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. 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 (K_B = 100 nM; K_M = 1 μM) described here as well as a lower affinity system and a higher affinity system. The RFC appears to transport reduced folate and various derivatives 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 Major Facilitator (MFS) Superfamily.
References:
Austin, M.U., W.S. Liau, K. Balamurugan, B. Ashokkumar, H.M. Said, and C.W. LaMunyon. (2010). Knockout of the folate transporter folt-1 causes germline and somatic defects in C. elegans. BMC Dev Biol 10: 46.
Bai, M., Q. Shen, Y. Wu, Z. Ma, Y. Wang, M. Chen, D. Liu, and L. Zhou. (2024). Evaluation of transport mechanisms of methotrexate in human choriocarcinoma cell lines by LC-MS/MS. J Pharm Biomed Anal 247: 116268.
Balamurugan, K., B. Ashokkumar, M. Moussaif, J.Y. Sze, and H.M. Said. (2007). Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans. Am. J. Physiol. Cell Physiol. 293: C670-681.
Bowman, B.B., D.B. McCormick, and I.H. Rosenberg. (1989). Epithelial transport of water-soluble vitamins. Annu. Rev. Nutr. 9: 187-199.
Bukhari, F.J., H. Moradi, P. Gollapudi, H. Ju Kim, N.D. Vaziri, and H.M. Said. (2011). Effect of chronic kidney disease on the expression of thiamin and folic acid transporters. Nephrol Dial Transplant 26: 2137-2144.
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.
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.
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.
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.
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.
He, Q. and J. Li. (2023). The evolution of folate supplementation - from one size for all to personalized, precision, poly-paths. J Transl Int Med 11: 128-137.
Henderson, G.B. (1990). Folate-binding proteins. Annu. Rev. Nutr. 10: 319-335.
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.
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.
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.
Li, X., Y. Zhu, X. Zhang, X. An, M. Weng, J. Shi, S. Wang, C. Liu, S. Luo, and T. Zheng. (2022). An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP. J Clin Invest 132:.
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.
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.
Miyake, A., J. Kawasaki, H. Ngo, I. Makundi, Y. Muto, A.H. Khan, D.J. Smith, and K. Nishigaki. (2019). The reduced folate carrier: an entry receptor for a novel feline leukemia virus variant. J. Virol. [Epub: Ahead of Print]
Naushad, S.M., A.R. Rama Devi, T. Hussain, S.A. Alrokayan, M. Janaki Ramaiah, and V.K. Kutala. (2019). analysis of the structural and functional implications of R27H polymorphism. J Genet 98:.
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.
Sirotnak, F.M. and B. Tolner. (1999). Carrier-mediated membrane transport of folates in mammalian cells. Annu. Rev. Nutr. 19: 91-122.
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.
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.
Szemere, Z.K. and E.A. Murphy. (2024). Import of extracellular 2''-3''cGAMP by the folate transporter, SLC19A1, establishes an antiviral response that limits herpes simplex virus-1. Antiviral Res 230: 105989.
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.
Whitford, W., I. Hawkins, E. Glamuzina, F. Wilson, A. Marshall, F. Ashton, D.R. Love, J. Taylor, R. Hill, K. Lehnert, R.G. Snell, and J.C. Jacobsen. (2017). Compound heterozygous SLC19A3 mutations further refine the critical promoter region for biotin-thiamine-responsive basal ganglia disease. Cold Spring Harb Mol Case Stud 3:.
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.
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.
Zhang, Q., X. Zhang, Y. Zhu, P. Sun, L. Zhang, J. Ma, Y. Zhang, L. Zeng, X. Nie, Y. Gao, Z. Li, S. Liu, J. Lou, A. Gao, L. Zhang, and P. Gao. (2022). Recognition of cyclic dinucleotides and folates by human SLC19A1. Nature. [Epub: Ahead of Print]
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.
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.
Examples:
TC#	Name	Organismal Type	Example
2.A.48.1.1	Folate/folate derivative transporter, RFC1. Although oligomeric, each monomer provides the channel (Hou et al., 2010). It is downregulated in Chronic Kidney Disease (CKD) in heart, liver, and brain, causing malabsorption (Bukhari et al., 2011). 5-Aminoimidazole-4-carboxamide riboside (AICAR), an agent with diverse pharmacological properties, augments transport of folates and antifolates by promoting substrate:substrate antiport (Visentin et al. 2012). Its role in antifolate cancer chemotherapy has been reviewed (Matherly et al. 2014). In cats, this protein is the receptor for feline leukemia virus (feLV) (Miyake et al. 2019). The R27H polymorphism affects the secondary and tertiary structures of SLC19A1 with a loss of ligand-binding sites (Naushad et al. 2019). 2'3'-cyclic-GMP-AMP (cGAMP), a second messenger that activates the antiviral stimulator of IFN genes (STING), elicits an antitumoral immune response. SLC19A1 is an importer that transports extracellular cGAMP into the cytosol (Li et al. 2022), and binding of cyclic dinucleotides has been demonstrated (Zhang et al. 2022). Folate supplementation has been reviewed (He and Li 2023). FRα and multiple transporters such as PCFT, RFC, OAT4, and OATPs are likely involved in the uptake of methotraxate (MTX), whereas MDR1 and BCRP are implicated in the efflux of MTX from choriocarcinoma cells (Bai et al. 2024). Import of extracellular 2'-3'cGAMP occurs via the folate transporter, SLC19A1, and this establishes an antiviral response that limits herpes simplex virus-1 (Szemere and Murphy 2024).	Animals	SLC19A1 of Homo sapiens

2.A.48.1.2	Thiamine uptake transporter-1, THTR-1, the thiamine-responsive megaloblastic anemia (TRMA) protein (Manimaran et al. 2016). Downregulated in Chronic Kidney Disease (CKD) in heart, liver, and brain causing malabsorption (Bukhari et al., 2011).	Animals	SLC19A2 of Homo sapiens

2.A.48.1.3	The folate transporter, FolT-1 of 453 aas, with a Km for folate of 1.2μM and a Ki for Sulfasalazine of 0.1 mM. Folt-1 also transports reduced folate and substituted folate derivatives. The system is maximally expressed in the pharynx and intestine, and it is developmentallyl regulated. DIDS, a general anion inhibitor, also inhibits (Balamurugan et al., 2007). FOLT-1 function affects both the soma and the germline. folt-1(ok1460) hermaphrodites suffer severely diminished lifespan and germline defects that result in sterility. Germline defects associated with folate deficiency appear widespread in animals, being found in humans, mice, fruit flies, and nematodes (Austin et al. 2010).	Metazoa	*FolT-1 of Caenorhabditis elegans* (Q17766)**

2.A.48.1.4	Thiamine (and biotin) transporter-2, ThTr-2, of 496 aas and 12 TMSs. Downregulated in Chronic Kidney Disease (CKD) in heart, liver, and brain causing malabsorption (Bukhari et al., 2011). Activated by microtubules but not microfilaments (Subramanian et al. 2013). Activated by direct interaction with the 4 TMS protein, TM4 of the L6 family, member 4, TM4SF4 (P48230) (Subramanian et al. 2014). Mutations result in thiamine metabolism dysfunction syndrome 2, also known as biotin-thiamine-responsive basal ganglia disease (BTBGD). This neurometabolic disease typically presents in early childhood with progressive neurodegeneration, including confusion, seizures, and dysphagia, advancing to coma and death (Whitford et al. 2017).	Animals	SLC19A3, Thiamine transporter-2 of Homo sapiens

2.A.48.1.5	Slime mold RFC homologue	slime mold	*RFC homologue of Dictyostelium discoideum* (Q559K0)**

2.A.48.1.6	Giardia RFC homologue	Lower eukaryotic protist	*RFC homologue of Giardia lamblia* (A8BIM9)**

2.A.48.1.7	Folate transporter, Folt-2 of 424 aas and 12 TMSs in an apparent 5 + 5 or 6 + 6 TMS arrangement, probably the latter.		Folt2 of Caenorhabditis elegans

Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example
Examples:
TC#	Name	Organismal Type	Example