASS5 and fumonisin The the two of in the of LASS1 and LASS5 is the of In our we HeLa cells fumonisin as an the two studies (17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 19Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) used human cells fumonisin C18 or C16 ceramide The fumonisin and the of the were the It is that a different of is the the LASS1 and LASS5 are fumonisin (dihydro)ceramide as shown in our of cellular and and in the in vitro results of the study by (17Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), but is that fumonisin ceramide synthesis in a the of a such as human for and the the of for lipid