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The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6-amino acid substitution into the putative active site (amino acids 249 -254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464 -523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.The murine fatty acid transport protein (FATP1) 1 was identified and cloned by Schaffer and Lodish (1) from a 3T3-L1 adipocyte cDNA expression library and is localized to the plasma and other membranes of adipocytes and other target tissues such as brain, skeletal muscle, heart, and kidney (1). The FATP1 gene is conserved widely in biology from bacteria to mammals and is one of several putative transporters of fatty acids (1-4). Factors that control FATP1 gene expression have been investigated by a number of laboratories and reveal regulation by several effector systems: up-regulation during preadipocyte differentiation (1, 5) by peroxisome proliferator-activated receptors (6 -8) and by fasting (5) and down-regulation by insulin (9). Despite a growing body of knowledge relating to the control of FATP1 gene expression, studies on the FATP1 protein and its mechanistic role in fatty acid uptake have been limited (10).FATP1 exhibits broad-based amino acid similarity to a family of very long chain coenzyme A synthetases but exhibits only limited sequence similarity to the multigene family of long chain coenzyme A synthetases. Disruption of Saccharomyces cerevisiae FAT1, the yeast homologue to mammalian FATP1 (2), results in decreased fatty acid uptake, a substantial reduction in very long chain fatty acyl-CoA synthetase activity, and the accumulation of very long chain fatty acids (11, 12). Moreover, in animal cells, fatty acid uptake is diminished in cell lines overexpressing FATP1 if either endogenous ATP levels are depleted or if FATP1 is mutated (S250A) at a putative covalent AMP bin...

The keratinocyte lipid-binding protein (KLBP) has been identified on the basis of nucleotide sequence analysis of its cloned cDNA as a new member of the intracellular lipid-binding protein (iLBP) multigene family. To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was overexpressed and purified to homogeneity by a combination of acid extraction, gel permeation, and ion-exchange chromatographies. Purified KLBP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dissociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2). Using an assay based upon displacement of the bound fluorophore, KLBP was found to bind long chain fatty acids most avidly; oleic acid (18:1) bound with an apparent Kd of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM. As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K(d) of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at all. The protein did not bind prostaglandin E2 with any measurable affinity but did associate with eicosanoids such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE; K(d) of 848 +/- 211 nM) and 15-HPETE (Kd of 463 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HETE and 15-HETE (Kd of 1560 +/- 115 nM and greater than 4 microM, respectively). all-trans-Retinoic acid was a weak ligand for KLBP, binding with a Kd of 3600 nM, and all-trans-retinol did not displace 1,8-ANS. Molecular modeling of the KLBP sequence upon the X-ray crystal structures of several iLBP's suggested that the side chains of one or more cysteine residues may reside within the putative ligand-binding cavity. Consistent with this, sulfhydryl titration of purified KLBP with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modification by the fatty acid. These results describe the first purification and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a tertiary structure likely to be similar to other members of the iLBP multigene family.