Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis

Abstract

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln(58) had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.

The Region between Transmembrane Domains 1 and 2 of the Reduced Folate Carrier Forms Part of the Substrate-binding Pocket

“…3B). Thus, these results confirm the extracellular location of the regions containing residues 46, 179, 300, and 430 as described previously by other methods (8,9) and also indicate that residue 355 is exposed to the extracellular environment, an observation contrary to previous work involving N-glycosylation scanning mutagenesis (9). However, residue 355 may be in a cleft since it is not very reactive with biotin maleimide and is not that accessible to LYI, but is accessible to the smaller sized reagent MTSET.…”

“…This labeling could be partially prevented by pretreatment with the membrane-permeable reagent NEM, but not with the membrane-impermeable reagent LYI. This confirms the cytosolic location of regions containing residues 224 and 475 as previously determined (8,9) and also positions residue 152 in a similar location as predicted by the 12-transmembrane model.…”

“…Predicted topology of RFC. The topology is based on hydropathy plots and epitope insertion analysis (8,9). Circled residues indicate those conserved among hamster, human, rat, and mouse species.…”

“…Hydrophobicity analyses have indicated that the RFC protein has 12 transmembrane-spanning segments with the N and C termini located intracellularly and a large intracellular loop between transmembrane domain (TM) 6 and TM7 (8). Epitope insertions into the predicted major loops (8,9) and N-glycosylation scanning mutagenesis (9) support this model, although the latter study suggests that the C-terminal portion of the protein may demonstrate an alternative topology.…”

Resistance to antifolates

“…The validity of this secondary structure has been strengthened by hemaglutatinin epitope insertion analysis (Ferguson and Flintoff, 1999) although there remains a question regarding the topology of TMDs 9-12 (Liu and Matherly, 2002). A His27Arg polymorphism has been identified in the human carrier (Chango et al, 2000), but appears to have only minor functional importance (Whetstine et al, 2001).…”