Restoration of high-level transport activity by human reduced folate carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in reduced folate carrier function

Abstract

The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1-6 and TMDs 7-12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204-214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFC(D215-R263 Delta), hRFC(K204-R263 Delta) and hRFC(K204-R214 Delta) proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6 S )5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFC(K204-R263 Delta) restored low levels of transport (16-21% of the wild type). Insertion of the ThTr1 linkers into hRFC(D215-R263 Delta) at position 215 restored 60-80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.

The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

“…Deletion of major segments (49 or 60 amino acids; positions 215-263 and 204-263, respectively) from the loop domain connecting TMDs 6 and 7 of hRFC ( Fig. 3) also abolished transport (Liu et al, 2003). Interestingly, when these deleted loop segments in hRFC were replaced by the corresponding segment from the MFS homolog SLC19A2, which transports thiamine, transport was restored (Liu et al, 2003).…”

Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier

“…We used the bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library to search for proteins that may interact with the hRFC. The large intracellular loop between transmembranes 6 and 7 (amino acids 204 to 264) of hRFC was used as the bait in screening since it has been reported to play a critical role in function and expression of the RFC at the plasma membrane (23,32,36). The BacterioMatch II human intestinal cDNA library (primary size 1.67 ϫ 10 6 ; Stratagene) was screened with the generated recombinant pBT-hRFC plasmid.…”

“…The RFC protein is predicted to have 12 transmembrane domains with both the amino and carboxy termini predicted to be oriented intracellularly (7,14). The RFC protein is also predicted to contain a long intracellular loop between transmembrane domains 6 and 7, which is believed to play a role in function and expression of the carrier protein at the cell membrane (23,32,36). Furthermore, the backbone of the RFC polypeptide has been shown to be important for membrane targeting and expression of the protein (25).…”

Chapter 5 Structure and Function of the Reduced Folate Carrier

“…Interestingly, maximal transport activities for these insertional mutants were absolutely dependent on the presence of the highly conserved 204-214 peptide and deletion of the 204-214 segment alone completely abolished transport (Liu et al, 2003).…”

“…While deletions of 49 or 60 amino acids from the TMD6/TMD7 linker of hRFC (amino acids 215-263 and 204-263, respectively) completely ablated transport of MTX and 5-formyl tetrahydrofolate, replacement of the deleted segments with non-homologous 73 or 84 amino acid segments from another MFS protein, SLC19A2 (transports thiamine; 18% homologous to hRFC for the TMD6/TMD7 linker region) restored transport (Liu et al, 2003). Interestingly, maximal transport activities for these insertional mutants were absolutely dependent on the presence of the highly conserved 204-214 peptide and deletion of the 204-214 segment alone completely abolished transport (Liu et al, 2003).…”