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...

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