Molecular Cloning of cDNA Encoding Rat Very Long-chain Acyl-CoA Synthetase

Abstract

The cDNA encoding rat very long-chain acyl-CoA synthetase (VLACS) was cloned, using degenerative primers synthesized according to the partial amino acid sequences of the peptide fragments of the purified rat liver enzyme. The longest cDNA insert was 2972 base pairs with a 1860-base pair open reading frame encoding 620 amino acids. The calculated molecular mass of 70,692 daltons was consistent with size of the purified enzyme. In Northern blot analysis, a single band was detected at the position of about 3 kilobases, corresponding to the size of the cloned cDNA. cDNA-directed expression in Escherichia coli resulted in accumulation of expressed protein, as an inclusion body. An antibody was raised using this expressed protein to characterize the cDNA and the enzyme. The subcellular localization of VLACS in peroxisomes and microsomes was demonstrated in Western blot analysis. The specific activity and the substrate specificity of the cDNA expressed enzyme in COS-1 cells were consistent with those of the purified rat enzyme. The predicted amino acid sequence of VLACS had a high sequence similarity to fatty acid transport protein (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436), and was considered to have domains for adenylation and thioester formation. The entire structure of VLACS was dissimilar to that of long-chain acyl-CoA synthetase (Suzuki, H., Kawarabayashi, Y., Kondo, Y., Abe, T., Nishikawa, K., Kimura, S., Hashimoto, T., and Yamamoto, T. (1990) J. Biol. Chem. 265, 8681-8685), except for the domains. The cDNA encoding rat very long-chain acyl-CoA synthetase (VLACS) was cloned, using degenerative primers synthesized according to the partial amino acid sequences of the peptide fragments of the purified rat liver enzyme. The longest cDNA insert was 2972 base pairs with a 1860-base pair open reading frame encoding 620 amino acids. The calculated molecular mass of 70,692 daltons was consistent with size of the purified enzyme. In Northern blot analysis, a single band was detected at the position of about 3 kilobases, corresponding to the size of the cloned cDNA. cDNA-directed expression in Escherichia coli resulted in accumulation of expressed protein, as an inclusion body. An antibody was raised using this expressed protein to characterize the cDNA and the enzyme. The subcellular localization of VLACS in peroxisomes and microsomes was demonstrated in Western blot analysis. The specific activity and the substrate specificity of the cDNA expressed enzyme in COS-1 cells were consistent with those of the purified rat enzyme. The predicted amino acid sequence of VLACS had a high sequence similarity to fatty acid transport protein (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436), and was considered to have domains for adenylation and thioester formation. The entire structure of VLACS was dissimilar to that of long-chain acyl-CoA synthetase (Suzuki, H., Kawarabayashi, Y., Kondo, Y., Abe, T., Nishikawa, K., Kimura, S., Hashimoto, T., and Yamamoto, T. (1990) J. Biol. Chem. 265, 8681-8685), except for the domains. INTRODUCTIONIn mammals, very long-chain fatty acids (VLCFAs) 1The abbreviations used are: VLCFAsvery long-chain fatty acidsVLACSvery long-chain fatty acyl-CoA synthetaseX-ALDX-linked adrenoleukodystrophyLACSlong-chain acyl-CoA synthetaseALDPadrenoleukodystrophy proteinDEHPdi(2-ethylhexyl)phthalatePCRpolymerase chain reactionVLACS-Hisfusion protein of VLACS and histidine tagIPTGisopropyl-thio-β-DgalactopyranosidePAGEpolyacrylamide gel electrophoresisFATPfatty acid transport proteinbpbase pair(s)kbkilobase pair(s). containing more than 22 carbon atoms are oxidized mainly in peroxisomes but not in mitochondria (1Singh I. Moser A.B. Goldfischer S. Moser H.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4203-4207Crossref PubMed Scopus (324) Google Scholar, 2Singh H. Derwas N. Poulos A. Arch. Biochem. Biophys. 1987; 259: 382-390Crossref PubMed Scopus (76) Google Scholar). Activation of VLCFAs to their CoA thioesters is apparently a crucial step and this step is catalyzed by very long-chain acyl-CoA synthetase (VLACS). VLACS activity localizes in peroxisomes and microsomes but not in mitochondria (3Singh H. Poulos A. Arch. Biochem. Biophys. 1988; 266: 486-495Crossref PubMed Scopus (60) Google Scholar).X-linked adrenoleukodystrophy (X-ALD) is the most common hereditary disease among the peroxisomal diseases and is characterized by progressive demyelination of the white matter and by adrenocortical insufficiency (4Moser H.W. Moser A.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1989: 1511-1532Google Scholar, 5Moser H.W. Moser A.B. Smith K.D. Bergin A. Borel J. Shankroff J. Stine O.C. Merette C. Ott J. Kirvert W. Shapiro E. J. Inherited Metab. Dis. 1992; 15: 645-664Crossref PubMed Scopus (166) Google Scholar). A marked biochemical deterioration in subjects with this disease is a defect of oxidation of VLCFAs. Since fibroblasts from these patients showed a very low activity in formation of very long-chain fatty acyl-CoAs, it has been considered that a new synthetase, VLACS being different from long-chain acyl-CoA synthetase (6Suzuki H. Kawarabayashi Y. Kondo J. Abe T. Nishikawa K. Kimura S. Hashimoto T. Yamamoto T. J. Biol. Chem. 1990; 265: 8681-8685Abstract Full Text PDF PubMed Google Scholar) must be present, and that the phenotypic etiology of X-ALD may be derived from a defect of this enzyme (7Hashmi M. Stanley W. Singh I. FFBS Lett. 1986; 196: 247-250Crossref PubMed Scopus (145) Google Scholar, 8Lazo O. Contreras M. Hashmi M. Stanley W. Irazu C. Singh I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7647-7651Crossref PubMed Scopus (217) Google Scholar, 9Wanders R.J.A. van Roermund C.W.T. van Wijland M.J.A. Schutgens R.B.H. Schram A.W. Tager J.M. van den Bosch H. Schalkwijk C. J. Inherited Metab. Dis. 1988; 11: 173-177Crossref PubMed Scopus (30) Google Scholar). The adrenoleukodystrophy protein (ALDP) gene has been cloned, and it was thought to be a disease-causing candidate for X-ALD (10Mosser J. Dauar A.-M. Sarde C.O. Kioschis P. Fejil R. Moser H.W. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (988) Google Scholar). Various point mutations, deletions, and insertions in the gene have been identified in these patients (11Cartier N. Sarde C.O. Douar A.M. Mosser J. Mandel J.L. Aubourg P. Hum. Mol. Genet. 1993; 2: 1949-1951Crossref PubMed Scopus (57) Google Scholar, 12Uchiyama A. Suzuki Y. Song X.Q. Fukao T. Imamura A. Tomatsu S. Shimozawa N. Kondo N. Orii T. Biochem. Biophys. Res. Commun. 1994; 198: 632-636Crossref PubMed Scopus (39) Google Scholar, 13Kemp S. Ligtenberg M.J.L. van Geel B.M. Barth P.G. Wolterman R.A. Schoute F. Sarde C.O. Mandel J.L. van Oost B.A. Bolhuis P.A. Biochem. Biophys. Res. Commun. 1994; 202: 647-653Crossref PubMed Scopus (69) Google Scholar, 14Barcelo A. Giros M. Sarde C.O. Martinez B.A. Mandel J.L. Pampols T. Estivill X. Hum. Mol. Genet. 1994; 3: 1889-1890Crossref PubMed Scopus (27) Google Scholar, 15Fuchs S. Sarde C.O. Wedemann H. Schwinger E. Mandel J.L. Gal A. Hum. Mol. Genet. 1994; 3: 1903-1905Crossref PubMed Scopus (55) Google Scholar, 16Bergar J. Molzer B. Fae I. Brenhaimer H. Biochem. Biophys. Res. Commun. 1994; 205: 1638-1643Crossref PubMed Scopus (98) Google Scholar, 17Fanen P. Guidoux S. Sarde C.O. Mandel J.L. Goossens M. Aubourg P. J. Clin. Invest. 1994; 94: 516-520Crossref PubMed Scopus (76) Google Scholar, 18Barcelo A. Giros M. Sarde C.O. Pintos G. Mandel J.L. Pampols T. Estivill X. Hum. Genet. 1995; 95: 235-237Crossref PubMed Scopus (12) Google Scholar, 19Ligtenberg M.J.L. Kemp S. Sarde C.O. van Geel B.M. Kleijer W.J. Barth P.G. Mandel J.L. van Oost B.A. Bolhuis P.A. Am. J. Hum. Genet. 1995; 56: 44-50PubMed Google Scholar, 20Braun A. Ambach H. Kammerer S. Rolinski B. Stockler S. Rabl W. Gartner J. Zierz S. Roscher A.A. Am. J. Hum. Genet. 1995; 56: 854-861PubMed Google Scholar, 21Song X.Q. Fukao T. Suzuki Y. Imamura A. Uchiyama A. Shimozawa N. Kondo N. Orii T. Hum. Mol. Genet. 1995; 4: 1093-1094Crossref PubMed Scopus (12) Google Scholar). Retroviral transfer of normal ALDP cDNA into patient's fibroblasts enhanced VLCFA oxidation activity to the level found in control fibroblasts (22Cartier N. Lopez J. Moullier P. Rocchiccioli F. Rolland M.O. Jorge P. Mosser J. Mandel J.L. Bougneres P.F. Danos O. Aubourg P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1674-1678Crossref PubMed Scopus (100) Google Scholar). These data indicate that ALDP is responsible for at least a part of X-ALD. The function of ALDP and the relation between ALDP and VLCFA oxidation activity are unknown. It is thus important to examine the exact localization in cells and the role of VLACS, catalyzing a crucial reaction in peroxisomal VLCFA oxidation, and the relation between ALDP and VLACS, including possible participation of 70-kDa peroxisomal membrane protein (23Kamijo K. Taketani S. Yokota S. Osumi T. Hashimoto T. J. Biol. Chem. 1990; 265: 4534-4540Abstract Full Text PDF PubMed Google Scholar), assigned as one of the ATP-binding cassette transporters (10Mosser J. Dauar A.-M. Sarde C.O. Kioschis P. Fejil R. Moser H.W. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (988) Google Scholar, 23Kamijo K. Taketani S. Yokota S. Osumi T. Hashimoto T. J. Biol. Chem. 1990; 265: 4534-4540Abstract Full Text PDF PubMed Google Scholar).VLACS was recently purified from rat liver peroxisomal membrane (24Uchida Y. Orii T. Kondo N. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar). Although our attempts to prepare a specific antibody were unsuccessful, the purified VLACS did provide partial amino acid sequence data of protease-digested polypeptides, and this made it feasible to begin the cloning of VLACS cDNA. We report here the isolation and sequencing of cDNA encoding rat VLACS, a comparison of the sequence to those of the related enzymes, the subcellular localization, and the biosynthesis of functional VLACS in COS-1 cells.DISCUSSIONA full-length VLACS cDNA clone was obtained from a rat λgt 11 cDNA library, using degenerative primers designed based on partial amino acid sequences of peptide fragments from rat liver VLACS. This clone was confirmed to encode VLACS, sequence analysis revealed the existence of the regions corresponding to the peptide fragments from the purified VLACS, the calculated molecular mass of the coding region corresponded to that of the purified VLACS, the size of mRNA in Northern blot analysis was similar to that of the full-length clone, the DEHP-induction motif of the mRNA was similar to that of VLACS activity in rat liver (24Uchida Y. Orii T. Kondo N. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar), the antibody raised against cDNA-expressed protein in E. coli specifically cross-reacted with the purified VLACS, and the specific activities and the substrate specificity of the cDNA-expressed protein in COS-1 cells were similar to those of the purified VLACS, respectively.On considering subcellular localization of acyl-CoA synthases, distributions of LACS and VLACS seem to be important. LACS was found to localize in peroxisomes, mitochondrial outer membranes, and microsomes (39Miyazawa S. Hashimoto T. Yokota S. J. Biochem. (Tokyo). 1985; 98: 723-733Crossref PubMed Scopus (120) Google Scholar). VLACS was estimated to be present in peroxisomes, but absent in mitochondria due to subcellular distribution of lignoceroyl-CoA synthetase activity (24Uchida Y. Orii T. Kondo N. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar). In the present study, VLACS was detected only in peroxisomes and microsomes (Fig. 6), and the calculated specific lignoceroyl-CoA synthetase activities in peroxisomal and microsomal fractions were much the same value of the purified VLACS, which is compatible with previous observations (24Uchida Y. Orii T. Kondo N. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar).VLACS has been considered a peroxisomal membrane protein (24Uchida Y. Orii T. Kondo N. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar), as well as 70-kDa peroxisomal membrane protein and ALDP, therefore, VLACS is expected to have a targeting signal into peroxisomes. In mammalian peroxisomal proteins, peroxisomal targeting signals 1 and 2 were identified (37Miura S. Kasuya-Arai I. Mori H. Miyazawa S. Osumi T. Hashimoto T. Fujiki Y. J. Biol. Chem. 1992; 267: 14405-14411Abstract Full Text PDF PubMed Google Scholar, 38Osumi T. Tsukamoto T. Hata S. Yokota S. Miura S. Fujiki Y. Hijikata M. Miyazawa S. Hashimoto T. Biochem. Biophys. Res. Commun. 1991; 181: 947-954Crossref PubMed Scopus (230) Google Scholar) and they seem to be specific to peroxisomal matrix protein. VLACS, as well as 70-kDa peroxisomal membrane protein and ALDP, did not have these signals, hence a hitherto unknown targeting signal(s) specific to peroxisomal membrane proteins may exist. In hydropathy analysis (45Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16999) Google Scholar), VLACS has no typical transmembrane region (data not shown), unlike 70-kDa peroxisomal membrane protein and ALDP. These results can serve as a good base for future projects such as a topological study of VLACS on peroxisomal membrane proteins and interaction of VLACS with ALDP and other peroxisomal membrane proteins, and this will be important to understand the mechanism related to deficiency of the peroxisomal VLACS activity in X-ALD fibroblasts (8Lazo O. Contreras M. Hashmi M. Stanley W. Irazu C. Singh I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7647-7651Crossref PubMed Scopus (217) Google Scholar). VLACS cDNA and the anti-VLACS antibody will be good tools for such studies.VLACS, LACS, and FATP seem to belong to the firefly luciferase family as there are two specific domains. Interestingly, VLACS has high sequence similarity to FATP (Fig. 8). However, there was no sequence homology in the NH2-terminal region between VLACS and FATP (1-41 amino acids in VLACS and 1-69 amino acids in FATP, Fig. 8). The NH2-terminal sequence of FATP (Fig. 8, underlined) is thought to encode a signal sequence to cytoplasmic membranes. The poor homology seems to reflect the difference concerning subcellular localization of the two proteins.Proteins in the firefly luciferase family generally show a high sequence divergence in their entire region, except for the two conserved domains, if they have different functions (42Toh H. Protein Seq. Data Anal. 1991; 4: 111-117PubMed Google Scholar). Since VLACS and FATP have different functions, it is the first case in the family that a high similarity through broad regions was observed between the two proteins with different functions. An additionally isolated clone with FATP cDNA was shown to encode mouse LACS. Interestingly, this LACS was found to have function not only as activation of fatty acids but also as active uptake of long-chain fatty acids (41Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (736) Google Scholar). Attention needs to be given to whether VLACS also functions as VLCFAs transporter. INTRODUCTIONIn mammals, very long-chain fatty acids (VLCFAs) 1The abbreviations used are: VLCFAsvery long-chain fatty acidsVLACSvery long-chain fatty acyl-CoA synthetaseX-ALDX-linked adrenoleukodystrophyLACSlong-chain acyl-CoA synthetaseALDPadrenoleukodystrophy proteinDEHPdi(2-ethylhexyl)phthalatePCRpolymerase chain reactionVLACS-Hisfusion protein of VLACS and histidine tagIPTGisopropyl-thio-β-DgalactopyranosidePAGEpolyacrylamide gel electrophoresisFATPfatty acid transport proteinbpbase pair(s)kbkilobase pair(s). containing more than 22 carbon atoms are oxidized mainly in peroxisomes but not in mitochondria (1Singh I. Moser A.B. Goldfischer S. Moser H.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4203-4207Crossref PubMed Scopus (324) Google Scholar, 2Singh H. Derwas N. Poulos A. Arch. Biochem. Biophys. 1987; 259: 382-390Crossref PubMed Scopus (76) Google Scholar). Activation of VLCFAs to their CoA thioesters is apparently a crucial step and this step is catalyzed by very long-chain acyl-CoA synthetase (VLACS). VLACS activity localizes in peroxisomes and microsomes but not in mitochondria (3Singh H. Poulos A. Arch. Biochem. Biophys. 1988; 266: 486-495Crossref PubMed Scopus (60) Google Scholar).X-linked adrenoleukodystrophy (X-ALD) is the most common hereditary disease among the peroxisomal diseases and is characterized by progressive demyelination of the white matter and by adrenocortical insufficiency (4Moser H.W. Moser A.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1989: 1511-1532Google Scholar, 5Moser H.W. Moser A.B. Smith K.D. Bergin A. Borel J. Shankroff J. Stine O.C. Merette C. Ott J. Kirvert W. Shapiro E. J. Inherited Metab. Dis. 1992; 15: 645-664Crossref PubMed Scopus (166) Google Scholar). A marked biochemical deterioration in subjects with this disease is a defect of oxidation of VLCFAs. Since fibroblasts from these patients showed a very low activity in formation of very long-chain fatty acyl-CoAs, it has been considered that a new synthetase, VLACS being different from long-chain acyl-CoA synthetase (6Suzuki H. Kawarabayashi Y. Kondo J. Abe T. Nishikawa K. Kimura S. Hashimoto T. Yamamoto T. J. Biol. Chem. 1990; 265: 8681-8685Abstract Full Text PDF PubMed Google Scholar) must be present, and that the phenotypic etiology of X-ALD may be derived from a defect of this enzyme (7Hashmi M. Stanley W. Singh I. FFBS Lett. 1986; 196: 247-250Crossref PubMed Scopus (145) Google Scholar, 8Lazo O. Contreras M. Hashmi M. Stanley W. Irazu C. Singh I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7647-7651Crossref PubMed Scopus (217) Google Scholar, 9Wanders R.J.A. van Roermund C.W.T. van Wijland M.J.A. Schutgens R.B.H. Schram A.W. Tager J.M. van den Bosch H. Schalkwijk C. J. Inherited Metab. Dis. 1988; 11: 173-177Crossref PubMed Scopus (30) Google Scholar). The adrenoleukodystrophy protein (ALDP) gene has been cloned, and it was thought to be a disease-causing candidate for X-ALD (10Mosser J. Dauar A.-M. Sarde C.O. Kioschis P. Fejil R. Moser H.W. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (988) Google Scholar). Various point mutations, deletions, and insertions in the gene have been identified in these patients (11Cartier N. Sarde C.O. Douar A.M. Mosser J. Mandel J.L. Aubourg P. Hum. Mol. Genet. 1993; 2: 1949-1951Crossref PubMed Scopus (57) Google Scholar, 12Uchiyama A. Suzuki Y. Song X.Q. Fukao T. Imamura A. Tomatsu S. Shimozawa N. Kondo N. Orii T. Biochem. Biophys. Res. Commun. 1994; 198: 632-636Crossref PubMed Scopus (39) Google Scholar, 13Kemp S. Ligtenberg M.J.L. van Geel B.M. Barth P.G. Wolterman R.A. Schoute F. Sarde C.O. Mandel J.L. van Oost B.A. Bolhuis P.A. Biochem. Biophys. Res. Commun. 1994; 202: 647-653Crossref PubMed Scopus (69) Google Scholar, 14Barcelo A. Giros M. Sarde C.O. Martinez B.A. Mandel J.L. Pampols T. Estivill X. Hum. Mol. Genet. 1994; 3: 1889-1890Crossref PubMed Scopus (27) Google Scholar, 15Fuchs S. Sarde C.O. Wedemann H. Schwinger E. Mandel J.L. Gal A. Hum. Mol. Genet. 1994; 3: 1903-1905Crossref PubMed Scopus (55) Google Scholar, 16Bergar J. Molzer B. Fae I. Brenhaimer H. Biochem. Biophys. Res. Commun. 1994; 205: 1638-1643Crossref PubMed Scopus (98) Google Scholar, 17Fanen P. Guidoux S. Sarde C.O. Mandel J.L. Goossens M. Aubourg P. J. Clin. Invest. 1994; 94: 516-520Crossref PubMed Scopus (76) Google Scholar, 18Barcelo A. Giros M. Sarde C.O. Pintos G. Mandel J.L. Pampols T. Estivill X. Hum. Genet. 1995; 95: 235-237Crossref PubMed Scopus (12) Google Scholar, 19Ligtenberg M.J.L. Kemp S. Sarde C.O. van Geel B.M. Kleijer W.J. Barth P.G. Mandel J.L. van Oost B.A. Bolhuis P.A. Am. J. Hum. Genet. 1995; 56: 44-50PubMed Google Scholar, 20Braun A. Ambach H. Kammerer S. Rolinski B. Stockler S. Rabl W. Gartner J. Zierz S. Roscher A.A. Am. J. Hum. Genet. 1995; 56: 854-861PubMed Google Scholar, 21Song X.Q. Fukao T. Suzuki Y. Imamura A. Uchiyama A. Shimozawa N. Kondo N. Orii T. Hum. Mol. Genet. 1995; 4: 1093-1094Crossref PubMed Scopus (12) Google Scholar). Retroviral transfer of normal ALDP cDNA into patient's fibroblasts enhanced VLCFA oxidation activity to the level found in control fibroblasts (22Cartier N. Lopez J. Moullier P. Rocchiccioli F. Rolland M.O. Jorge P. Mosser J. Mandel J.L. Bougneres P.F. Danos O. Aubourg P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1674-1678Crossref PubMed Scopus (100) Google Scholar). These data indicate that ALDP is responsible for at least a part of X-ALD. The function of ALDP and the relation between ALDP and VLCFA oxidation activity are unknown. It is thus important to examine the exact localization in cells and the role of VLACS, catalyzing a crucial reaction in peroxisomal VLCFA oxidation, and the relation between ALDP and VLACS, including possible participation of 70-kDa peroxisomal membrane protein (23Kamijo K. Taketani S. Yokota S. Osumi T. Hashimoto T. J. Biol. Chem. 1990; 265: 4534-4540Abstract Full Text PDF PubMed Google Scholar), assigned as one of the ATP-binding cassette transporters (10Mosser J. Dauar A.-M. Sarde C.O. Kioschis P. Fejil R. Moser H.W. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (988) Google Scholar, 23Kamijo K. Taketani S. Yokota S. Osumi T. Hashimoto T. J. Biol. Chem. 1990; 265: 4534-4540Abstract Full Text PDF PubMed Google Scholar).VLACS was recently purified from rat liver peroxisomal membrane (24Uchida Y. Orii T. Kondo N. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar). Although our attempts to prepare a specific antibody were unsuccessful, the purified VLACS did provide partial amino acid sequence data of protease-digested polypeptides, and this made it feasible to begin the cloning of VLACS cDNA. We report here the isolation and sequencing of cDNA encoding rat VLACS, a comparison of the sequence to those of the related enzymes, the subcellular localization, and the biosynthesis of functional VLACS in COS-1 cells.