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2.A.48 The Reduced Folate Carrier (RFC) Family

Proteins of the RFC family have been characterized only from animals, but homologues can also be found in other eukaryotes such as slime molds (2.A.48.3.1) and Giardia (2.A.48.4.1). They have been sequenced from several mammals and from the worm, Caenorhabditis elegan, as well as the fly, Drosophila melanogaster. Humans have at least two RFC family paralogues, and C. elegans has three. All homologues exhibit a high degree of sequence similarity with each other. They are usually 500-600 amino acyl residues long and possess 12 putative transmembrane α-helical spanners (TMSs). Evidence for a 12 TMS topology has been published for a human RFC. RFCs take up folate, reduced folate, derivatives of reduced folate and the drug, methotrexate. Residues in the first TMS and in the region between TMSs 1, 2 and 11 appear to play a role in substrate recognition (Flintoff et al., 2003; Hou et al., 2005). The large cytoplasmic loop between TMSs 6 and 7 is required for stability and efficient transport.  The reduced folate carrier (RFC) is cytotoxic to animal cells under conditions of severe folate deprivation (Ifergan et al., 2008).

Mammals possess at least three folate transporters: the RFC (KB = 100 nM; KM = 1 μM) described here as well as a lower affinity system and a higher affinity system. The RFC appears to transport reduced folate by an energy-dependent, pH-dependent, Na+-independent mechanism. Folate:H+ symport, folate:OH- antiport and folate:anion antiport mechanisms have been proposed. Intracellular anions are able to promote folate derivative uptake. A bidirectional anion antiport mechanism for RFC family members is favored. In support of this notion, RFC1 has been shown to catalyze efflux of thiamin pyrophosphate (TPP) (Zhao et al., 2001; Visentin et al., 2012).

The human thiamine transporter is a member of the RFC family. The transporter is highly specific for thiamine and is not inhibited by other organic cation. It transports thiamine by a Na+-independent pmf-dependent process. Folates are not substrates of this system.

The generalized transport reaction(s) catalyzed by the proteins of the RFC family is/are probably:

Folate derivative (out) + anion (in) ⇌ folate derivative (in) + anion (out).

or

Thiamine (out) + nH+ (out) ⇌ thiamine (in) + nH+ (in)

This family belongs to the: MFS Superfamily.

References associated with 2.A.48 family:

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Dixon, K.H., B.C. Lanpher, J. Chiu, K. Kelly, and K.H. Cowan. (1994). A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells. J. Biol. Chem. 269: 17-20. 8276792
Drori S., G. Jansen, R. Mauritz, G.J, Peters, and Y.G. Assaraf. (2000). Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake. J. Biol. Chem. 275: 30855-30863. 10899164
Dutta, B., W. Huang, M. Molero, R. Kekuda, F.H. Leibach, L.D. Devoe, V. Ganapathy, and P.D. Prasad. (1999). Cloning of the human thiamine transporter, a member of the folate transporter family. J. Biol. Chem. 274: 31925-31929. 10542220
Ferguson, P.L. and W.F. Flintoff. (1999). Topological and functional analysis of the human reduced folate carrier by hemagglutinin epitope insertion. J. Biol. Chem. 274: 16269-16278. 10347183
Flintoff, W.F., F.M.R. Williams, and H. Sadlish. (2003). The region between transmembrane domains 1 and 2 of the reduced folate carrier forms part of the substrate-binding pocket. J. Biol. Chem. 278: 40867-40876. 12909642
Henderson, G.B. (1990). Folate-binding proteins. Annu. Rev. Nutr. 10: 319-335. 2166548
Hou, Z., C. Cherian, J. Drews, J. Wu, and L.H. Matherly. (2010). Identification of the minimal functional unit of the homo-oligomeric human reduced folate carrier. J. Biol. Chem. 285: 4732-4740. 20018840
Hou, Z., S.E. Stapels, C.L. Haska, and L.H. Matherly. (2005). Localization of a substrate binding domain of the human reduced folate carrier to transmembrane domain 11 by radioaffinity labeling and cysteine-substituted accessibility methods. J. Biol. Chem. 280: 36206-36213. 16115875
Ifergan, I., G. Jansen, and Y.G. Assaraf. (2008). The Reduced Folate Carrier (RFC) Is Cytotoxic to Cells under Conditions of Severe Folate Deprivation: RFC AS A DOUBLE EDGED SWORD IN FOLATE HOMEOSTASIS. J. Biol. Chem. 283: 20687-20695. 18499665
Manimaran, P., V.S. Subramanian, S. Karthi, K. Gandhimathi, P. Varalakshmi, R. Ganesh, A. Rathinavel, H.M. Said, and B. Ashokkumar. (2016). Novel nonsense mutation (p.Ile411Metfs*12) in the SLC19A2 gene causing Thiamine Responsive Megaloblastic Anemia in an Indian patient. Clin Chim Acta 452: 44-49. 26549656
Matherly LH., Wilson MR. and Hou Z. (2014). The major facilitative folate transporters solute carrier 19A1 and solute carrier 46A1: biology and role in antifolate chemotherapy of cancer. Drug Metab Dispos. 42(4):632-49. 24396145
Rodionov, D.A., P. Hebbeln, A. Eudes, J. ter Beek, I.A. Rodionova, G.B. Erkens, D.J. Slotboom, M.S. Gelfand, A.L. Osterman, A.D. Hanson, and T. Eitinger. (2009). A novel class of modular transporters for vitamins in prokaryotes. J. Bacteriol. 191: 42-51. 18931129
Sirotnak, F.M. and B. Tolner. (1999). Carrier-mediated membrane transport of folates in mammalian cells. Annu. Rev. Nutr. 19: 91-122. 10448518
Subramanian, V.S., J.S. Marchant, I. Parker, and H.M. Said. (2003). Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms. J. Biol. Chem. 278: 3976-3984. 12454006
Subramanian, V.S., S.M. Nabokina, and H.M. Said. (2014). Association of TM4SF4 with the human thiamine transporter-2 in intestinal epithelial cells. Dig Dis Sci 59: 583-590. 24282057
Visentin, M., R. Zhao, and I.D. Goldman. (2012). Augmentation of reduced folate carrier-mediated folate/antifolate transport through an antiport mechanism with 5-aminoimidazole-4-carboxamide riboside monophosphate. Mol Pharmacol 82: 209-216. 22554803
Williams, F.M.R., R.C. Murray, T.M. Underhill, and W.F. Flintoff. (1994). Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake. J. Biol. Chem. 269: 5810-5816. 8119923
Wong, S.C., S.A. Proefke, A. Bhushan, and L.H. Matherly. (1995). Isolation of a human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective chinese hamster ovary cells. J. Biol. Chem. 270: 17468-17475. 7615551
Zhao, R., F. Gao, Y. Wang, G.A. Diaz, B.D. Gelb, and I.D. Goldman. (2001). Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells. J. Biol. Chem. 276: 1114-1118. 11038362
Zhao, R., Y.G. Assaraf, and I.D. Goldman. (1998). A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function. J. Biol. Chem. 273: 19065-19071. 9668089