Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis.
Site-directed mutagenesis was used to characterize the functional role of lysine-411, a conserved amino acid located in putative transmembrane domain (TMD) 11 of the human reduced folate carrier (hRFC). Lysine-411 was mutagenized to arginine, glutamate, and leucine, and the mutant constructs (K411R-, K411E-, and K411L-hRFC, respectively) were transfected into hRFC-deficient K562 cells. The mutant hRFC constructs were all expressed at high levels and restored 22-36% of the methotrexate (MTX) transport level in wild-type (K43-6) hRFC transfectants. Although 5-formyl tetrahydrofolate (5-CHO-H(4)PteGlu) uptake levels for both the K411E- and K411L-hRFCs were also impaired (approximately 33% and 28%, respectively), a complete restoration of the wild-type level was observed for K411R-hRFC. While loss of MTX transport activity for the K411R-hRFC transfectant was associated with an incomplete restoration of MTX sensitivity compared to K43-6 cells, these cells were similarly sensitive to Tomudex. The K411R-hRFC transfectants showed an approximately threefold decreased growth requirement for 5-CHO-H(4)PteGlu compared to K43-6 cells. The 5-CHO-H(4)PteGlu transport stimulation observed for the wild-type carrier in chloride-free buffer was also observed for K411R-hRFC, however, this response was decreased for the K411E- and K411L-hRFCs. The preservation of low levels of transport for the K411E- and K411L-hRFCs suggest that the amino acid at position 411 does not directly participate in the binding of anionic hRFC substrates. However, a functionally important role for a basic amino acid at position 411 was, nonetheless, implied by the increased MTX transport for wild-type hRFC over the K411 mutant hRFCs, and the highly selective uptake of 5-CHO-H(4)PteGlu over MTX for K411R-hRFC.
Witt
Teah L
TL
Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 E. Warren Ave., Detroit, MI 48201, USA.
Matherly
Larry H
LH
D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D058978:Reduced Folate Carrier Protein; D013379:Substrate Specificity; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D008239:Lysine