Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier.
The reduced folate carrier (RFC1) plays a major role in the delivery of folates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane domains. This article explores the role of these residues in transport function by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines transfected with R131L, R155L, or R366L all lacked activity, despite high levels of protein expression in the plasma membrane, suggesting the crucial role of these amino acid residues in RFC1 function. In several mutant carriers, R26L, R42L, and K332L, there was little or no change in the influx K(t) value for MTX or influx K(i) value for folic acid. However, the R26L, R42L, and K332L carriers had decreased affinity for reduced folates. This was most prominent for K404L, which had 11- and 4-fold increases in influx K(i) for 5-methyl-THF and 5-formyl-THF, respectively, compared with L1210 cells. The marked influx stimulation observed with wild-type carrier when extracellular chloride was decreased was significantly diminished when influx was mediated by the K404L carrier, but was only slightly decreased with the R26L, R42L, and K332L mutants. This suggested that the K404 residue may be a major site of inhibition by chloride in the wild-type carrier. These studies indicate the important role that some positively charged residues within transmembrane domains of RFC1 play in RFC1 function.
Sharina
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IG
Department of Integrative Biology, and the Institute of Molecular Medicine, University of Texas, Houston, Texas, USA.
Zhao
R
R
Wang
Y
Y
Babani
S
S
Goldman
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ID
D000818:Animals; D000838:Anions / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D058978:Reduced Folate Carrier Protein; D014162:Transfection
D000838:Anions; D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548700:Slc19a1 protein, mouse; C492292:Slc19a2 protein, mouse; D005492:Folic Acid; D001120:Arginine; D008239:Lysine; D008727:Methotrexate