Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: identification of a novel variant of isoform 6

The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.     

Characterization of the porcine acyl-CoA synthetase long-chain 4 gene and its association with growth and meat quality traits

Summary Long-chain acyl-CoA synthetase (ACSL) catalyses the formation of long-chain acyl-CoA from fatty acid, ATP and CoA, activating fatty acids for subsequent reactions. Long-chain acyl-CoA synthetase thus plays an essential role in both lipid biosynthesis and fatty acid degradation. The ACSL4 gene was evaluated as a positional candidate gene for the quantitative trait loci (QTL) located between SW2456 and SW1943 on chromosome X. We have sequenced 4906 bp of the pig ACSL4 mRNA. Sequence analysis allowed us to identify 10 polymorphisms located in the 3'-UTR region and to elucidate two ACSL4 haplotypes. Furthermore, a QTL and an association study between polymorphisms of the ACSL4 gene and traits of interest were carried out in an Iberian x Landrace cross. We report QTL that have not been previously identified, and we describe an association of the ACSL4 polymorphisms with growth and percentage of oleic fatty acid. Finally, we have determined allelic frequencies in 140 pigs belonging to the Iberian, Landrace, Large White, Meishan, Pietrain, Duroc, Vietnamese, Peccary and Babirusa populations.     

Role of ACSL4 in the chemical-induced cell death in human proximal tubule epithelial HK-2 cells

Acyl-CoA synthetase long-chain family member 4 (ACSL4) activates polyunsaturated fatty acids (PUFAs) to produce PUFA-derived acyl-CoAs, which are utilised for the synthesis of various biological components, including phospholipids (PLs). Although the roles of ACSL4 in non-apoptotic programmed cell death ferroptosis are well-characterised, its role in the other types of cell death is not fully understood. In the present study, we investigated the effects of ACSL4 knockdown on the levels of acyl-CoA, PL, and ferroptosis in the human normal kidney proximal tubule epithelial (HK-2) cells. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses revealed that the knockdown of ACSL4 markedly reduced the levels of PUFA-derived acyl-CoA, but not those of other acyl-CoAs. In contrast with acyl-CoA levels, the docosahexaenoic acid (DHA)-containing PL levels were preferentially decreased in the ACSL4-knockdown cells compared with the control cells. Cell death induced by the ferroptosis inducers RSL3 and FIN56 was significantly suppressed by treatment with ferrostatin-1 or ACSL4 knockdown, and, unexpectedly, upon treating with a necroptosis inhibitor. In contrast, ACSL4 knockdown failed to suppress the other oxidative stress-induced cell deaths initiated by cadmium chloride and sodium arsenite. In conclusion, ACSL4 is involved in the biosynthesis of DHA-containing PLs in HK-2 cells and is specifically involved in the cell death induced by ferroptosis inducers.     

Crystal structure of mouse carnitine octanoyltransferase and molecular determinants of substrate selectivity

Carnitine acyltransferases have crucial functions in fatty acid metabolism. Members of this enzyme family show distinctive substrate preferences for short-, medium- or long-chain fatty acids. The molecular mechanism for this substrate selectivity is not clear as so far only the structure of carnitine acetyltransferase has been determined. To further our understanding of these important enzymes, we report here the crystal structures at up to 2.0-A resolution of mouse carnitine octanoyltransferase alone and in complex with the substrate octanoylcarnitine. The structures reveal significant differences in the acyl group binding pocket between carnitine octanoyltransferase and carnitine acetyltransferase. Amino acid substitutions and structural changes produce a larger hydrophobic pocket that binds the octanoyl group in an extended conformation. Mutation of a single residue (Gly-553) in this pocket can change the substrate preference between short- and medium-chain acyl groups. The side chains of Cys-323 and Met-335 at the bottom of this pocket assume dual conformations in the substrate complex, and mutagenesis studies suggest that the Met-335 residue is important for catalysis.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

Mouse cardiac acyl coenzyme a synthetase 1 deficiency impairs Fatty Acid oxidation and induces cardiac hypertrophy

Long-chain acyl coenzyme A (acyl-CoA) synthetase isoform 1 (ACSL1) catalyzes the synthesis of acyl-CoA from long-chain fatty acids and contributes the majority of cardiac long-chain acyl-CoA synthetase activity. To understand its functional role in the heart, we studied mice lacking ACSL1 globally (Acsl1(T-/-)) and mice lacking ACSL1 in heart ventricles (Acsl1(H-/-)) at different times. Compared to littermate controls, heart ventricular ACSL activity in Acsl1(T-/-) mice was reduced more than 90%, acyl-CoA content was 65% lower, and long-chain acyl-carnitine content was 80 to 90% lower. The rate of [(14)C]palmitate oxidation in both heart homogenate and mitochondria was 90% lower than in the controls, and the maximal rates of [(14)C]pyruvate and [(14)C]glucose oxidation were each 20% higher. The mitochondrial area was 54% greater than in the controls with twice as much mitochondrial DNA, and the mRNA abundance of Pgc1α and Errα increased by 100% and 41%, respectively. Compared to the controls, Acsl1(T-/-) and Acsl1(H-/-) hearts were hypertrophied, and the phosphorylation of S6 kinase, a target of mammalian target of rapamycin (mTOR) kinase, increased 5-fold. Our data suggest that ACSL1 is required to synthesize the acyl-CoAs that are oxidized by the heart, and that without ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids lead to mTOR activation and cardiac hypertrophy without lipid accumulation or immediate cardiac dysfunction.     

An Essential Role of the N-Terminal Region of ACSL1 in Linking Free Fatty Acids to Mitochondrial β-Oxidation in C2C12 Myotubes

Free fatty acids are converted to acyl-CoA by long-chain acyl-CoA synthetases (ACSLs) before entering into metabolic pathways for lipid biosynthesis or degradation. ACSL family members have highly conserved amino acid sequences except for their N-terminal regions. Several reports have shown that ACSL1, among the ACSLs, is located in mitochondria and mainly leads fatty acids to the β-oxidation pathway in various cell types. In this study, we investigated how ACSL1 was localized in mitochondria and whether ACSL1 overexpression affected fatty acid oxidation (FAO) rates in C2C12 myotubes. We generated an ACSL1 mutant in which the N-terminal 100 amino acids were deleted and compared its localization and function with those of the ACSL1 wild type. We found that ACSL1 adjoined the outer membrane of mitochondria through interaction of its N-terminal region with carnitine palmitoyltransferase-1b (CPT1b) in C2C12 myotubes. In addition, overexpressed ACSL1, but not the ACSL1 mutant, increased FAO, and ameliorated palmitate-induced insulin resistance in C2C12 myotubes. These results suggested that targeting of ACSL1 to mitochondria is essential in increasing FAO in myotubes, which can reduce insulin resistance in obesity and related metabolic disorders.     

Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids.

The primary ligands of human serum albumin (HSA), an abundant plasma protein, are non-esterified fatty acids. In vivo, the majority of fatty acids associated with the protein are unsaturated. We present here the first high-resolution crystal structures of HSA complexed with two important unsaturated fatty acids, the monounsaturated oleic acid (C18:1) and the polyunsaturated arachidonic acid (C20:4). Both compounds are observed to occupy the seven binding sites distributed across the protein that are also bound by medium and long-chain saturated fatty acids. Although C18:1 fatty acid binds each site on HSA in a conformation almost identical with that of the corresponding saturated compound (C18:0), the presence of multiple cis double bonds in C20:4 induces distinct binding configurations at some sites. The observed restriction on binding configurations plausibly accounts for differences in the pattern of binding affinities for the primary sites between polyunsaturated fatty acids and their saturated or monounsaturated counterparts.     

Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP-forming, EC ) catalyzes the esterification of fatty acids to CoA thioesters for further metabolism and is hypothesized to play a pivotal role in the coupled transport and activation of exogenous long-chain fatty acids in Escherichia coli. Previous work on the bacterial enzyme identified a highly conserved region (FACS signature motif) common to long- and medium-chain acyl-CoA synthetases, which appears to contribute to the fatty acid binding pocket. In an effort to further define the fatty acid-binding domain within this enzyme, we employed the affinity labeled long-chain fatty acid [(3)H]9-p-azidophenoxy nonanoic acid (APNA) to specifically modify the E. coli FACS. [(3)H]APNA labeling of the purified enzyme was saturable and specific for long-chain fatty acids as shown by the inhibition of modification with increasing concentrations of palmitate. The site of APNA modification was identified by digestion of [(3)H]APNA cross-linked FACS with trypsin and separation and purification of the resultant peptides using reverse phase high performance liquid chromatography. One specific (3)H-labeled peptide, T33, was identified and following purification subjected to NH(2)-terminal sequence analysis. This approach yielded the peptide sequence PDATDEIIK, which corresponded to residues 422 to 430 of FACS. This peptide is immediately adjacent to the region of the enzyme that contains the FACS signature motif (residues 431-455). This work represents the first direct identification of the carboxyl-containing substrate-binding domain within the adenylate-forming family of enzymes. The structural model for the E. coli FACS predicts this motif lies within a cleft separating two distinct domains of the enzyme and is adjacent to a region that contains the AMP/ATP signature motif, which together are likely to represent the catalytic core of the enzyme.     

Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.     

Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.     

Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells

Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors α and β (LXRα and LXRβ), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.     

Overexpressed FATP1, ACSVL4/FATP4 and ACSL1 Increase the Cellular Fatty Acid Uptake of 3T3-L1 Adipocytes but Are Localized on Intracellular Membranes

Long chain acyl-CoA synthetases are essential enzymes of lipid metabolism, and have also been implicated in the cellular uptake of fatty acids. It is controversial if some or all of these enzymes have an additional function as fatty acid transporters at the plasma membrane. The most abundant acyl-CoA synthetases in adipocytes are FATP1, ACSVL4/FATP4 and ACSL1. Previous studies have suggested that they increase fatty acid uptake by direct transport across the plasma membrane. Here, we used a gain-of-function approach and established FATP1, ACSVL4/FATP4 and ACSL1 stably expressing 3T3-L1 adipocytes by retroviral transduction. All overexpressing cell lines showed increased acyl-CoA synthetase activity and fatty acid uptake. FATP1 and ACSVL4/FATP4 localized to the endoplasmic reticulum by confocal microscopy and subcellular fractionation whereas ACSL1 was found on mitochondria. Insulin increased fatty acid uptake but without changing the localization of FATP1 or ACSVL4/FATP4. We conclude that overexpressed acyl-CoA synthetases are able to facilitate fatty acid uptake in 3T3-L1 adipocytes. The intracellular localization of FATP1, ACSVL4/FATP4 and ACSL1 indicates that this is an indirect effect. We suggest that metabolic trapping is the mechanism behind the influence of acyl-CoA synthetases on cellular fatty acid uptake.     

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.     

Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 +/- 3 vs. 67 +/- 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 +/- 6 vs. 100 +/- 6 nmol/g protein in controls, P < 0.05) and increased [(14)C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 +/- 4 vs. 20 +/- 2 mumol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-(14)C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.     

Identification of potential physiological activators of protein phosphatase 5

The protein serine/threonine phosphatase designated PP5 has little basal activity, and physiological activators of the enzyme have never been identified. Purified PP5 can, however, be activated by partial proteolysis or by the binding of supraphysiological concentrations of polyunsaturated long-chain fatty acids to its tetratricopeptide repeat (TPR) domain. To test whether activation of PP5 by polyunsaturated but not saturated fatty acids was an artifact of the lower solubility of saturated fatty acids, the effects of fatty acyl-CoA esters were examined. Saturated and unsaturated long-chain fatty acids are both freely water-soluble when esterified to CoA. Long-chain fatty acyl-CoA esters activated PP5 at physiological concentrations, with the saturated compounds being more effective. We investigated the effects of chain length and of the CoA moiety on PP5 activation. Chains of 16 carbons or more were required for optimal activation, with no activation observed below 10 carbons. On the basis of competition studies using acetyl-CoA, the function of the CoA moiety appeared to be to increase solubility of the fatty acyl moiety rather than to interact with a specific binding site. These data suggested that long-chain fatty acid-CoA esters might be physiological activators of PP5 and point to a potential link between fatty acid metabolism and signal transduction via this enzyme. Because heat shock protein 90 is also known to bind to the TPR domain of PP5 via its C-terminal domain (C90), we investigated its effect on PP5 activity. C90 activated the enzyme approximately 10-fold. Thus, we have identified two potential physiological activators of PP5.     

Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases

Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.     

Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol

The acyl-CoA synthetase 4 (ACSL4) has been implicated in carcinogenesis and neuronal development. Acyl-CoA synthetases are essential enzymes of lipid metabolism, and ACSL4 is distinguished by its preference for arachidonic acid. Two human ACSL4 isoforms arising from differential splicing were analyzed by ectopic expression in COS cells. We found that the ACSL4_v1 variant localized to the inner side of the plasma membrane including microvilli, and was also present in the cytosol. ACSL4_v2 contains an additional N-terminal hydrophobic region; this isoform was located at the endoplasmic reticulum and on lipid droplets. A third isoform was designed de novo by appending a mitochondrial targeting signal. All three ACSL4 variants showed the same specific enzyme activity. Overexpression of the isoenzymes increased cellular uptake of arachidonate to the same degree, indicating that the metabolic trapping of fatty acids is independent of the subcellular localization. Remarkably, phospholipid metabolism was changed by ACSL4 expression. Labeling with arachidonate showed that the amount of newly synthesized phosphatidylinositol was increased by all three ACSL4 isoenzymes but not by ACSL1. This was dependent on the expression level and the localization of the ACSL4 isoform. We conclude that in our model system exogenous fatty acids are channeled preferentially towards phosphatidylinositol by ACSL4 overexpression. The differential localization of the endogenous isoenzymes may provide compartment specific precursors of this anionic phospholipid important for many signaling processes.     

The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae.

Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta-oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium-chain fatty acids are activated inside peroxisomes hby the acyl-CoA synthetase Faa2p. On the other hand, long-chain fatty acids are imported from the cytosolic pool of activated long-chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.