The fatty acid transport proteins (FATP) and longchain acyl coenzyme A synthetase (ACSL) proteins have been shown to play a role in facilitating long-chain fatty acid (LCFA) transport in mammalian cells under physiologic conditions. The involvement of both FATP and ACSL proteins is consistent with the model of vectorial acylation, in which fatty acid transport is coupled to esterification. This study was undertaken to determine whether the functions of these proteins are coordinated through a protein-protein interaction that might serve as a point of regulation for cellular fatty acid transport. We demonstrate for the first time that FATP1 and ACSL1 coimmunoprecipitate in 3T3-L1 adipocytes, indicating that these proteins form an oligomeric complex. The efficiency of FATP1 and ACSL1 coimmunoprecipitation is unaltered by acute insulin treatment, which stimulates fatty acid uptake, or by treatment with isoproterenol, which decreases fatty acid uptake and stimulates lipolysis. Moreover, inhibition of ACSL1 activity in adipocytes impairs fatty acid uptake, suggesting that esterification is essential for fatty acid transport. Together, our findings suggest that a constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation. A central aspect of adipocyte biology is the ability of these specialized cells to efficiently take up and store longchain fatty acids (LCFAs) in response to nutritional and hormonal cues. In the "fed" state, higher serum levels of insulin stimulate an increase in fatty acid uptake and triglyceride storage in adipose tissue. On the other hand, during "starvation," b-adrenergic receptor agonists, such as epinephrine, stimulate pathways that activate hormonesensitive lipase in adipocytes, resulting in the hydrolysis of triglycerides and the release of free fatty acids and glycerol. In obesity and the metabolic syndrome, dysregulation of these processes may contribute to increases in serum free fatty acids and the genesis of type 2 diabetes (1, 2).The movement of LCFAs across the plasma membrane is tightly coupled to thioesterification, a process referred to as vectorial acylation (3). Early evidence for this mechanism came from genetic studies of Escherichia coli, in which fatty acid transport requires FadL, an outer membrane LCFA transporter, and FadD, an inner membraneassociated acyl CoA synthetase (4, 5). Vectorial acylation provides cells with an efficient means of rapidly metabolizing incoming fatty acids and contributes to decreasing the intracellular concentration of free fatty acids to favor import. Thioesterification of LCFAs is also an essential initial metabolic step for many downstream metabolic pathways of LCFA use, such as b-oxidation or triglyceride synthesis. The coupled transport and metabolism of LCFAs is analogous to glucose transport in mammalian cells, a process in which GLUT4-mediated translocation of glucose across the plasma membrane is coordinated with rapid phosphorylation by hexokin...
The 63-kDa murine fatty acid transport protein 1 (FATP1) was cloned on the basis of its ability to augment fatty acid import when overexpressed in mammalian cells. The membrane topology of this integral plasma membrane protein does not resemble that of polytopic membrane transporters for other substrates. Western blot analysis of 3T3-L1 adipocytes that natively express FATP1 demonstrate a prominent 130-kDa species as well as the expected 63-kDa FATP1, suggesting that this protein may participate in a cell surface transport protein complex. To test whether FATP1 is capable of oligomerization, we expressed functional FATP1 molecules with different amino-or carboxyl-terminal epitope tags in fibroblasts. These epitope-tagged proteins also form apparent higher molecular weight species. We show that, when expressed in the same cells, differentially tagged FATP1 proteins co-immunoprecipitate. The region between amino acid residues 191 and 475 is sufficient for association of differentially tagged truncated FATP1 constructs. When wild type FATP1 and the non-functional s250a FATP1 mutant are co-expressed in COS7 cells, mutant FATP1 has dominant inhibitory function in fatty acid uptake assays. Taken together, these results are consistent with a model in which FATP1 homodimeric complexes play an important role in cellular fatty acid import.
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