The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase

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 syntheta se, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzym atic activity was analyzed. In addition, mutations were generated in two do mains conserved in acyl-CoA synthetases: a 6-amino acid substitution into t he putative active site (amino acids 249-254) generating mutant M1 and a 59 -amino acid deletion into a conserved C-terminal domain (amino acids 464-52 3) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracell ular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 8 -fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long c hain 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 i n very long chain acyl-CoA specific activity (C24:0/C16:0). These results i ndicate that FATP1 is a very long chain acyl-CoA synthetase and suggest tha t a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.