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• W2000961724 abstract "Activation of fatty acids to their coenzyme A derivatives is necessary for subsequent metabolism. Very long-chain fatty acids, which accumulate in tissues of patients with X-linked adrenoleukodystrophy, are activated by very long-chain acyl-CoA synthetase (VLCS) normally found in peroxisomes and microsomes. We identified a candidate yeast VLCS gene (FAT1), previously identified as encoding afatty acid transport protein, by its homology to rat liver peroxisomal VLCS. Disruption of this gene decreased, but did not abolish, cellular VLCS activity. Fractionation studies showed that VLCS activity, but not long-chain acyl-CoA synthetase activity, was reduced to about 40% of wild-type level in both 27,000 × g supernatant and pellet fractions. Separation of organelles in the pellet fraction by density gradient centrifugation revealed that VLCS activity was associated with peroxisomes and microsomes but not mitochondria. FAT1deletion strains exhibited decreased growth on medium containing dextrose, oleic acid, and cerulenin, an inhibitor of fatty acid synthesis. FAT1 deletion strains grown on either dextrose or oleic acid medium accumulated very long-chain fatty acids. Compared with wild-type yeast, C22:0, C24:0, and C26:0 levels were increased approximately 20-, 18-, and 3-fold in deletion strains grown on dextrose, and 2-, 7-, and 5-fold in deletion strains grown on oleate. Long-chain fatty acid levels in wild-type and deletion strains were not significantly different. All biochemical defects in FAT1deletion strains were restored to normal after functional complementation with the FAT1 gene. The level of VLCS activity measured in both wild-type and deletion yeast strains transformed with FAT1 cDNA paralleled the level of expression of the transgene. The extent of both the decrease in peroxisomal VLCS activity and the very long-chain fatty acid accumulation in the yeast FAT1 deletion model resembles that observed in cells from X-linked adrenoleukodystrophy patients. These studies suggest that the FAT1 gene product has VLCS activity that is essential for normal cellular very long-chain fatty acid homeostasis. Activation of fatty acids to their coenzyme A derivatives is necessary for subsequent metabolism. Very long-chain fatty acids, which accumulate in tissues of patients with X-linked adrenoleukodystrophy, are activated by very long-chain acyl-CoA synthetase (VLCS) normally found in peroxisomes and microsomes. We identified a candidate yeast VLCS gene (FAT1), previously identified as encoding afatty acid transport protein, by its homology to rat liver peroxisomal VLCS. Disruption of this gene decreased, but did not abolish, cellular VLCS activity. Fractionation studies showed that VLCS activity, but not long-chain acyl-CoA synthetase activity, was reduced to about 40% of wild-type level in both 27,000 × g supernatant and pellet fractions. Separation of organelles in the pellet fraction by density gradient centrifugation revealed that VLCS activity was associated with peroxisomes and microsomes but not mitochondria. FAT1deletion strains exhibited decreased growth on medium containing dextrose, oleic acid, and cerulenin, an inhibitor of fatty acid synthesis. FAT1 deletion strains grown on either dextrose or oleic acid medium accumulated very long-chain fatty acids. Compared with wild-type yeast, C22:0, C24:0, and C26:0 levels were increased approximately 20-, 18-, and 3-fold in deletion strains grown on dextrose, and 2-, 7-, and 5-fold in deletion strains grown on oleate. Long-chain fatty acid levels in wild-type and deletion strains were not significantly different. All biochemical defects in FAT1deletion strains were restored to normal after functional complementation with the FAT1 gene. The level of VLCS activity measured in both wild-type and deletion yeast strains transformed with FAT1 cDNA paralleled the level of expression of the transgene. The extent of both the decrease in peroxisomal VLCS activity and the very long-chain fatty acid accumulation in the yeast FAT1 deletion model resembles that observed in cells from X-linked adrenoleukodystrophy patients. These studies suggest that the FAT1 gene product has VLCS activity that is essential for normal cellular very long-chain fatty acid homeostasis. Metabolism of fatty acids by either catabolic or anabolic pathways requires initial activation to their coenzyme A (CoA) 1The abbreviations used are: CoA, coenzyme A; VLCFA, very long-chain fatty acid; VLCS, very long-chain acyl-CoA synthetase; rVLCS, rat VLCS; mFATP, mouse fatty acid transport protein; LCS, long-chain acyl-CoA synthetase; FAT1 and Fat1p,S. cerevisiae fatty acid transporter/VLCS gene and protein, respectively; FAA and Faap, S. cerevisiae fatty acid activation gene and protein, respectively; XALD, X-linked adrenoleukodystrophy; ALDP, adrenoleukodystrophy protein; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); MES, 4-morpholineethanesulfonic acid. thioesters, a reaction catalyzed by acyl-CoA synthetase (1Watkins P.A. Prog. Lipid Res. 1997; 36: 55-83Crossref PubMed Scopus (156) Google Scholar). Differences in substrate chain length specificity and subcellular localization distinguish the various acyl-CoA synthetases present in cells from all species examined, including animals, yeast, bacteria, and plants (1Watkins P.A. Prog. Lipid Res. 1997; 36: 55-83Crossref PubMed Scopus (156) Google Scholar). Long-chain fatty acids (C12–C20) are activated by long-chain acyl-CoA synthetases (LCSs) found in mitochondria, peroxisomes, and microsomes (1Watkins P.A. Prog. Lipid Res. 1997; 36: 55-83Crossref PubMed Scopus (156) Google Scholar), reflecting the primary roles of these organelles in fatty acid β-oxidation (mitochondria and peroxisomes) and complex lipid synthesis (microsomes). In contrast, activation of very long-chain fatty acids (VLCFA; C22 or longer) by very long-chain acyl-CoA synthetase (VLCS) takes place only in peroxisomal and microsomal subcellular fractions of rat (2Singh H. Poulos A. Arch. Biochem. Biophys. 1988; 266: 486-495Crossref PubMed Scopus (60) Google Scholar), human (3Lazo 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), and yeast (4Kalish J.E. Chen C.I. Gould S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1995; 206: 335-340Crossref PubMed Scopus (13) Google Scholar) cells. Lack of mitochondrial VLCS prevents this organelle from catabolizing VLCFA via β-oxidation, thus relegating this function to the peroxisome. VLCFA β-oxidation is impaired in cells from patients with the progressive neurodegenerative disease X-linked adrenoleukodystrophy (XALD), resulting in elevated plasma and tissue concentrations of saturated VLCFA (5Moser H.W. Smith K.D. Moser A.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 2325-2349Google Scholar). Biochemical studies revealed that VLCS activity was decreased in peroxisomal, but not microsomal, subcellular fractions of fibroblasts from XALD patients (3Lazo 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, 6Wanders R.J.A. van Roermund C.W.T. van Wijland M.J.A. Schutgens R.B.H. van den Bosch H. Schram A.W. Tager J.M. Biochem. Biophys. Res. Commun. 1988; 153: 618-624Crossref PubMed Scopus (136) Google Scholar). However, further investigation of the molecular defect in XALD revealed that key steps in peroxisomal VLCFA metabolism are still poorly understood. The product of the gene defective in XALD (ALDP) is not VLCS, but rather is a peroxisomal membrane protein whose existence was previously unknown (7Mosser J. Douar A.M. Sarde C.O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (1004) Google Scholar). Studies in many laboratories, including our own (8Kok F. Neumann S. Sarde C.O. Zheng S. Wu K.H. Wei H.M. Bergin J. Watkins P.A. Gould S.J. Moser H.W. Mandel J.L. Smith K.D. Hum. Mutat. 1995; 6: 104-115Crossref PubMed Scopus (97) Google Scholar), clearly demonstrated that XALD patients have mutations in the ALD gene, and complementation studies confirmed that transfection of XALD cells with the ALD gene restores their ability to catabolize VLCFA (9Braiterman L.T. Zheng S. Watkins P.A. Geraghty M.T. Johnson G. McGuinness M.C. Moser A.B. Smith K.D. Hum. Mol. Genet. 1998; 7: 239-247Crossref PubMed Scopus (84) Google Scholar, 10Cartier 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). While studies of XALD suggest that ALDP is required for VLCFA activation, the biochemical relationship between ALDP and VLCS has not yet been elucidated. Thus, to further our understanding of peroxisomal VLCFA activation, we are investigating this process in yeast. Yeasts are unique model organisms for the study of peroxisomal fatty acid β-oxidation, since they lack the mitochondrial β-oxidation pathway found in higher animals. They have been valuable for identification of proteins subsequently shown to be defective in human disorders of peroxisome biogenesis (11Shimozawa N. Tsukamoto T. Suzuki Y. Orii T. Shirayoshi Y. Mori T. Fujiki Y. Science. 1992; 255: 1132-1134Crossref PubMed Scopus (313) Google Scholar, 12Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (387) Google Scholar, 13Yahraus T. Braverman N. Dodt G. Kalish J.E. Morrell J.C. Moser H.W. Valle D. Gould S.J. EMBO J. 1996; 15: 2914-2923Crossref PubMed Scopus (161) Google Scholar, 14Braverman N. Steel G. Obie C. Moser A. Moser H. Gould S.J. Valle D. Nat. Genet. 1997; 15: 369-376Crossref PubMed Scopus (359) Google Scholar, 15Chang C.C. Lee W.H. Moser H. Valle D. Gould S.J. Nat. Genet. 1997; 15: 385-388Crossref PubMed Scopus (128) Google Scholar, 16Reuber B.E. Germain-Lee E. Morrell J.C. Geisbrecht B. Collins C. Ameritunga R. Moser H.W. Valle D. Gould S.J. Am. J. Hum Genet. 1997; 61: A54Google Scholar). We previously reported that, as in mammalian tissues, peroxisomes and microsomes isolated from the yeast Pichia pastoris contain VLCS activity (4Kalish J.E. Chen C.I. Gould S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1995; 206: 335-340Crossref PubMed Scopus (13) Google Scholar). The yeast Saccharomyces cerevisiae has been used by Gordon and co-workers to identify and characterize four fattyacid activation (FAA1–4) genes that encode acyl-CoA synthetases (17Knoll L.J. Johnson D.R. Gordon J.I. J. Biol. Chem. 1994; 269: 16348-16356Abstract Full Text PDF PubMed Google Scholar, 18Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (138) Google Scholar). They examined the fatty acid substrate specificity of the protein products of three of theseFAA genes (Faa1p, Faa2p, and Faa3p) in detail; while Faa3p weakly activated VLCFA, Faa1p and Faa2p had no detectable activity with these substrates (17Knoll L.J. Johnson D.R. Gordon J.I. J. Biol. Chem. 1994; 269: 16348-16356Abstract Full Text PDF PubMed Google Scholar). Faa4p was reported to be functionally interchangeable with Faa1p and thus is not likely to be a VLCS (18Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (138) Google Scholar). Based on growth studies of yeast mutants in which all fourFAA genes were disrupted, Gordon and colleagues concluded that at least one other FAA gene exists in S. cerevisiae (18Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (138) Google Scholar). Recently, Hashimoto and co-workers purified an enzyme with VLCS activity from rat liver peroxisomes (rVLCS) (19Uchida Y. Kondo N. Orii T. Hashimoto T. J. Biochem. ( Tokyo ). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar) and subsequently cloned and sequenced its cDNA (20Uchiyama A. Aoyama T. Kamijo K. Uchida Y. Kondo M. Orii T. Hashimoto T. J. Biol. Chem. 1996; 271: 30360-30365Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). We identified an open reading frame on S. cerevisiae chromosome II (YBR041W; locus FAT1) with homology to the rVLCS amino acid sequence. Both rVLCS and theFAT1 gene product, Fat1p, are homologous to mouse fatty acid transport protein (mFATP), described by Schaffer and Lodish (21Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (744) Google Scholar). Faergeman et al. (22Faergeman N.J. DiRusso C.C. Elberger A. Knudsen J. Black P.N. J. Biol. Chem. 1997; 272: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) independently identified theFAT1 (fatty acidtransporter 1) gene by virtue of the homology of Fat1p to mFATP. These investigators found that deletion ofFAT1 resulted in decreased rates of cellular uptake of labeled long-chain fatty acid, decreased cellular uptake of fluorescent fatty acids, and decreased uptake and incorporation of labeled oleic acid into cellular phospholipids (22Faergeman N.J. DiRusso C.C. Elberger A. Knudsen J. Black P.N. J. Biol. Chem. 1997; 272: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Although they noted amino acid sequence similarities between Fat1p and acyl-CoA synthetases, these investigators found that deletion of the FAT1 gene did not affect LCS activity (22Faergeman N.J. DiRusso C.C. Elberger A. Knudsen J. Black P.N. J. Biol. Chem. 1997; 272: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). However, they did not investigate either the role of Fat1p in VLCFA metabolism or the subcellular localization of this protein. In this report, we present evidence that Fat1p has VLCS activity and is present in both peroxisomes and a 27,000 ×g supernatant fraction of S. cerevisiae. Furthermore, deletion of the FAT1 gene resulted in accumulation of VLCFA in yeast cells similar to that observed in plasma and tissues of XALD patients. These findings support the hypothesis that Fat1p is a VLCS whose activity is critical for normal cellular VLCFA homeostasis. Degenerate oligonucleotide primers (5′-GGYTCRTCYTTYTCNACRTC-3′ and 5′-ACNGGNACNCCRTANACRTT-3′) corresponding to two peptide fragments (referred to as internal sequences 1 and 2) of the rVLCS (19Uchida Y. Kondo N. Orii T. Hashimoto T. J. Biochem. ( Tokyo ). 1996; 119: 565-571Crossref PubMed Scopus (48) Google Scholar) were used to amplify by PCR a 348-base pair product using a liver cDNA library from a Gemfibrozil-treated rat as the template. Treatment of rats with the peroxisome proliferator Gemfibrozil was previously found to increase peroxisomal rVLCS activity by about 2-fold. 2P. A. Watkins, unpublished observations. The PCR product, containing an open reading frame with 45% amino acid identity to mFATP (21Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (744) Google Scholar), was labeled with [32P]dCTP and used as a probe to screen the rat liver cDNA library. One clone (0.7 kb) containing an open reading frame of 220 amino acids was obtained; the nucleotide sequence of this clone was subsequently found to be identical to the carboxyl terminus of the rVLCS cDNA sequence later published (20Uchiyama A. Aoyama T. Kamijo K. Uchida Y. Kondo M. Orii T. Hashimoto T. J. Biol. Chem. 1996; 271: 30360-30365Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The 220-amino acid open reading frame was compared with all open reading frames of the complete S. cerevisiae genome (Saccharomyces Genome Data Base) using the BLAST algorithm (23Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). Only one likely candidate for a homologous yeast protein was identified (smallest sum probability = 9e-24). This gene (open reading frame YBR041W; locus FAT1) is found on chromosome II and is identical to that subsequently described by Faergeman et al. (22Faergeman N.J. DiRusso C.C. Elberger A. Knudsen J. Black P.N. J. Biol. Chem. 1997; 272: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The SWISS-PROT Data base entry for the protein (Fat1p) encoded by the FAT1 gene noted similarity to enzymes that act via an ATP-dependent covalent binding of AMP to their substrate. S. cerevisiae strain YPH680 (MATα his3Δ200 leu2Δ1 lys2Δ202 trp1Δ1 ura3–52) isogenic to YPH681 (MATa), a derivative of S288C (24Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google Scholar), was the strain used for PCR-mediated disruption of the FAT1 gene. Cells were grown at 30 °C in rich medium containing dextrose as primary carbon source (YPD, consisting of 10 g/liter yeast extract (Life Technologies, Inc.), 20 g/liter Bacto-peptone (Difco), and 20 g/liter dextrose (Sigma)). For induction of peroxisomes, yeasts were grown in YPD to an A 600 of 1. Cells were harvested by centrifugation at 3000 × g for 5 min, resuspended to an A 600 of 0.5 in medium containing oleic acid as primary carbon source (YPOLT, where 0.2% (w/v) oleic acid (Sigma) solubilized with 0.02% (w/v) Tween 40 (Sigma) replaced dextrose), and grown for 12–14 h at 30 °C (25Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar). Yeast growth characteristics were assessed on YPD/agar plates supplemented with 500 μmoleic acid and 0.033% (w/v) Tween 40, with or without 25 μm cerulenin (Sigma); 5 μl of cells diluted to anA 600 of 0.01 were applied to plates and incubated for 16 h at 24 and 30 °C. S. cerevisiae genomic DNA was isolated (26Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1979Google Scholar) and used as a template for PCR amplification of the FAT1 gene. PCR amplification was performed using the Expand Long Template PCR System (Boehringer Mannheim) with forward primer 5′-TACTGTCACCTTGATGAGCGAC-3′ and reverse primer 5′-TCGATGGCTTCCCAATCAG-3′. Thirty-five cycles of 94 °C (20 s), 57 °C (60 s), and 68 °C (150 s) were performed. The resulting 2.2-kb amplicon containing the FAT1 gene (from −196 to +1990, where +1 is the first nucleotide of the translation initiation codon) was TA-cloned into pCR2.1 (Invitrogen) to produce pJFL3301. AHindIII/XbaI fragment from pJFL3301 was then cloned into the low copy number (CEN) vector pRS416 (27Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), producing pJFL3302. DNA sequencing of pJFL3302 was performed at the Johns Hopkins University Genetics Core Facility using the fluorescent dideoxy terminator method of cycle sequencing on an Applied Biosystems Inc. 373a automated DNA sequencer, following ABI protocols. Comparison of the sequence of pJFL3302 with that of FAT1 as contained in the Saccharomyces Genome Data base revealed that the data base sequence contained a CpC dinucleotide insertion at bp 1857–1858. Four subsequent attempts to amplify an 896-bp fragment containing this region using forward primer 5′-GGTGAAGGTTGCTTATGGTAACGG-3′ and the reverse primer described above and PCR kits from three different manufacturers (Boehringer Mannheim, Promega, and Life Technologies, Inc.) yielded products with sequence identical to that contained in pJFL3302, ruling out random PCR-generated error. These findings suggested that the data base sequence may be in error. To verify this possibility, reverse transcription-PCR of wild-type yeast RNA was carried out as described below. RNA was treated with DNase I prior to cDNA synthesis. A 595-bp fragment of the FAT1 gene containing the putative CpC deletion region was amplified using the forward primer 5′-TTTCCTTGATAGAATGGGTG-3′ and reverse primer 5′-CCTTTCCATATGTTATTAGG-3′. Sequencing of this fragment confirmed that the FAT1 transcript also lacked the CpC dinucleotide. Thus, we concluded that the data base sequence was in error, and this has been communicated to the curator of the Saccharomyces Genome Data base. Computer-assisted translation of our correctedFAT1 sequence yielded an open reading frame of 669 amino acids ending in -IKL, a yeast peroxisome targeting signal (28Elgersma Y. Vos A. van den Berg M. van Roermund C.W. van der Sluijs P. Distel B. Tabak H.F. J. Biol. Chem. 1996; 271: 26375-26382Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The correct nucleotide sequence of FAT1 and the predicted amino acid sequence of Fat1p are shown in Fig. 1. Computer-assisted translation of the coding sequence of pJFL3302 revealed that this plasmid encodes amino acids 1–662 of Fat1p plus 11 amino acids (EAEFQHTGGRY-COOH) derived from vector sequence. Since this construct did not contain the entire open reading frame ofFAT1, we amplified by PCR a fragment containing the true 3′-end of the gene from genomic DNA. A 1-kb fragment was amplified using forward primer 5′-GGTGGAGGTTGCTTATGGTAACGG-3′, reverse primer 5′-CCTTTCCATATGTTATTAGG-3′, and Taq polymerase (Promega). Amplicons were TA-cloned into the pGEM-T Easy vector (Promega) to produce pJFL3305. A 223-bp Eco47III/SalI fragment of pJFL3305 was introduced into pJFL3302 to generate pJFL3307, which contains full-length FAT1 cDNA in the pRS416 (CEN) vector. The nucleotide sequence of pJFL3307 was correct with the exception of a silent mutation in amino acid Ser595. In complementation experiments, FAT1 deletion strains were transformed with pJFL3302 or pJFL3307 (26Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1979Google Scholar). For overexpression studies, the complete FAT1 sequence was transferred to a high copy 2μ vector. A 2.2-kbEco47III/ApaI fragment was transferred from pJFL3301 to pJFL3305 to form pJFL3306; the complete FAT1open reading frame was excised from pJFL3306 with SpeI andXhoI and transferred to the 2μ vector pRS426 (27Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to form pSJW981. Here, we refer to the sequence contained in pJFL3302 asFAT1–1 and that contained in pJFL3307 and pSJW981 asFAT1. PCR-mediated gene disruption of the FAT1 gene was performed by the method of Lorenz et al. (29Lorenz M.C. Muir R.S. Lim E. McElver J. Weber S.C. Gene ( Amst. ). 1995; 158: 113-117Crossref PubMed Scopus (257) Google Scholar). A forward primer consisting of 40 nucleotides of FAT1 sequence upstream of the initiation ATG and 20 left primer sequence-specific nucleotides for pRS plasmids (5′-TCTATATCGTTGAACTTTTAATAGGCTGCGAATACCGACTCTGTGCGGTATTTCACACCG-3′) and a reverse primer consisting of 40 nucleotides of FAT1sequence downstream of the stop codon (as contained in theSaccharomyces Genome Data base; nucleotides 1871–1910) plus 20 right primer sequence-specific nucleotides for pRS plasmids (5′-TCCAAACCCTTTGGTAATTTTTGCTCTCTATAAACCTTCTAGATTGTACTGAGAGTGCAC-3′) were used to amplify pRS303 using a two-step program (24Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google Scholar). SevenHIS prototrophs were selected following transformation of YPH680 with this PCR product (26Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1979Google Scholar). PCR using the forward and reverse primers used to clone FAT1 (described above) and genomic DNA prepared from wild-type and candidate gene disruption strains was performed to confirm disruption of FAT1. PCR products were separated by electrophoresis on 1% agarose gels. Four of the seven strains contained a PCR product of the expected size (1231 bp) forFAT1 disruption. The expected 2186-bp PCR product was observed in wild-type strains. Peroxisomes were induced in wild-type and gene deletion yeast strains by growth in YPOLT medium as described above. Spheroplasts were prepared, homogenized, and fractionated into 27,000 × g supernatant and organelle pellet as described by Crane et al. (25Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar). In some experiments, the organelle pellet was further fractionated by centrifugation on linear Nycodenz (sold as Accudenz by Accurate Chemical and Scientific) gradients (30Erdmann R. Blobel G. J. Cell Biol. 1995; 128: 509-523Crossref PubMed Scopus (236) Google Scholar). 0.5 ml of Maxidens (sold as Purdenz by Accurate Chemical and Scientific) was placed at the bottom of a 13-ml Beckman Quickseal tube, followed by a 10.5-ml linear gradient of 15% Nycodenz and 0.25 m sucrose (top) to 42.5% Nycodenz and 0.125m sucrose (bottom) in 5 mm MES, pH 6.0, 1 mm EDTA, 1 mm KCl, 0.1% ethanol. The organelle pellet in homogenization buffer (5 mm MES, pH 6.0, 0.6m sorbitol, 0.5 mm EDTA, 1 mm KCl, 0.1% ethanol) was layered on top of the Nycodenz, and tubes were centrifuged for 75 min at 174,000 × g in a vertical ultracentrifuge rotor. Fractions of 0.5 ml were collected from the bottom of the tube and assayed for acyl-CoA synthetase activity (below) and marker enzyme activity as described previously (4Kalish J.E. Chen C.I. Gould S.J. Watkins P.A. Biochem. Biophys. Res. Commun. 1995; 206: 335-340Crossref PubMed Scopus (13) Google Scholar). Because protein cannot be quantitated accurately in Nycodenz solutions, and because this compound had been shown to inhibit other fatty acid metabolic pathways (31Singh I. Pahan K. Dhaunsi G.S. Lazo O. Ozand P. J. Biol. Chem. 1993; 268: 9972-9979Abstract Full Text PDF PubMed Google Scholar), in some experiments, peak peroxisomal and mitochondrial fractions were pooled and dialyzed against 10 mm MES, pH 7.0, prior to assay. Acyl-CoA synthetase activity was measured essentially as described previously (32Watkins P.A. Ferrell Jr., E.V. Pedersen J.I. Hoefler G. Arch. Biochem. Biophys. 1991; 289: 329-336Crossref PubMed Scopus (75) Google Scholar). Labeled fatty acids, [1-14C]lignoceric acid (C24:0; American Radiolabeled Chemicals) and [1-14C]palmitic acid (C16:0; NEN Life Science Products) were solubilized in α-cyclodextrin (Sigma) and incubated with enzyme for 20 min at 30 °C in 40 mmTris-Cl, pH 7.5, containing 10 mm ATP, 5 mmMgCl2, 0.2 mm CoA, and 0.2 mmdithiothreitol. Labeled acyl-CoAs were extracted from the reaction mixture by the method of Dole (33Dole V.P. J. Clin. Invest. 1956; 35: 150-154Crossref PubMed Scopus (1598) Google Scholar) and quantitated in a Beckman LS 3801 liquid scintillation counter. Protein was determined by the method of Lowry et al. (34Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Total RNA was isolated from spheroplasts prepared from yeast, grown in either YPD or YPOLT medium, using an RNeasy kit (Qiagen). cDNA synthesis was from 1.0 μg of RNA (pretreated with DNase I; Boehringer Mannheim) from yeast grown under each condition and utilized 20 ng of (dT)15 (Boehringer Mannheim) and 100 ng of hexanucleotides (Sigma) as primers. The reaction was carried out at 42 °C for 1 h with 20 units of Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim), buffer supplied with the enzyme, 20 units of RNasin (Promega), and 125 μm each dNTP. After treatment at 95 °C for 3 min, an equal aliquot of each reverse transcription reaction was used as PCR template with forward primer 5′-TTTCCTTGATAGAATGGGTG-3′ and reverse primer 5′-CCAATCAGCAGCGGTCAAGA-3′ designed to amplify a 424-bp fragment ofFAT1 cDNA. An additional PCR was carried out using forward primer 5′-CCCAGATCTATGAGCAGTCGTGTGTGCTAC-3′ and reverse primer 5′-CCCGAGCTCAGTTGTGAAGCACTTTAGTTAC-3′ (kindly provided by Dr. M. Geraghty) designed to amplify a 1116-bp fragment containing YOR180C (locus EDH2), a peroxisomal enoyl-CoA hydratase gene with a consensus upstream oleate response element. Thirty-five cycles of 94 °C (45 s), 55 °C (45 s), and 72 °C (60 s) were performed using the Expand High Fidelity PCR system (Boehringer Mannheim). Yeast grown in either YPD (250A 600) or YPOLT (60 A 600) were harvested by centrifugation, washed three times with deionized water, suspended in 0.5 ml water, and subjected to sonic disruption. After determination of the protein concentration by the method of Miller (35Miller G.L. Anal. Chem. 1959; 31: 964Crossref Scopus (1) Google Scholar), total lipids were extracted by a modification of the method of Folch et al. (36Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1957; 226: 457-509Abstract Full Text PDF Google Scholar), and total fatty acids were quantitated by gas chromatography as their methyl esters using the method of Moser and Moser (37Moser H.W. Moser A.B. Hommes F.A. Techniques in Diagnostic Human Biochemical Genetics. Wiley-Liss, New York1991: 177-191Google Scholar). A cDNA library from the liver of a rat treated with the peroxisome proliferator Gemfibrozil was screened, and a 660-bp fragment containing a 220-amino acid open reading frame thought to be the carboxyl terminus of rat VLCS was cloned as described under “Experimental Procedures.” The sequence of this fragment was 100% identical to that of authentic rat liver VLCS, which was subsequently published by Uchiyama et al. (20Uchiyama A. Aoyama T. Kamijo K. Uchida Y. Kondo M. Orii T. Hashimoto T. J. Biol. Chem. 1996; 271: 30360-30365Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The 220-amino acid sequence was compared with all open reading frames of the completeS. cerevisiae genome using the BLAST algorithm. Only one likely candidate for a h" @default.
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• W2000961724 title "Disruption of the Saccharomyces cerevisiae FAT1 Gene Decreases Very Long-chain Fatty Acyl-CoA Synthetase Activity and Elevates Intracellular Very Long-chain Fatty Acid Concentrations" @default.
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