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• W2159868923 abstract "Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC 6.2.1.3) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5–2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations. Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC 6.2.1.3) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5–2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations. acyl-CoA synthetase diacylglycerol acyltransferase endoplasmic reticulum glycerol-3-phosphate acyltransferase N-ethylmaleimide mitochondria-associated membrane phosphatidylethanolamine methyltransferase The first step in long chain fatty acid use in mammals requires the ligation of fatty acid with coenzyme A (CoA). This reaction, catalyzed by acyl-CoA synthetase (ACS,1 EC 6.2.1.3), produces acyl-CoAs, which are primary substrates for energy use via β-oxidation and for the synthesis of triacylglycerol, phospholipids, cholesterol esters, and sphingomyelin, and are the source of signaling molecules like ceramide, diacylglycerol, and arachidonic acid (1Faergman N.J. Knudsen J. Biochem. J. 1997; 323: 1-12Crossref PubMed Scopus (576) Google Scholar, 2Prentki M. Corkey B.E. Diabetes. 1996; 45: 273-283Crossref PubMed Scopus (0) Google Scholar). Acyl-CoAs up-regulate uncoupling protein in brown fat and key enzymes of glycolysis, gluconeogenesis, and β-oxidation; are essential for vesicle trafficking; and play a critical role in the transport of fatty acids into cells by making transport unidirectional. Protein esterification with myristate and palmitate anchors proteins to specific membranes and enables them to function correctly (3Yamashita A. Sugiura T. Waku K. J. Biochem. ( Tokyo ). 1997; 122: 1-16Crossref PubMed Scopus (233) Google Scholar). Thus, acyl-CoAs participate in a large number of cellular reactions that involve lipid synthesis, energy metabolism, and regulation, but how acyl-CoAs are partitioned or directed toward these diverse synthetic, degradative, and signaling pathways is not understood. Currently, five different rat ACS cDNAs have been cloned, each the product of a different gene (4Suzuki H. Kawarabayasi 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, 5Fujino T. Yamamoto T. J. Biochem. ( Tokyo ). 1992; 111: 197-203Crossref PubMed Scopus (120) Google Scholar, 6Fujino T. Kang M.-J. Suzuki H. Iijima H. Yamamoto T. J. Biol. Chem. 1996; 271: 16748-16752Crossref PubMed Scopus (160) Google Scholar, 7Kang M.-J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2880-2884Crossref PubMed Scopus (203) Google Scholar, 8Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.-J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. ( Tokyo ). 1998; 124: 679-685Crossref PubMed Scopus (123) Google Scholar). Rat ACS1–5 share a common structural architecture and are further classified into two subfamilies based on amino acid identity and fatty acid preference (4Suzuki H. Kawarabayasi 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, 5Fujino T. Yamamoto T. J. Biochem. ( Tokyo ). 1992; 111: 197-203Crossref PubMed Scopus (120) Google Scholar, 6Fujino T. Kang M.-J. Suzuki H. Iijima H. Yamamoto T. J. Biol. Chem. 1996; 271: 16748-16752Crossref PubMed Scopus (160) Google Scholar, 7Kang M.-J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2880-2884Crossref PubMed Scopus (203) Google Scholar, 8Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.-J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. ( Tokyo ). 1998; 124: 679-685Crossref PubMed Scopus (123) Google Scholar). ACS1, ACS2, and ACS5 make up one subfamily with about 60% homology to one another, and ACS3 and ACS4 make up a second subfamily with about 70% homology to each other and 30% similarity to ACS1. Within each subfamily, the ACS isoforms differ in their mRNA size, tissue distribution, and transcriptional regulation. ACS1, ACS4, and ACS5 are all expressed in liver. Studies with the ACS inhibitors triacsin and troglitazone suggested that long chain acyl-CoAs are functionally channeled toward specific metabolic fates. In most of these studies, de novoglycerolipid synthesis was severely inhibited, whereas phospholipid reacylation and ketone bodies formation was less impaired. Inhibition of fatty acid incorporation into cholesterol esters varied with cell type, being completely blocked in human fibroblasts and only moderately decreased in hepatocytes. For example, in hepatocytes, triacsin C did not alter oxidation of pre-labeled intracellular lipid, but did inhibit triacylglycerol synthesis 40% and 70% in hepatocytes isolated from starved and fed rats, respectively (9Muoio D.M. Lewin T.M. Weidmar P. Coleman R.A. Am. J. Physiol. 2000; 279: E1366-E1373Crossref PubMed Google Scholar). Additionally, in hepatocytes isolated from fasted rats, troglitazone blocked incorporation of oleate into triacylglycerol, but not into phospholipid (10Fulgencio J.P. Kohl C. Girard J. Pegorier J.P. Diabetes. 1996; 45: 1556-1562Crossref PubMed Google Scholar). In addition, troglitazone inhibited ketone body production. In human fibroblasts, triacsin C blocked the incorporation of [3H]glycerol into phospholipid by 80% and the incorporation into triacylglycerol by 99%, indicating severely impaired acylation of glycerol 3-phosphate, lysophosphatidic acid, and diacylglycerol via the de novosynthetic pathway from glycerol 3-phosphate (11Igal R.A. Wang P. Coleman R.A. Biochem. J. 1997; 324: 529-534Crossref PubMed Scopus (123) Google Scholar). Incorporation of [14C]oleate into triacylglycerol was also blocked 95%, consistent with impaired acylation via the de novo pathway; however, incorporation into phospholipids was not impaired, suggesting that separate pools of acyl-CoAs exist and that the reacylation pathway is functionally separate from de novo glycerolipid synthesis. Taken as a whole, these studies suggest that there are functionally independent acyl-CoA pools within cells, and that acyl-CoAs might be channeled toward specific fates rather than being freely available for all possible enzymatic reactions. Yeast provide clear evidence for functionally different ACS-linked pathways. In Candida lipolytica, studies using ACS mutants indicate that ACS I activates exogenous fatty acids for glycerolipid synthesis and ACS II activates them for β-oxidation (12Kamiryo T. Nishkawa Y. Mishina M. Terao M. Numa S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4390-4394Crossref PubMed Scopus (42) Google Scholar). InSaccharomyces cerevisiae, the ACS proteins Faa1p and Faa4p account for 99% of yeast C14-CoA and C16-CoA activity (13Knoll L.J. Johnson D.R. Gordon J.I. J. Biol. Chem. 1995; 270: 10861-10867Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and activate exogenously derived fatty acids destined for phospholipid synthesis (14Knoll L.J. Johnson D.R. Gordon J.I. J. Biol. Chem. 1994; 269: 16348-16356Abstract Full Text PDF PubMed Google Scholar). Faa4p is specifically needed for myristoylation of protein substrates (15Ashrafi K. Farazi T.A. Gordon J.I. J. Biol. Chem. 1998; 273: 25864-25874Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and Faa2p is required for peroxisomal β-oxidation (16Hettema E.H. van Roermund C.W.T. Distel B. van den Berg M. Vilela C. Rodrigues-Pousada C. Wanders R.J.A. Tabak H.F. EMBO J. 1996; 15: 3813-3822Crossref PubMed Scopus (248) Google Scholar, 17Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (137) Google Scholar). Yeast ACS isoforms are also differentially inhibited by triacsin C (18Knoll L.J. Schall O.F. Suzuki I. Gokel G.W. Gordon J.I. J. Biol. Chem. 1995; 270: 20090-20097Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). From these data, Gordon's group (17Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (137) Google Scholar) concluded that there are differences in location or accessibility of those acyl-CoAs that are derived from endogenous synthesis and those acyl-CoAs formed from exogenously provided fatty acids. Thus, genetic studies in yeast link specific ACS isoforms to different pathways that use acyl-CoAs. We examined ACS1, ACS4, and ACS5 in rat liver, which contains a variety of pathways that use acyl-CoAs, in order to determine whether the subcellular locations, inhibition by specific inhibitors, and nutritional regulation might link the different ACS isoforms with different metabolic pathways. Our data indicate that ACS1, ACS4, and ACS5 are present in different subcellular membranes; that ACS activity is inhibited by triacsin C, troglitazone, and NEM to varying degrees in these subcellular fractions; and that nutritional changes regulate each ACS isoform independently. [2-3H]Glycerol and [9,10-3H]palmitate were from Amersham Pharmacia Biotech.Glycerol, palmitoyl-CoA, ATP, and bovine serum albumin (essentially fatty acid-free) were from Sigma. Triacsin C (>95% pure) was from Biomol. Troglitazone was the gift of Dr. Steven Jacobs, GlaxoSmithKline. A polyclonal antibody to rat ACS1 was the gift of Dr. Paul Watkins, Kennedy Krieger Institute. Animal protocols were approved by the University of North Carolina (UNC), Chapel Hill, NC and University of Alberta Institutional Animal Care and Use Committees. Male and female (150 g) Harlan Sprague-Dawley rats were housed on a 12-h/12-h light/dark cycle with free access to water. Control animals had free access to Purina rat chow. Fasted animals were sacrificed after being without food for 48 h. Refed rats were sacrificed after being fed Purina rat chow or a high sucrose diet (69.5% sucrose, Dyets, Inc.) for 24 h after a 48-h fast. For subcellular localization experiments, liver cytosol, microsomes, rough and smooth endoplasmic reticulum, mitochondria-associated membrane (MAM), and mitochondria were isolated from male rats by a method (19Croze E.M. Morre D.J. J. Cell. Physiol. 1984; 119: 46-57Crossref PubMed Scopus (65) Google Scholar) modified by Vance (20Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar). Liver total membrane fraction, microsomes, and mitochondria were isolated from female rats by differential centrifugation (21Fleischer S. McIntyre J.O. Vidal J.C. Methods Enzymol. 1979; 55: 32-39Crossref PubMed Scopus (73) Google Scholar) in the presence of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) for subcellular localization and nutritional regulation experiments. Fractions were stored in aliquots at −80 °C. Protein concentrations were determined by the BCA method (Pierce) using bovine serum albumin as the standard. Peptides corresponding to regions of rat ACS1, ACS4, and ACS5 that show poor amino acid conservation (ACS1, MEVHELFRYFRMPELIDIR; ACS4, EIHSMQSVEELGSKPENSSI; ACS5, KCGIEMLSLHDAENL) were synthesized, purified, and coupled to keyhole limpet hemocyanin in the UNC/PMBB Micro Protein Chemistry Facility. Rabbit antibodies to these peptides and to purified mitochondrial GPAT were raised commercially in New Zealand White rabbits (ImmunoDynamics, La Jolla, CA). Antibodies to PEMT2 were made as described previously (22Cui Z. Vance J.E. Chen M.H. Voelker D.R. Vance D.E. J. Biol. Chem. 1993; 268: 16655-16663Abstract Full Text PDF PubMed Google Scholar). Proteins were separated on an 8% (or 12% for PEMT2) polyacrylamide gel containing 1% SDS and transferred to a polyvinylidene difluoride membrane (Bio-Rad). For chemiluminescent detection, the immunoreactive bands were visualized by incubating the membrane with horseradish peroxidase-conjugated goat anti-rabbit IgG and PicoWest reagents (Pierce). For quantitation, the polyvinylidene difluoride membrane was incubated with 0.5 μ Ci of 125I-Protein A (ICN), exposed to a phosphor screen, and quantified with the Molecular Dynamics Storm 840 and ImageQuant software. Diacylglycerol acyltransferase was assayed at 23 °C with 200 μm sn-1,2-diolein and 25 μm [3H]palmitoyl-CoA (23Coleman R.A. Bell R.M. J. Biol. Chem. 1980; 255: 7681-7687Abstract Full Text PDF PubMed Google Scholar), acyl-CoA synthetase was assayed at 37 °C with 5 mm ATP, 250 μm CoA, and 50 μm[3H]palmitate (24Banis R.J. Tove S.B. Biochim. Biophys. Acta. 1974; 348: 210-220Crossref PubMed Scopus (43) Google Scholar), and glycerol 3-phosphate acyltransferase (GPAT) was assayed at 23 °C with 300 μm [3H]glycerol-3-P and 112.5 μm palmitoyl-CoA in the presence or absence of 2 mm N-ethylmaleimide to inhibit the microsomal isoform (25Coleman R.A. Haynes E.B. J. Biol. Chem. 1983; 258: 450-465Abstract Full Text PDF PubMed Google Scholar). Microsomal GPAT was estimated by subtracting theN-ethylmaleimide-resistant activity (mitochondrial GPAT) from the total. All assays measured initial rates. [3H]Palmitoyl-CoA (26Merrill A.H.J. Gidwitz S. Bell R.M. J. Lipid Res. 1982; 23: 1368-1373Abstract Full Text PDF PubMed Google Scholar) and [3H]glycerol 3-phosphate (27Chang Y.-Y. Kennedy E.P. J. Lipid Res. 1967; 8: 447-455Abstract Full Text PDF PubMed Google Scholar) were synthesized enzymatically. The relative distribution of ACS activity in rat liver is 7% in peroxisomes, 20% in mitochondria, and 73% in microsomes (28Krisans S.K. Mortensen R.M. Lazarow P.B. J. Biol. Chem. 1980; 255: 9599-9607Abstract Full Text PDF PubMed Google Scholar). Since measurements of ACS activity do not distinguish among the different ACS isoforms, we localized each isoform in order to determine whether it is evenly distributed or, instead, located in a specific subcellular membrane. We raised isoform-specific rabbit antibodies against unique peptides present in ACS1, ACS4, or ACS5 because an antibody raised against purified ACS1 not only recognized purified recombinant ACS1, but also recognized purified recombinant ACS4 and ACS5 (Fig.1 A). The cross-reactivity is most likely due to the high degree of sequence similarity found among ACS1, ACS4, and ACS5. The specificity of each of our peptide antibodies was verified by immunoblot analysis of each isoform specific antibody against purified recombinant rat ACS1, ACS4, and ACS5, each with a C-terminal Flag epitope (29Kim J.-H. Lewin T.M. Coleman R.A. J. Biol. Chem. 2001; 276: 24667-24673Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Peptide antibodies for ACS4 and ACS5 each recognized only the correct ACS isoform (Fig. 1, C andD). The ACS1 peptide antibody failed to recognize recombinant ACS1-Flag (Fig. 1 B) because the initial two amino acids of the N-terminal sequence had been altered to clone the recombinant protein. However, the recombinant ACS1-Flag protein detected by the Flag antibody and the major band in rat liver microsomes detected by the ACS1 peptide antibody migrate to the same position, showing that the ACS1 peptide antibody recognizes native ACS1 (Fig. 1 E). Rat liver was fractionated by two different methods. One method used a sucrose gradient to further purify microsomes into ER1 and ER2 fractions that are enriched in rough and smooth ER, respectively (19Croze E.M. Morre D.J. J. Cell. Physiol. 1984; 119: 46-57Crossref PubMed Scopus (65) Google Scholar,20Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar). A Percoll gradient separated crude mitochondria into MAM and purified mitochondria (19Croze E.M. Morre D.J. J. Cell. Physiol. 1984; 119: 46-57Crossref PubMed Scopus (65) Google Scholar, 20Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar). The second method used only differential centrifugation (21Fleischer S. McIntyre J.O. Vidal J.C. Methods Enzymol. 1979; 55: 32-39Crossref PubMed Scopus (73) Google Scholar) and produced mitochondria that had very little microsomal contamination and microsomes that primarily contained ER membranes, as well as the MAM fraction. The purity of each fraction was ascertained by enzymatic assays for the ER enzymes DGAT and NEM-sensitive GPAT, and for the mitochondrial enzyme NEM-resistant GPAT and an by immunoblots for mitochondrial GPAT and for PEMT, the MAM marker. These markers showed that the microsome fraction was free of mitochondrial contamination because little NEM-resistant GPAT activity was present (Table I) and no mitochondrial GPAT protein was detected by immunoblotting (Fig.2 B). In the ER and MAM fractions purified using gradients, 11–28% of the GPAT activity was resistant to NEM (Table I), indicating that these fractions were somewhat contaminated with mitochondria. No PEMT was detected in the ER and purified mitochondria by immunoblot analysis (Fig. 2 B), indicating that these fractions were not contaminated with the MAM fraction. Very low DGAT and NEM-sensitive GPAT activities were measured in the purified mitochondria (Table I), indicating little contamination with ER. DGAT activity was enriched in the MAM fraction compared with ER, consistent with previous results (40Rusinol A.E. Cui Z. Chen M.H. Vance J.E. J. Biol. Chem. 1994; 269: 27494-27502Abstract Full Text PDF PubMed Google Scholar). The microsome fraction (100,000 × g pellet) also has high DGAT activity, and it contains the MAM fraction, as evidenced by the presence of the MAM-specific marker, PEMT (Fig. 2 B).Table IDGAT and GPAT activity in liver subcellular fractionsSpecific activity nmol/min/mg(n = 3)DGATGPAT1-aGPAT specific activity assayed in the absence of NEM.Mitochondrial GPAT1-bRemaining GPAT activity following treatment of sample with 2 mm NEM.Microsomes1-c100,000 × g pellet from supernatant fraction after isolation of crude mitochondria.1.72 ± 0.221.27 ± 0.160.06 ± 0.01 (4.7%)1-dPercent of GPAT specific activity assayed in the absence of NEM.ER11-eFraction isolated from sucrose gradient.0.31 ± 0.152.35 ± 0.230.51 ± 0.19 (22%)1-dPercent of GPAT specific activity assayed in the absence of NEM.ER21-eFraction isolated from sucrose gradient.0.59 ± 0.191.41 ± 0.090.29 ± 0.02 (20%)1-dPercent of GPAT specific activity assayed in the absence of NEM.MAM1-fFraction isolated from Percoll gradient.0.94 ± 0.072.05 ± 0.350.61 ± 0.32 (30%)1-dPercent of GPAT specific activity assayed in the absence of NEM.Crude mitochondria1-g10,000 × gpellet.0.38 ± 0.091.20 ± 0.070.64 ± 0.05 (54%)1-dPercent of GPAT specific activity assayed in the absence of NEM.Pure mitochondria1-fFraction isolated from Percoll gradient.0.19 ± 0.131.10 ± 0.351.00 ± 0.22 (91%)1-dPercent of GPAT specific activity assayed in the absence of NEM.1-a GPAT specific activity assayed in the absence of NEM.1-b Remaining GPAT activity following treatment of sample with 2 mm NEM.1-c 100,000 × g pellet from supernatant fraction after isolation of crude mitochondria.1-d Percent of GPAT specific activity assayed in the absence of NEM.1-e Fraction isolated from sucrose gradient.1-f Fraction isolated from Percoll gradient.1-g 10,000 × gpellet. Open table in a new tab The location of ACS1, ACS4, and ACS5 in rat liver was determined by Western blot analysis of various subcellular fractions. ACS1 (68 kDa) was strongly detected in the microsomal fraction, which comprises rough ER, smooth ER, and MAM (Fig. 2 A). The molecular mass is smaller than that predicted by the cDNA sequence (78 kDa), but in agreement with the size of the recombinant ACS1 run in the same manner (Fig. 1). A prominent ACS1 band was also detected in the cytosol, but cytosolic ACS specific activity was only 2.6% that of microsomal ACS specific activity (3.5 versus 132 nmol/min/mg of protein). Although ACS1 was present in crude mitochondria, which contain the MAM fraction, no ACS1 band was observed in purified mitochondria (Fig.2 A), as we reported previously (9Muoio D.M. Lewin T.M. Weidmar P. Coleman R.A. Am. J. Physiol. 2000; 279: E1366-E1373Crossref PubMed Google Scholar). ACS4 (74 kDa) was enriched in the MAM fractions from rat liver (Fig. 2 A) and Chinese hamster ovary cells (data not shown), and was only very weakly detected in microsomal and mitochondrial fractions. In some preparations, ACS4 was detected as a doublet with a larger 75-kDa protein (data not shown), consistent with alternative start sites as described for the human ACS4 homologue (30Cao Y. Murphy K.J. McIntyre T.M. Zimmerman G.A. Prescott S.M. FEBS Lett. 2000; 467: 263-267Crossref PubMed Scopus (58) Google Scholar). The ACS5-specific antiserum detected a protein of 76 kDa in the mitochondrial fraction (Fig. 2 A), 73- and 74.5-kDa proteins in ER, and a 74.5-kDa protein in cytosol and MAM fractions (Fig. 2 A). The 76-kDa protein agrees with the size predicted by the cDNA sequence and migrates to the same position as recombinant ACS5. Taken together, these data indicate that ACS1, ACS4, and ACS5 each have unique distributions in liver subcellular membranes. Since ACS1, ACS4, and ACS5 were present in different subcellular membranes and the recombinant proteins expressed inEscherichia coli were inhibited to different extents by triacsin C, thiazolidinediones, and NEM (29Kim J.-H. Lewin T.M. Coleman R.A. J. Biol. Chem. 2001; 276: 24667-24673Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), we hypothesized that these inhibitors would affect ACS activity differently in each fraction. Triacsin C (10 μm), which inhibits purified recombinant ACS1 and ACS4 by 60%, and does not inhibit ACS5 (29Kim J.-H. Lewin T.M. Coleman R.A. J. Biol. Chem. 2001; 276: 24667-24673Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), decreased ACS activity in microsomes, mitochondria, and MAM by 60% (Fig. 3). This result is surprising because the only ACS we identified in mitochondria was ACS5, which is resistant to inhibition by triacsin C (29Kim J.-H. Lewin T.M. Coleman R.A. J. Biol. Chem. 2001; 276: 24667-24673Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The mitochondria we assayed were free of contamination by the MAM fraction as determined by the absence of PEMT on an immunoblot (data not shown). Therefore, the observed inhibition suggests that mitochondria contain a hitherto undescribed triacsin-sensitive ACS. Troglitazone and NEM, two specific ACS4 inhibitors, were more selective inhibitors of ACS activity in liver subcellular fractions. Troglitazone at 10 μm decreased ACS activity 25% in MAM, but had no effect on activity in the microsomal or mitochondrial fractions (Fig.4). With 50 μmtroglitazone, ACS activity was inhibited 45% in MAM, again with little effect on microsomal or mitochondrial ACS activity. NEM, a second ACS4 inhibitor, decreased ACS activity 50% in MAM and only 27% in the smooth ER fraction (Fig. 5). These results are consistent with the immunolocalization of ACS4 preferentially to the MAM fraction.Figure 5N-Ethylmaleimide inhibits ACS activity in mitochondria-associated membrane. ER1 (enriched in rough ER) and MAM fractions (0.5–1.5 μg of protein) were pre-incubated in the absence or presence of 5 mm NEM for 10 min on ice prior to ACS assay. Control specific activities (100%): ER1 = 181 nmol/min/mg; MAM = 397 nmol/min/mg.View Large Image Figure ViewerDownload (PPT) If ACS1, ACS4, and ACS5 are linked to different metabolic pathways, one might expect that each isoform would be regulated differently under conditions of fasting and re-feeding. ACS activity and protein expression were measured in liver microsomes and mitochondria isolated by differential centrifugation from control rats, from rats fasted for 48 h, and from rats fasted for 48 h and then refed either Purina rat chow or a high sucrose diet for 24 h. The microsomes contained MAM, and the mitochondria were free of contamination by MAM as determined by immunoblot with antibody against PEMT, the MAM-specific marker (data not shown). ACS activity, assayed with palmitate as the fatty acid substrate, was similar in microsomes and in mitochondria isolated from control rats (18.5 ±3.5 and 14.3 ± 2.9, respectively), 48-h fasted rats (21.8 ± 0.9 and 11.9 ± 2.9, respectively), and rats fed Purina rat chow for 24 h after a 48-h fast (15.1 ± 4.5 and 14.2 ± 2.4, respectively). Although total ACS activity was unchanged by nutritional status, ACS1 protein expression increased 1.8- and 2-fold with Purina chow and high sucrose re-feeding, respectively (Fig.6). ACS4 protein expression also increased with re-feeding (50% with Purina chow diet and 64% with sucrose diet). ACS5 protein expression in mitochondria did not appear to be altered by re-feeding. After a 48-h fast, ACS1 protein expression decreased 14%, ACS4 protein decreased 47%, whereas ACS5 protein expression increased 82% in mitochondria (Fig. 6). Long chain acyl-CoA synthetase catalyzes the initial step required for oxidation, elongation, and desaturation of fatty acids; for the synthesis of complex lipids and acylated proteins; and for a variety of signals that regulate cellular metabolism (1Faergman N.J. Knudsen J. Biochem. J. 1997; 323: 1-12Crossref PubMed Scopus (576) Google Scholar, 2Prentki M. Corkey B.E. Diabetes. 1996; 45: 273-283Crossref PubMed Scopus (0) Google Scholar). It had been thought that the ACSs synthesize a common pool of acyl-CoAs, which move freely within cell membrane monolayers and have equal access to the numerous metabolic pathways in which they participate. However, genetic studies in yeast link specific ACS isoforms to different pathways that use acyl-CoAs (12Kamiryo T. Nishkawa Y. Mishina M. Terao M. Numa S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4390-4394Crossref PubMed Scopus (42) Google Scholar, 13Knoll L.J. Johnson D.R. Gordon J.I. J. Biol. Chem. 1995; 270: 10861-10867Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Knoll L.J. Johnson D.R. Gordon J.I. J. Biol. Chem. 1994; 269: 16348-16356Abstract Full Text PDF PubMed Google Scholar, 15Ashrafi K. Farazi T.A. Gordon J.I. J. Biol. Chem. 1998; 273: 25864-25874Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Hettema E.H. van Roermund C.W.T. Distel B. van den Berg M. Vilela C. Rodrigues-Pousada C. Wanders R.J.A. Tabak H.F. EMBO J. 1996; 15: 3813-3822Crossref PubMed Scopus (248) Google Scholar, 17Johnson D.R. Knoll L.J. Levin D.E. Gordon J.I. J. Cell Biol. 1994; 127: 751-762Crossref PubMed Scopus (137) Google Scholar). In addition, in cultured cells, inhibitors of ACS (triacsin and troglitazone) selectively alter the synthesis and oxidation of cellular lipids, suggesting that the ACS isoforms might be differentially inhibited, and that triacsin-sensitive isoforms might be functionally linked to de novo glycerolipid synthesis whereas triacsin-resis" @default.
• W2159868923 created "2016-06-24" @default.
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• W2159868923 creator A5021007054 @default.
• W2159868923 creator A5055571562 @default.
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• W2159868923 date "2001-01-01" @default.
• W2159868923 modified "2023-10-13" @default.
• W2159868923 title "Acyl-CoA Synthetase Isoforms 1, 4, and 5 Are Present in Different Subcellular Membranes in Rat Liver and Can Be Inhibited Independently" @default.
• W2159868923 cites W1453647352 @default.
• W2159868923 cites W1495373525 @default.
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