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• W2006023613 abstract "In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions inFAA1 and FAA4 or FAA1 andFAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C1-BODIPY-C12 indicating a fatty acid transport defect. By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, andFAA4 in the faa1Δ faa4Δ strain and encoding FAA1 and FAT1 in thefaa1Δ fat1Δ strain. MulticopyFAA4 could not suppress the growth defect in thefaa1Δ fat1Δ strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Δfaa1Δ and faa1Δ faa4Δstrains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2μ plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches. First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity. Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p. In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions inFAA1 and FAA4 or FAA1 andFAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C1-BODIPY-C12 indicating a fatty acid transport defect. By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, andFAA4 in the faa1Δ faa4Δ strain and encoding FAA1 and FAT1 in thefaa1Δ fat1Δ strain. MulticopyFAA4 could not suppress the growth defect in thefaa1Δ fat1Δ strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Δfaa1Δ and faa1Δ faa4Δstrains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2μ plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches. First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity. Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p. fatty acid translocase fatty acid transport protein phosphate-buffered saline 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid Biological membranes are complex in both their protein and lipid compositions. This complexity is essential and contributes to the barrier function of the membrane and to selectively regulated transport of molecules into and out of the cell. Unlike hydrophilic molecules such as sugars and amino acids, hydrophobic fatty acids are able to dissolve in the membrane, and as a consequence, the processes governing their regulated movement across membranes are likely to be quite distinct. Recent investigations into the problem of fatty acid transport have intensified due to findings that exogenous fatty acids influence a number of important cellular functions, including signal transduction and transcriptional control. To date, several distinct membrane-bound and membrane-associated proteins have been identified as components of fatty acid import systems in eukaryotic cells. Most notable among these are fatty acid translocase (FAT,1 the murine homologue to CD36) (1Abumrad N.A. el-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. J. Biol. Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar, 2Abumrad N.A. Coburn C. Ibrahimi A. Biochim. Biophys. Acta. 1999; 1441: 4-13Crossref PubMed Scopus (210) Google Scholar), fatty acid transport protein (FATP) (3Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (743) Google Scholar), and fatty acyl-CoA synthetase (3Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (743) Google Scholar, 4DiRusso C.C. Black P.N. Mol. Cell. Biochem. 1999; 192: 41-52Crossref PubMed Google Scholar, 5Klein K. Steinberg R. Fiethen B. Overath P. Eur. J. Biochem. 1971; 19: 442-450Crossref PubMed Scopus (143) Google Scholar, 6Frerman F.E. Bennett W. Arch. Biochem. Biophys. 1973; 159: 434-443Crossref PubMed Scopus (25) Google Scholar). FAT was identified following protein modification using sulfo-N-succinimidyl oleate (7Harmon C.M. Luce P. Beth A.H. Abumrad N.A. J. Membr. Biol. 1991; 121: 261-268Crossref PubMed Scopus (165) Google Scholar), whereas FATP and fatty acyl-CoA synthetase were both identified using expression cloning (3Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (743) Google Scholar). Both FAT and FATP have been claimed to be fatty acid transport proteins (1Abumrad N.A. el-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. J. Biol. Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar, 8Schaffer J.E. Lodish H.F. Trends Cardiovasc. Med. 1995; 4: 218-224Crossref Scopus (48) Google Scholar, 9Stahl A. Hirsch D.J. Gimeno R.E. Punreddy S. Ge P. Watson N. Patel S. Kotler M. Raimondi A. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Despite these claims, there is controversy surrounding the classification of FAT/CD36 and FATP asbona fide integral membrane-bound fatty acid transporters (10Hamilton J.A. Johnson R.A. Corkey B. Kamp F. J. Mol. Neurosci. 2001; 16: 99-108Crossref PubMed Scopus (91) Google Scholar). Indeed, there are gnawing questions as to whether these proteins actually function as components of a fatty acid delivery system (i.e. FAT/DC36) or as components of a utilization driven fatty acid import system (i.e. FATP), which also includes fatty acyl-CoA synthetase (2Abumrad N.A. Coburn C. Ibrahimi A. Biochim. Biophys. Acta. 1999; 1441: 4-13Crossref PubMed Scopus (210) Google Scholar, 4DiRusso C.C. Black P.N. Mol. Cell. Biochem. 1999; 192: 41-52Crossref PubMed Google Scholar, 8Schaffer J.E. Lodish H.F. Trends Cardiovasc. Med. 1995; 4: 218-224Crossref Scopus (48) Google Scholar, 10Hamilton J.A. Johnson R.A. Corkey B. Kamp F. J. Mol. Neurosci. 2001; 16: 99-108Crossref PubMed Scopus (91) Google Scholar, 11Færgeman N.J. Black P.N. Zhao X.D. Knudsen J. DiRusso C.C. J. Biol. Chem. 2001; 276: 37051-37059Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In this regard, proteins identified as required for fatty acid transport may function not as transport proteins per se but in an alternative manner, perhaps by promoting selectivity and specificity of fatty acid delivery to downstream metabolic events. The best characterized fatty acid transport system is that found inEscherichia coli (4DiRusso C.C. Black P.N. Mol. Cell. Biochem. 1999; 192: 41-52Crossref PubMed Google Scholar). In this case, the specific integral outer membrane protein, FadL, is required for long-chain fatty acid binding and transport across that membrane. The fatty acid ligands must then traverse the bacterial periplasmic space and the inner membrane. No inner membrane proteins have been identified that are required for this process. On the basis of studies defining the energetics of fatty acid transport, we suggested protonated fatty acids flip across the inner membrane and are subsequently abstracted from the inner membrane concomitant with activation by fatty acyl-CoA synthetase (12Azizan A. Sherin D. DiRusso C.C. Black P.N. Arch. Biochem. Biophys. 1999; 365: 299-306Crossref PubMed Scopus (20) Google Scholar). In this manner, exogenous fatty acids are metabolically trapped as CoA thioesters upon transport, which in turn generates a concentration gradient further driving the system. Overath and colleagues (5Klein K. Steinberg R. Fiethen B. Overath P. Eur. J. Biochem. 1971; 19: 442-450Crossref PubMed Scopus (143) Google Scholar) coined the term “vectorial acylation” to describe this process at the time they identified the structural gene for the E. coli fatty acyl-CoA synthetase (fadD). This postulate was initially expanded by Frerman and Bennett (6Frerman F.E. Bennett W. Arch. Biochem. Biophys. 1973; 159: 434-443Crossref PubMed Scopus (25) Google Scholar) and subsequently by our laboratory (4DiRusso C.C. Black P.N. Mol. Cell. Biochem. 1999; 192: 41-52Crossref PubMed Google Scholar, 12Azizan A. Sherin D. DiRusso C.C. Black P.N. Arch. Biochem. Biophys. 1999; 365: 299-306Crossref PubMed Scopus (20) Google Scholar) as the underlying mechanism driving long-chain fatty acid transport in bacteria. Although at the time the model of vectorial acylation was proposed the bacterial fatty acid transporter FadL had not been identified, our subsequent studies have clearly shown that both FadL and fatty acyl-CoA synthetase are required for fatty acid transport in E. coli. By using the yeast Saccharomyces cerevisiae as a model eukaryotic system, we have recently shown the fatty acyl-CoA synthetases Faa1p or Faa4p function in the fatty acid transport system presumably by activating exogenous fatty acids concomitant with transport (11Færgeman N.J. Black P.N. Zhao X.D. Knudsen J. DiRusso C.C. J. Biol. Chem. 2001; 276: 37051-37059Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This finding presents somewhat of a conundrum as we have also shown that long-chain fatty acid import in yeast requires Fat1p, the yeast orthologue of the murine FATP1 (13Færgeman 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). One of the central questions we are now faced with is to determine the mechanisms by which Fat1p and fatty acyl-CoA synthetase (Faa1p and/or Faa4p) work in concert to promote fatty acid import. A similar situation appears to be operational in murine adipocytes, where there are data supporting a functional association of mmFATP1 with fatty acyl-CoA synthetase (3Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (743) Google Scholar,15Gargiulo C.E. Stuhlsatz-Krouper S.M. Schaffer J.E. J. Lipid Res. 1999; 40: 881-892Abstract Full Text Full Text PDF PubMed Google Scholar). We suggest vectorial acylation is one general mechanism of fatty acid import, which functions to promote the regulated import and metabolic trapping of exogenous long-chain fatty acids. In our prior investigations into fatty acid import in yeast, we used reverse genetic approaches to demonstrate this process requires the yeast orthologue of mmFATP (Fat1p) and fatty acyl-CoA synthetase (Faa1p or Faa4p) (11Færgeman N.J. Black P.N. Zhao X.D. Knudsen J. DiRusso C.C. J. Biol. Chem. 2001; 276: 37051-37059Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 13Færgeman 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, 14DiRusso C.C. Connell E.J. Færgeman N.J. Knudsen J. Hansen J.K. Black P.N. Eur. J. Biochem. 2000; 267: 4422-4433Crossref PubMed Scopus (41) Google Scholar). Despite the information gleaned from these studies, there are no data demonstrating these proteins function cooperatively in a physical complex, and there is no information as to whether there are additional proteins involved in mediating the regulated import of exogenous long-chain fatty acids. In the present work, we sought to identify additional components required for fatty acid transport and to confirm the importance of Fat1p and fatty acyl-CoA synthetase (Faa1p and Faa4p) by using a genetic approach. A valuable molecular-genetic method for the identification of participants in multicomponent cellular processes is the selection of plasmid-encoded multicopy extragenic suppressors (16Rhine J. Methods Enzymol. 1991; 194: 239-251Crossref PubMed Scopus (82) Google Scholar). The rationale behind this approach is that the altered phenotype resulting from a deficiency in one participant can be suppressed by overexpression of another participant required for the same process (16Rhine J. Methods Enzymol. 1991; 194: 239-251Crossref PubMed Scopus (82) Google Scholar). In this manner, we sought to identify plasmid-encoded multicopy extragenic suppressors of the deficiency in fatty acid import caused by deletion ofFAT1 and/or FAA1 and FAA4. We report that plasmids encoding Fat1p, Faa1p, and Faa4p were identified in a screen for multicopy extragenic suppressors of the transport and activation deficiency of a faa1Δ faa4Δ strain, and plasmids encoding only Fat1p and Faa1p were identified as multicopy extragenic suppressors of the transport deficiency of afaa1Δ fat1Δ strain. Additional biochemical evidence is provided demonstrating Fat1p and acyl-CoA synthetase interact in a physical complex. This work establishes for the first time a genetic, physical, and functional linkage between Fat1p and fatty acyl-CoA synthetase and substantiates the hypothesis that these proteins, perhaps exclusively, are required for long-chain fatty acid transport in yeast. The S. cerevisiaestrains used in this study are listed in TableI. The fat1Δ::G418 mutation was introduced by transformation of the strain of interest with linear DNA generated by amplification of the kanamycin resistance cassette (resulting in G418 resistance) using oligonucleotides complementary to both FAT1 and the cassette as described (17Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1361) Google Scholar). The oligonucleotide for the coding strand was 5′-CACTGTCAAGAAGGGCAAGAAGGCAGCAGTATGGCTTGGGCATAGGCCACTAGTGGATCTG-3′, and the oligonucleotide for the template strand was 5′-CCACTGGATCATTCGTAAGTGATCCTGAAACAAACCATTCAGCAGCTGAAGCTTCGTACGC-3′. Chromosomal replacement of the native gene was confirmed by Southern analysis of chromosomal DNA from the transformants by comparison to DNA obtained from the parental strain. Yeast strains were transformed by the lithium acetate method (18Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1704) Google Scholar).Table IYeast strains used in this studyNameRelevant genotypeComplete genotype (Ref.)YB332Wild typeMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 (30Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (138) Google Scholar)YB497faa1ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 faa1Δ::HIS3 (30Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (138) Google Scholar)YB524faa4ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 faa4Δ:LYS2 (30Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (138) Google Scholar)LS2020fat1ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 fat1Δ::G418 (this study)YB525faa1Δfaa4ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 faa1Δ::HIS3 faa4Δ:LYS2 (31Duronio R.J. Knoll L.J. Gordon J.I. J. Cell Biol. 1992; 117: 515-529Crossref PubMed Scopus (59) Google Scholar)LS2086faa1Δfat1ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 faa1Δ::HIS3 fat1Δ::G418 (this study)LS2087faa4Δfat1ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 faa4Δ:LYS2 fat1Δ::G418 (this study)LS2089faa1Δfaa4Δfat1ΔMATa ura3–52 his3Δ200 ade2–101 lys2–801 leu2–3,112 faa1Δ::HIS3 faa4::LYS2 fat1Δ::G418 (this study) Open table in a new tab YPDA consisted of 1% yeast extract, 2% peptone, 2% dextrose, and 20 mg/liter adenine hemisulfate. Yeast-supplemented minimal media contained 0.67% yeast nitrogen base (YNB), 2% dextrose, adenine (20 mg/liter), uracil (20 mg/liter), and amino acids as required (arginine, tryptophan, methionine, histidine, and tyrosine (20 mg/liter); lysine (30 mg/liter); and leucine (100 mg/liter)). To assess growth when fatty-acid synthase was inhibited, cells were grown on YNBD or YPDA plates supplemented with 45 μm cerulenin and 100 μm oleic acid unless otherwise indicated. Growth in liquid culture and on plates was at 30 °C. Yeast extract, yeast peptone, and yeast nitrogen base were obtained from Difco. Oleic acid was obtained from Sigma. 3H- or14C-labeled fatty acids were from PerkinElmer Life Sciences and American Radiochemicals. C1-BODIPY-C12 was purchased from Molecular Probes. Enzymes required for all DNA manipulations were from Promega, Invitrogen, New England Biolabs, U. S. Biochemical Corp., or Roche Molecular Biochemicals. Anti-V5 antibody and anti-T7 antibodies were purchased from Invitrogen and Novagen, respectively. Anti-Pma1p was the gift of Dr. Günther Daum (Technische Universität Graz, Graz, Austria). Cells of the faa1Δfaa4Δ strain or faa1Δ fat1Δ strain were rendered competent using lithium acetate as noted above, transformed with a yeast multicopy library in YEp24, and transformants selected on YNBD containing the appropriate supplements but lacking uracil (19Carlson M. Botstein D. Cell. 1982; 28: 145-154Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Thirty thousand individual Ura+ transformants were selected from the library and were screened for growth following replica plating on YPD plates containing 45 μm cerulenin and 100 μm oleic acid (YPD-CER-OLE). Transformants that were able to grow on YPD-CER-OLE were colony-purified on the same media and phenotypes validated on YPD-CER-OLE. Plasmids were isolated from those that retained positive growth on all three media and retransformed into the faa1Δ faa4Δ andfaa1Δ fat1Δ strains. Additionally, the same plasmids were propagated in the E. coli strain DH5α, purified using QiaPrep columns (Qiagen), and sequenced using two plasmid-specific primers flanking the insert (upstream, 5′-GGAGCCACTATCGACTACGC-3′; downstream, 5′-CCTGTGGCGCCGGTGATG-3′) using an Applied Biosystems automated fluorescence DNA sequencer. The sequences obtained were compared with the Saccharomycesgenome data base for identification. Fatty acid import was assessed using confocal laser scanning microscopy to detect accumulation of the fluorescent long-chain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12) as described previously (13Færgeman 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). Following growth under selective conditions, cells were harvested, washed with phosphate-buffered saline (PBS) and resuspended in 0.1 volume of PBS. All steps were performed at room temperature. Washed cells were incubated with 10 μmC1-BODIPY-C12 for 60 s, washed with PBS containing 50 μm fatty acid-free bovine serum albumin (two times), PBS, resuspended in PBS, and visualized on an NORAN-OZ confocal laser scanning microscopy, interfaced with a Nikon Diaphot 200 inverted microscope equipped with a PlanApo ×60, 1.4 NA oil-immersion objective lens. The instrument settings for brightness, contrast, laser power, and slit size were optimized for the brightest sample to ensure that the confocal laser scanning microscopy was set for its full dynamic range. The same settings were used for all subsequent image collections. Cells were grown from overnight cultures in YNBD (with appropriate supplements) and grown toA 600 of 1.0. Following growth, cells were harvested by centrifugation, washed twice with PBS, and resuspended to a density of 1.2 × 109 cells/ml in 200 mmTris-HCl, pH 8.0, 4 mm EDTA, 5 mm2-mercaptoethanol, 10% glycerol, 0.01% Triton X-100, 0.5 mm phenylmethylsulfonyl fluoride, 4 μmpepstatin A, and 8 μm leupeptin. The cells were lysed by vigorously vortexing the cell suspension containing glass beads for 1 min, 5 times at 0 °C. Samples were clarified by centrifugation (1,500 × g, 5 min, 4 °C), and supernatants were used to assess fatty acyl-CoA synthetase activities as described (20Black P.N. Zhang Q. Weimar J.D. DiRusso C.C. J. Biol. Chem. 1997; 272: 4896-4903Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The reaction mixtures contained 200 mm Tris-HCl, pH 7.5, 2.5 mm ATP, 8 mm MgCl2, 2 mm EDTA, 20 mm NaF, 0.01% Triton X-100, fatty acid dissolved in 10 mg/ml α-cyclodextrin (final concentrations of fatty acids were 50 μm), 0.5 mm coenzyme A, and cell extract in a total volume of 0.5 ml. The reactions were initiated by the addition of coenzyme A, incubated at 30 °C for 20 min, and terminated by the addition of 2.5 ml of isopropyl alcohol, n-heptane, 1 mH2SO4 (40:10:1). The radioactive fatty acid was removed by organic extraction using n-heptane. Acyl-CoA formed during the reaction remained in the aqueous fraction and was quantified by scintillation counting. Protein concentrations in the cell extracts were determined using the Bradford assay and bovine serum albumin as a standard (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216204) Google Scholar). The values presented represent the average from at least three independent experiments performed in duplicate. All experiments were subjected to analysis of variance (StatView, SAS Institute, Inc.). The sequence encoding the C-terminal 125 amino acids (residues 545–669) of Fat1p was cloned in-frame to the T7 epitope tag of the yeast expression vector YEpGALSET983 to generate YEpDB213. The resulting T7Fat1p125C fusion was expressed under the control of the GAL10 promoter. To test for negative dominance, YEpDB213 was transformed into YB332. Cells transformed with the vector (YEpGALSET983) served as a control. The cells were pre-grown in YNBD (without leucine) overnight. The culture was harvested by centrifugation and resuspended to a cell density of 0.1 A 600 in 50 ml of YNB containing 2% galactose and 2% raffinose to induce expression ofT7Fat1p125C. When the density reached 1.0A 600, cells were harvested by centrifugation, washed once in PBS, and resuspended in 1 ml of breaking buffer (200 mm Tris, pH 8.0, 4 mm EDTA, 10% glycerol, 5 mm β-mercaptoethanol, 0.01% Triton X-100, 0.5 mm phenylmethylsulfonyl fluoride, 4 μmpepstatin A, and 8 μm leupeptin). The cells were lysed by vortexing with glass beads and assayed for long-chain acyl-CoA synthetase activity as detailed above. The yeast two-hybrid system was used to test Faa1p-Fat1p interaction (22Bartel P.L. Fields S. Bartel F. The Yeast Two Hybrid System. Oxford University Press, Oxford, UK1997Google Scholar). The bait plasmid vector used was pEG202; the trap plasmid vector was pJG4-5, and the reporter plasmid was pSH18-34T. To generate the full-length Faa1p-bait fusion protein, the coding sequence of FAA1 was amplified using the upstream primer 5′-AGACCCATGGATGGTTGCTCAATATACCG-3′ and the downstream primer 5′-AAATGTTGGCGGCCGCAGACGAACTATAAACGGC-3′. The amplified DNA fragment was cleaved with NcoI and NotI and ligated into pEG202 cleaved with the same enzymes. For the trap plasmids, a single primer was used to amplify DNA at the 3′ end of the gene including the termination codon encoding amino acid 669, 5′-GAACATCCTCGAGTAATTTAATTGTTTGTGC-3′, whereas unique primers were used to amplify DNA at the 5′ ends. These included 5′-TTTTTAGCGCGCAATACTAAAGGCACTCCG-3′ to generate a peptide from amino acids 169 to 669 of Fat1p (Fat1p500C) and 5′-GAAGATGAATTCACGGCCAGTAACAAAGAAC-3′ to generate a peptide from amino acids 544 to 669 of Fat1p (Fat1p125C). The amplified DNA fragments were digested with the appropriate restriction enzymes and ligated into pJG4-5. To test interaction, the Faa1p bait plasmid and the target trap plasmid of interest were transformed into yeast strain W303B carrying the reporter plasmid pSH18-34T. The reporter plasmid pSH18-34T contains thelacZ gene encoding β–galactosidase driven by a promoter controlled by eight LexA operators. To maintain each plasmid, transformants were selected and maintained on YNBD media lacking uracil (for pSH18-34T), histidine (for the pEG202-derived bait), and tryptophan (for the pJG4-5-derived traps). Expression of β-galactosidase activity was measured using the liquid assay employing o-nitrophenyl β-d-galactopyranoside as substrate as described previously (23Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). For these experiments, cells were grown overnight in YNBD (without uracil, histidine, or tryptophan) and subcultured to A 600 0.02–0.1 in 10 ml of YNB containing 2% galactose and 2% raffinose. Growth was continued until the A 600 reached 0.5–1.0, at which time was stopped by placing the cultures on ice. Aliquots of cells (1 ml) were harvested by centrifugation (14,000 rpm for 3 min). The cell pellets were resuspended in 200 μl of 0.1 m Tris, pH 7.5, containing 0.05% Triton X-100. The sample was frozen on dry ice and stored at −80 °C prior to assay. All experiments defining β-galactosidase activities were performed in duplicate at least five times as described previously (23Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar); the data were analyzed using pairedt-tests against cells containing the bait (Faa1p), the trap vector (pJG4-5), and the reporter (StatView, SAS Institute, Inc.). To identify a protein complex containing Fat1p and Faa1p or Faa4p, plasmids were constructed expressing each protein fused to a peptide tag, which is recognized by a commercially available antibody. Full-length Fat1p tagged with a T7 epitope was constructed in the vector YEpGALSET983 to generate plasmid YEpDB204. The coding sequence of FAT1 was amplified using the upstream primer 5′-GCGGAGCTCATGTCTCCCATACAGGTTGTTG-3′ and the downstream primer 5′-CGCGGTACCATGCTCTAATGGAAAGGTAC-3′. The amplified DNA fragment was cleaved with SacI and KpnI and ligated into YEpGALSET983 cleaved with the same restriction enzymes. Expression clones encoding full-length Faa1p or Faa4p tagged at the C terminus with a V5 epitope were obtained from Invitrogen (GeneStormTM clones pYES2/YOR317w and pYES2/YMR246w, respectively). The plasmid pair encoding the proteins of interest (e.g. T7Fat1p and V5Faa1p or V5Faa4p) was transformed into the fat1Δ faa1Δ strain to test Fat1p-Faa1p interaction or the fat1Δfaa4Δ strain to test Fat1p-Faa4p interaction. The cells were pre-grown in YNBD without leucine and uracil to maintain both plasmids; cells were subsequently subcultured to 0.1A 600 in 75 ml of YNB containing 2% galactose and 2% raffinose (without leucine and uracil) to induce expression of the epitope-tagged target proteins. When the cell density reached 1.0A 600, the cells were harvested, washed once with PBS, and resuspended in 1.5 ml of lysis buffer containing 50 mm Tris, pH 7.5, and 150 mm NaCl. The cells were lysed by vortexing with glass beads on ice as detailed above. The glass beads were pelleted by centrifugation (2,000 rpm, 2 min, 4 °C). The supernatant was removed to a new tube, and Triton X-100 was added to a final concentration of 1%, and the mixture was incubated on ice for 45 min. The sample was clarified by centrifugation (4,000 rpm, 15 min, 4 °C). The resultant supernatant was split into three 0.5-ml aliquots (∼0.7 mg/ml); 2 μg of anti-T7 or 2 μg of anti-V5 antibodies was added to the first two, and an equal volume of lysis buffer was added to the third as a control (protein A-Sepharose bead control). The samples were incubated with gentle rotation overnight at 4 °C. Protein A-Sepharose beads (50 μl of 50% slurry) were added to each sample, which were then incubated for 2 h with gentle rotation at 4 °C. The protein A-Sepharose beads (containing the antigen-antibody complex) were pelleted by centrifugation (1,000 rpm, 1 min, 4 °C) and subsequently washed 5 times in 50 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100. The final pellets containing the protein A-Sepharose beads/antigen-antibody complex were resuspended in 70 μl of SDS sample buffer. Samples were boiled 5 min, and the proteins from" @default.
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