Substitution of Alanine for Serine 250 in the Murine Fatty Acid Transport Protein Inhibits Long Chain Fatty Acid Transport

Abstract

The murine fatty acid transport protein (FATP) was identified on the basis of its ability to facilitate uptake of long chain fatty acids (LCFAs) when expressed in mammalian cells. To delineate FATP domains important for transport function, we cloned the human heart FATP ortholog. Comparison of the human, murine, and yeast amino acid sequences identified a highly conserved motif, IYTSGTTGXPK, also found in a number of proteins that form adenylated intermediates. We demonstrate that depletion of intracellular ATP dramatically reduces FATP-mediated LCFA uptake. Furthermore, wildtype FATP specifically binds [␣-32 P]azido-ATP. Introduction of a serine to alanine substitution (S250A) in the IYTSGTTGXPK motif produces an appropriately expressed and metabolized mutant FATP that demonstrates diminished LCFA transport function and decreased [␣-32 P]azido-ATP binding. These results are consistent with a mechanism of action for FATP involving ATP binding that is dependent on serine 250 of the IYTSGTTGXPK motif.The precise mechanism of long chain fatty acid (LCFA) 1 transport is not well understood. In mammalian cells such as myocytes and adipocytes, LCFA uptake is efficient and highly regulated. Experiments demonstrating specific, saturable LCFA uptake that is inhibited by prior protease treatment of the cell surface suggested that LCFAs are transported by a protein-mediated mechanism (1-5). CD36 and mitochondrial aspartate aminotransferase were initially proposed to facilitate LCFA transport because they are capable of binding LCFAs (6 -10). More recently, we identified the fatty acid transport protein (FATP) on the basis of its function in LCFA uptake. We isolated the cDNA encoding the murine FATP using an expression cloning strategy to screen a 3T3-L1 adipocyte cDNA library for cDNAs that increase LCFA uptake when expressed in mammalian cells (11). FATP may function as an LCFA transporter that facilitates bi-directional LCFA movement across the plasma membrane (12, 13).FATP is a 63-kDa integral plasma membrane protein. Stable overexpression of FATP confers a 4-fold increase in initial rates of LCFA uptake with a K m of 0.2 M for oleic acid, comparable to the K m for oleic acid uptake by 3T3-L1 adipocytes (11). FATP facilitates uptake of saturated and mono-enoic LCFAs with 14 -22 carbons, suggesting that it has broad specificity with respect to fatty acid chain length and degree of saturation (11). 2 FATP expression in cultured adipocytes is inhibited by insulin (13). In mice, FATP expression is induced during fasting in adipose and heart tissue, suggesting that FATP may be important not only for uptake of LCFAs into tissues with a metabolic requirement for this substrate, but also for efflux of LCFAs from adipocytes during lipolysis. Disruption of the gene encoding the Saccharomyces cerevisiae (yeast) ortholog, Fat1p, results in a 2-3-fold decrease in the rate of oleate uptake and impaired growth of yeast in which de novo fatty acid synthesis is inhibited (14).The mechanism of action of FATP is unknown. Hydropathy analys...

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

“…Importantly, the LCFA pool was not increased but also similarly reduced, consistent with the model that the ACSL activity of FATP1 is critical for facilitating LCFA influx. These results are in agreement with the observations of StuhlsatzKrouper, Bennett, and Schaffer (43), who have shown that mutation of FATP1 serine 250 to alanine not only reduced the catalytic activity of the enzyme but also eliminated LCFA influx, implying that catalysis is functionally linked to uptake. In contrast to FATP1 kd adipocytes, FATP4 kd adipocytes revealed no change in basal or insulin-stimulated cellular fatty acid influx.…”

Characterization of the Acyl-CoA Synthetase Activity of Purified Murine Fatty Acid Transport Protein 1

“…Subsequent studies have demonstrated that the overexpression of FATP1, FATP2, or FATP4 in COS1 cells increases cell-associated acyl-CoA synthetase activities (11,26,27). Moreover, mutation of serine 250 to alanine significantly reduced fatty acid uptake, suggesting that esterification of fatty acids is coupled to influx (28). The current study was undertaken to evaluate the catalytic properties of the FATP1 acyl-CoA synthetase reaction.…”

Oligomerization of the Murine Fatty Acid Transport Protein 1

“…Antibodies to native FATP1 sequences were generated as previously described (2,14). Cell culture reagents were from Invitrogen.…”

“…Expression Constructs-PCR was used to insert amino-terminal HA, amino-terminal Myc, and carboxyl-terminal FLAG tags into ⌬U3FATP1 or ⌬U3FATP1s250a mutant retroviral constructs (14). Retrovirus was generated and used to transduce cells as previously described (17).…”

“…Fatty Acid Uptake Assays-Parental and retroviral-transduced NIH 3T3 cells were assayed for fatty acid uptake as previously described (14). Flow cytometric analysis of samples of 10 4 propidium iodidenegative (live) cells was carried out in triplicate using a BD Biosciences FACScan.…”