Characterization of the Acyl-CoA Synthetase Activity of Purified Murine Fatty Acid Transport Protein 1*

Fatty acid transport protein 1 (FATP1) is an ∼63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16–24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.

The mechanism by which fatty acids enter cells has been the source of considerable debate. Similar to other hydrophobic compounds, fatty acids are able to cross membranes by passive diffusion in a protein-independent mechanism (1, 2) driven by the concentration gradient on either side of the membrane. Whether passive diffusion within the biological context is sufficient to supply the amount of fatty acids required for tissues with high lipid flux remains to be determined. A second view contends that fatty acids traverse the plasma membrane via specific protein-mediated transporters (3,4). Studies in multiple tissue types, including cardiomyocytes and adipocytes (5,6), support the hypothesis that fatty acid transport occurs by a saturable protein-mediated mechanism and several candidate proteins have been identified.
A variety of proteins have been implicated in protein-mediated fatty acid import. These include the fatty acid-binding protein from the plasma membrane (FABP PM ) 1 and the fatty acid translocase (FAT/CD36) as well as the fatty acid transport protein family of molecules (FATP) (reviewed in Ref. 7). FATP1 is a member of a large family of related proteins from diverse organisms that increase fatty acid import when expressed in cultured cells (8,9). In mammalian cells, FATP isoforms 1-6 have been identified based on sequence similarity and have distinct tissue-specific distributions of expression (9). The yeast homologue (Fat1p) has been shown by genetic and biochemical analyses to be required for fatty acid uptake (10).
FATP1 shares sequence similarity with the family of acyl-CoA synthetases and has been demonstrated to increase very long chain acyl-CoA synthetase activity of extracts when expressed in COS1 cells (11). FATP1 contains two functional motifs: motif 1, which contains a putative site for the binding of ATP (via serine 250) that is part of the acyl-CoA synthetase reaction mechanism, and motif 2, which is proposed to be a ligand (fatty acid) binding domain (11)(12)(13). Overexpression of FATP1 in human embryonic kidney 293 cells results in an increased rate of fatty acid influx for both C18:1 and C24:0, which are channeled to triacylglycerol biosynthesis (14). As such, fatty acid transport protein 1 has been proposed to be a bifunctional protein capable of facilitating transbilayer movement of fatty acids and their metabolic activation (esterification) with coenzyme A.
To characterize the FATP1 acyl-CoA synthetase reaction, murine FATP1 was engineered to contain a C-terminal Myc-His tag, expressed in COS1 cells, and purified to homogeneity by nickel affinity chromatography. Additionally, the well studied long chain acyl-CoA synthetase 1 (ACS1) was cloned, expressed, and purified similarly to make a comparative analysis. Kinetic analysis of purified FATP1 and ACS1 was carried out for two model fatty acid substrates (C16:0 and C24:0), ATP, and CoA. These data lay the foundation for the biochemical dissection of FATP1-dependent fatty acid uptake and metabolic activation and provide evidence consistent with the model for vectoral acylation of fatty acids at the plasma membrane. Generation of FATP1 and ACS1 Recombinant Adenovirus-A recombinant adenovirus expressing both the green fluorescent protein (controlled by the cytomegalovirus promoter) and either murine FATP1 or ACS1 were constructed by recombination in Escherichia coli using the methods described by He and colleagues (15). The resulting construct was recombined into pADEasy in E. coli BJ5183 cells recreating the replication-deficient adenovirus genome. Linear constructs of the recombinant adenovirus were transfected (LipofectAMINE, Invitrogen) into 293 cells (American Type Culture Collection, Manassas, VA) to allow packaging and amplification of the adenovirus. Large-scale adenovirus preparations from twenty 10-cm plates of infected cells were propagated until ϳ50% of the cells were lysed. The cells and medium were collected, and the remaining cells were lysed by three freeze-thaw cycles. The medium was centrifuged at 20,000 ϫ g for 10 min to pellet the cellular debris, and the supernatant containing virus particles was recovered and frozen in aliquots at Ϫ70°C.

Reagents-[
Protein Expression of Recombinant FATP1 and ACS1 Proteins in COS1 Cells-FATP1 and ACS1 His-tagged proteins were expressed in COS1 cells using adenovirus infection. Prior to infection, COS1 cells were plated in 10-cm plates and grown to ϳ80% confluency at 37°C in a 5% CO 2 incubator. For infection, the effective concentration of infectious adenoviral particles was experimentally determined by monitoring green fluorescent protein expression and COS1 cell viability 72 h post-infection. Adenovirus particles that yielded ϳ90 -100% infection were delivered in 8 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum/plate. 72 h post-transfection, cells were harvested by centrifugation, immediately frozen, and stored at Ϫ70°C.
Affinity Purification of Recombinant FATP1 and ACS1 Proteins-COS1 cells expressing FATP1 or ACS1 were thawed in buffer A (150 mM Tris-HCl, pH 7.5, 250 mM sucrose, and 150 mM NaCl), subjected to five freeze-thaw cycles, and solubilized with 2% Triton X-100 for 4 h at 4°C. The soluble fraction was separated from debris by centrifugation at 100,000 ϫ g for 1 h at 4°C and recovered, and glycerol was added to a final concentration of 20%. For purification of recombinant proteins, the nickel affinity matrix beads (Qiagen) were equilibrated with buffer A plus 30 mM imidazole and the bound protein eluted with five column volumes of buffer A plus 150 mM imidazole. Eluates were pooled, aliquoted, and stored at Ϫ70°C until use. FATP1 and ACS1 migrated as single bands on a 12% SDS-polyacrylamide gel and were judged to be at least 90% pure by Coomassie Blue staining. The level of murine FATP1 enrichment obtained from the Ni 2ϩ column purification was ϳ2000-fold as assessed by comparison of the specific activity of the loaded fraction (0.5 nmol/min/mg for C16:0) to the eluted fraction (1000 nmol/min/mg for C16:0). To determine the amount of purified protein, samples were precipitated (16) and protein concentration was determined by the bicinchoninic acid method (Pierce) with bovine serum albumin as the standard.
Fatty Acyl-CoA Synthetase Assays-Samples were assayed for acyl-CoA synthetase activity by the conversion of [ 3 H]palmitate or [ 3 H]lignocerate to their CoA derivatives by a modified method from Nagamatsu et al. (17). All of the kinetic studies reported used 0.5-2 g of purified FATP1 or ACS1 for 2 min at pH 7.5 and 30 mM NaCl in 250 l of a buffer containing 20 M fatty acid, 100 mM Tris-HCl, pH 7.5, 10 mM ATP, 5 mM MgCl 2 , 200 M CoA, and 200 M dithiothreitol. The addition of enzyme purified in the presence of 2% Triton X-100 resulted in a final concentration of 0.4% Triton X-100 in all of the standard assays. Reactions were terminated with the addition of 1.25 ml of isopropyl alcohol:heptane:H 2 SO 4 (40:10:1, v/v/v), 0.5 ml of H 2 0, and 0.75 ml of heptane to facilitate organic phase separation. The aqueous phase was extracted three times with 0.75 ml of heptane to remove unreacted fatty acids, and the radioactivity was determined by liquid phase scintillation counting. The enzyme activity was stable in 20% glycerol and was retained for 2 months without significant loss of activity when stored at Ϫ70°C.
Competition Studies-Because of the unavailability of commercially radiolabeled fatty acids, the lipid substrate specificity of the purified FATP1 was indirectly determined by competitive enzymatic inhibition analysis. In these studies, unlabeled fatty acids at a fixed concentration of 15 M was added to either [ 3 H]palmitic acid or [ 3 H]lignoceric acid and the rate of reaction was monitored. A selectivity series for fatty preference was generated by determining the fraction of acyl-CoA synthetase activity at [S] 0.5 . The detergent-like effects of fatty acids are not significant because 0.4% Triton X-100 is present in the standard assays.

RESULTS
Expression and Purification of Recombinant FATP1 Protein-Previous studies in cellular extracts have suggested that FATP1 is an acyl-CoA synthetase (11). However, an alternate possibility is that FATP1 activates a latent endogenous acyl-CoA synthetase present in cells that results in increased fatty acid esterification. Alternatively, the fatty acid transport activity of FATP1 could lead to an influx of fatty acids that in turn generates signaling molecules, which regulate the expression of an acyl-CoA synthetase gene. To demonstrate that FATP1 exhibits intrinsic fatty acid CoA synthetase activity and to kinetically characterize the properties of such a reaction, murine FATP1 was purified and studied. To facilitate purification, FATP1 was C-terminal tagged with a Myc epitope and a polyhistidine tract. The purification involved detergent extraction of FATP1 from the membrane with Triton X-100 and chromatography on Ni 2ϩ -agarose resin. Fig. 1 presents the SDS-PAGE analysis of fractions collected during a typical FATP1 purification. The purified enzyme was essentially homogenous as shown by SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of ϳ68 kDa (Fig. 1), a value that agrees well with the calculated molecular mass from the deduced amino acid sequence of the cDNA (63 kDa) plus that of the Myc-His epitope and linker amino acids.
Optimization of the FATP1 Acyl-CoA Synthetase Reaction-An analysis of very long chain acyl-CoA synthetase activity enriched from rat liver microsomes demonstrated an inhibitory effect of Triton X-100 on the activity (18). To deter- mine whether FATP1 acyl-CoA synthetase activity was sensitive to Triton X-100, purified enzyme was assayed with increasing concentration detergent. FATP1 activity (for both C16:0 and C24:0 esterification) was sensitive to Triton X-100 in the reaction buffer (Fig. 2). To establish standard reaction conditions, 0.4% Triton X-100 in the final assay buffer was adopted as routine. This level of Triton X-100 partially inhibited both FATP1 catalytic activity but was in a region of the progress curve that was relatively insensitive to small changes in detergent concentrations.
The FATP1 acyl-CoA synthetase activity was optimized with respect to several standard reaction parameters for both long chain (C16:0) and very long chain (C24:0) fatty acids. As shown in Fig. 3, the activity was dependent on and proportional to the amount of enzyme added to the reaction (Fig. 3A) as well as the time of reaction (Fig. 3B). In addition, the pH sensitivity of the reaction was evaluated (Fig. 3C) as was the influence of ionic strength (Fig. 3D). The purified FATP1 has broad pH dependence with 6.5-8.0 being generally optimal, and there was little effect on enzymatic activity when the NaCl concentration was varied from 30 to 150 mM; however, the activity was reduced by 80% at 500 mM. Importantly, optimization utilizing C16:0 as a substrate yielded results that were essentially identical to those obtained with C24:0 as a substrate.
Kinetic Properties of FATP1-The fatty acid esterification properties of purified FATP1 were measured for two model lipid substrates as well as for CoA and ATP. The apparent K m values of the purified enzyme were determined for palmitic acid (C16:0), lignoceric acid (C24:0), ATP, and CoA at 37°C (Fig. 4). FATP1 demonstrated high affinity toward its substrates and co-substrates, exhibiting a K m of 21 M for C16:0, 13 M for C24:0, 850 M for ATP, and 8.3 M for CoA (Table I). It should be stressed that because these studies are done in the presence of detergent, the free concentration of fatty acids cannot be determined and the K m values reported for each fatty acid represent apparent values assuming that all of the lipid was available to the enzyme. Because the solubility of lignoceric acid and palmitic acid vary greatly, this assumption is probably incorrect but represents an experimentally tractable method for analyzing the data and comparing one enzyme to another. A catalytic constant of 1.5 s Ϫ1 at 37°C was calculated from the molecular mass of the purified enzyme (68 kDa) and the maximal specific activity of 1000 nmol/min/mg for C16:0.
The kinetic properties of purified ACS1 protein were evaluated and compared with those for FATP1. The kinetic profile obtained in this study closely resembled that for both purified native ACS1 and FLAG-tagged ACS1 (Fig. 4) (15). Purified His-tagged ACS1 demonstrated the expected high affinities toward C16:0, ATP, and CoA with K m values of 33, 320, and 6.4 M, respectively (Table I). Interestingly, ACS1 also utilized very long chain fatty acids (C24:0) with a K m of 18 M. The maximal velocity of ACS1 for C16:0 was approximately 3200 nmol/min/mg but was greatly reduced for C24:0 to 240 nmol/ min/mg. Consistent with the reduced velocity for very long chain fatty acids, ACS1 was 10-fold more active toward C16:0 than C24:0 as demonstrated by V max /K m values of 100 and 15, respectively. FATP1 exhibited similar activities toward C16:0 (V max /K m of 6) and increased specificity for C24:0 (V max /K m of 17).
Fatty Acid Specificity of FATP1-Because a large number of different fatty acids are not commercially available in radiolabeled form, the fatty acid substrate specificity of the purified FATP1 was indirectly determined by a competitive enzymatic inhibition by the addition of unlabeled fatty acids in the reaction in addition to either [ 3 H] palmitic acid or [ 3 H] lignoceric acid (Fig. 5). Esterification of labeled lignoceric acid was effectively inhibited by the addition of 15 M of various long chain fatty acids with the rank order of C18:1 Ͼ C20:4 Ͼ C16:0 Ͼ C12. Similarly, the conversion of [ 3 H]palmitate to palmitoyl-CoA was decreased by the addition of very long chain fatty acids with the rank order of C22:0 ϭ C20:0 Ͼ C24:0 Ͼ C16:0. As  with the other kinetic analysis, because of the differing solubilities of the various competing fatty acids, true K i values cannot be determined and a simple selectivity series is presented. These results are consistent with a model for FATP1 having a single fatty acid binding domain that is utilized for esterification of both long and very long chain fatty acids.
FATP1 Is Subject to Feedback Inhibition-Reports of the cellular concentration of long chain acyl-CoA esters vary between 5 and 160 M (19,20). To determine whether FATP1 is regulated by feedback inhibition, increasing concentrations of palmitoyl-CoA were titrated into the standard reaction conditions (Fig. 6) and the activity of FATP1 evaluated. At a concentration of 10 M palmitoyl-CoA, the FATP1 acyl-CoA synthetase reaction was inhibited by ϳ20 -25%, whereas 100 M inhibited the reaction by greater than 60% for both C16:0 and C24:0. Although to a slightly less degree, a similar inhibition was observed for ACS1.
FATP1 Is Insensitive to Triacsin C Inhibition-Triacsin C has been reported to be a potent competitive inhibitor of ACS1 and ACS4 (21). However, studies in crude cellular extracts suggested that FATP1 may not be similarly affected (14). To test whether triacsin C inhibits purified FATP1, various concentrations were added to the standard reaction mixture (Fig.  7) and the conversion of C16:0 and C24:0 was evaluated. Surprisingly, triacsin C had no effect on FATP1 acyl-CoA synthetase activity, whereas ACS1 was inhibited in the reported dose-dependent manner with an IC 50 of 1 M. Troglitazone at concentrations up to 10 M inhibited FATP1-dependent acylation of C16:0 or C24:0 by only 20% (data not shown).

DISCUSSION
Initial studies to identify proteins that facilitate fatty acid transport into mammalian cells focused on the ability of the protein to bind fatty acids. FABP PM was purified by oleateagarose affinity chromatography from hepatocytes, adipocytes, intestinal epithelial cells, and cardiomyocytes and was proposed to play a role in fatty acid import (22). Partial protein sequencing of FABP PM revealed an identity with mitochondrial aspartate aminotransferase. Furthermore, antibodies to mitochondrial aspartate aminotransferase were able to inhibit uptake of oleate into 3T3-L1 adipocytes, further supporting the conclusion that these proteins are identical (23). CD36, also known as FAT, was identified based on its ability to bind a cross-linking sulfonsuccinimidyl fatty acid derivative (24). Influx of fatty acid analogs is reduced in the heart, skeletal muscle, and adipose tissues in CD36-null mice (25). CD36 also functions as a multi-ligand scavenger receptor, binding oxidized low density lipoproteins, thrombospondin, and collagen. The additional functions of FABP PM and FAT complicate their interpretation and role in fatty acid uptake.
The FATP family of proteins was originally identified by the ability to facilitate fatty acid uptake using a fluorescent fatty acid internalization assay (8). Sequence analysis of cloned FATP family members revealed two sequence motifs indicative of acyl-CoA synthetases. Subsequent studies have demonstrated that the overexpression of FATP1, FATP2, or FATP4 in COS1 cells increases cell-associated acyl-CoA synthetase activities (11,26,27). Moreover, mutation of serine 250 to alanine significantly reduced fatty acid uptake, suggesting that esterification of fatty acids is coupled to influx (28). The current study was undertaken to evaluate the catalytic properties of the FATP1 acyl-CoA synthetase reaction. Here we demonstrate that purified FATP1 has an intrinsic acyl-CoA synthetase activity with broad specificity for both long and very long chain fatty acids. FATP1 is a low velocity enzyme compared with ACS1, the traditional enzyme believed to esterify fatty acids broadly in cells. Although ACS1 prefers C16:0 over C24:0, FATP1 has comparable specific activities toward C16:0 and C24:0. This observation with purified FATP1 was masked in previous studies utilizing COS cell extracts attributed to a high level of endogenous long chain acyl-CoA synthetase activity. The FATP1 K m values measured for CoA and ATP are similar to those measured for ACS1.
The fundamental question arising from the observations that FATP1 functions in both fatty acid transport and fatty acid activation is whether fatty acid import is driven by this intrinsic acyl-CoA synthetase activity (vectoral acylation) or whether these activities are distinct and the enzyme is bifunctional. Using site-directed mutagenesis of the FATP1 orthologue in yeast Fat1p, fatty acid uptake and fatty acid activation are generally but not universally coupled processes (29). Four FAT1 alleles distinguish fatty acid transport and very long chain acyl-CoA synthetase activity, suggesting separable functions. Interestingly, the FAT1 T260A mutant exhibited only moderately reduced fatty acid import and acyl-CoA synthetase activity, whereas the corresponding mutant in murine FATP1, T252G, eliminates fatty acid uptake. Further analysis of the molecular determinants that characterize the two activities will be needed to critically evaluate the relationship between transport and catalysis.
Recently, Zou et al. (30) demonstrated in Saccharomyces cerevisiae that the yeast orthologues of FATP1 and ACS1, Fat1p, and Faa1p or Faa4p function cooperatively to form a physical complex and mediate the import of exogenous long chain fatty acids. This conclusion was supported by several lines of biochemical evidence. Firstly, a C-terminal truncated peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long chain acyl-CoA synthetase activity. Secondly, a fusion protein of Fat1p as the bait and Faa1p as the trap were active partners when tested in the yeast two-hybrid system. Finally, Fat1p co-immunoprecipitated with Faa1p and Faa4p when expressed together. Interestingly, FATP1 has been linked to ACS1 by both functional assays and cellular localization in adipocytes (8,31), suggesting that they may also form a physical complex to facilitate fatty acid transport in mammalian cells through vectoral acylation. Preliminary experiments with FATP1 and ACS1 co-expressed into COS cells suggest no significant kinetic differences in FATP1 acyl-CoA synthetase activity when assayed in the presence of triacsin C. 2 In summary, these results demonstrate that FATP1 exhibits intrinsic acyl-CoA synthetase activity and is a broad substrate enzyme. This report represents the first characterization of the enzymatic activity of the protein in purified form. For long chain fatty acids, the prototypical ACS1 enzyme exhibits a higher V max /K m ratio while for very long chain fatty acid the enzymes are comparable. This finding may suggest that ACS family members should functionally compensate for disruptions of FATP and that loss of the fatty acid transport protein would not be metabolically significant. However, striking evidence from FATP4 null mice (32) that exhibit a wrinkle-free phenotype reminiscent of essential fatty acid deficiency sug-gests unique specialized roles for the fatty acid transport proteins and that their physiological significance cannot be overlooked. Additional experimentation will be required to dissect the molecular roles of the FATP transport and esterification activity as well as their metabolic functions in vivo.