Abstract
Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.
Highlights
Studies in multiple tissue types, including cardiomyocytes and adipocytes [1], support the hypothesis that fatty acid transport occurs by a saturable, protein-mediated mechanism, and several candidate proteins responsible for fatty acid uptake have been identified
The objective of the study was to determine whether Fatty acid transport protein 4 (FATP4) is an acyl-CoA synthetase, to assess the substrate specificity and intrinsic catalytic efficacy of the purified protein, and to compare those properties to FATP1 in order to determine whether fatty acid transport proteins have similar
FATP4 was C-terminally tagged with a FLAG epitope and over-expressed in COS1 cells by a recombinant adenoviral infection method
Summary
Studies in multiple tissue types, including cardiomyocytes and adipocytes [1], support the hypothesis that fatty acid transport occurs by a saturable, protein-mediated mechanism, and several candidate proteins responsible for fatty acid uptake have been identified. Fat1p functions in concert with acyl-CoA synthetases (Faa1p and/or Faa4p) to mediate fatty acid influx [6, 7]; from a combination of molecular and cellular studies it has been inferred that they function as part of a fatty acid import complex, such an entity has not been demonstrated. Watkins and colleagues [9] have shown that FATP3 is an acyl-CoA synthetase but that the protein does not facilitate fatty acid internalization into cultured cells These results suggest that the acyl-CoA synthetase and transport functions of FATPs might be separable functions. The S250A mutant of murine FATP1, as well as multiple yeast FAT1 mutants lacking acyl-CoA synthetase, activity exhibits greatly diminished fatty acid influx, supporting the hypothesis that fatty acid uptake into cells is linked, at least in part, to their esterification with coenzyme A in a process termed vectoral acylation [11, 12]. We present data characterizing FATP4 as a high velocity enzyme with specificity for long and very long chain fatty acids as well as the acyl-CoA synthetase activity of tissues from wild type and FATP4 ablated mice, suggesting that in vivo, the transport and/or acyl-CoA synthetase activity for very long chain fatty acids is critical for FATP4 function
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