Substitution of alanine for serine 250 in the murine fatty acid transport protein inhibits long chain fatty acid transport.
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, wild-type FATP specifically binds [alpha-32P]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 [alpha-32P]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.
Stuhlsatz-Krouper
S M
SM
Center for Cardiovascular Research, Department of Internal Medicine and the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110-1010, USA.
Bennett
N E
NE
Schaffer
J E
JE
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000378:metabolism; D001692:Biological Transport; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003486:Cyanides / Q000494:pharmacology; D018076:DNA, Complementary; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*
D001386:Azides; D002352:Carrier Proteins; D003486:Cyanides; D018076:DNA, Complementary; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C493572:Slc27a4 protein, mouse; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D012694:Serine; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000409:Alanine