The Fatty Acid Transport Protein (FATP1) Is a Very Long Chain Acyl-CoA Synthetase

Natalie Ribarik Coe,Anne Johnston Smith,Brigitte I Frohnert,Paul A Watkins,David A Bernlohr

doi:10.1074/jbc.274.51.36300

Natalie Ribarik Coe, Anne Johnston Smith + Show 3 more

Open Access

https://doi.org/10.1074/jbc.274.51.36300

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Abstract

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.

Highlights

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]
Since the mechanism of fatty acid activation with coenzyme A requires the formation of an enzyme-adenylate intermediate and uptake is linked to the presence of the AMP binding site, we hypothesized that FATP1 may be a plasma membrane very long chain acyl-CoA synthetase
These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that fatty acid uptake in mammalian cells is mediated by esterification-coupled influx, similar to the mechanism used by bacteria [13, 14]

Summary

Introduction

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]. Since the mechanism of fatty acid activation with coenzyme A requires the formation of an enzyme-adenylate intermediate and uptake is linked to the presence of the AMP binding site, we hypothesized that FATP1 may be a plasma membrane very long chain acyl-CoA synthetase. To test the hypothesis that FATP1 is a plasma membrane very long chain acyl-CoA synthetase, COS1 cells were transfected with an epitope-tagged FATP1 cDNA construct.

Results

Conclusion