2.A.39 The Nucleobase:Cation Symporter-1 (NCS1) Family

The NCS1 family consists of over 1000 currently sequenced proteins derived from Gram-negative and Gram-positive bacteria, archaea, yeast, fungi and plants. The bacterial and yeast proteins are widely divergent and do not cluster closely on the NCS1 family phylogenetic tree. B. subtilis possesses two paralogues of the NCS1 family, and S. cerevisiae has several. Two of the yeast proteins (Dal4 and Fur4) cluster tightly together, and three other S. cerevisiae proteins, one of which is the thiamin permease, Thi10, and another of which is the nicotinamide riboside transporter, Nrt1 (Belenky et al., 2008), also cluster tightly together. The latter three proteins are likely to be closely-related thiamin permease isoforms. The yeast cytosine-purine and vitamin B6 transporters cluster loosely together (24% identity; e-50 (Stolz and Vielreicher, 2003). The bacterial proteins are derived from several Gram-negative and Gram-positive species. These proteins exhibit limited sequence similarity with the xanthine permease, PbuX, of Bacillus subtilis which is a member of the NCS2 family. The two families are therefore probably related.

Proteins of the NCS1 family are 419-635 amino acyl residues long and possess twelve putative transmembrane α-helical spanners (TMSs). At least some of them have been shown to function in uptake by substrate:H+ symport. In these respects, and with respect to substrate specificity, these proteins resemble the symporters of the NCS2 family, providing further evidence that the two families represent distant constituents of a single superfamily. The two families probably arose by an early gene duplication event that occurred long before divergence of the three major kingdoms of life. It is possible that they are distant constituents of the MFS (2.A.1).

The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Weyand et al. 2008 reported the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport (Weyand et al. 2008).

NCS1 proteins are H+/Na+ symporters specific for the uptake of purines, pyrimidines and related metabolites. Krypotou et al. 2015 studied the origin, diversification and substrate specificities of fungal NCS1 transporters, suggesting that the two fungal NCS1 subfamilies, Fur and Fcy, and plant homologues, originated through independent horizontal transfers from prokaryotes.  Expansion by gene duplication led to functional diversification of fungal NCS1 porters. They characterized all Fur proteins in Aspergillus nidulans. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. They predicted and confirmed that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selectivity gate. Their evolutionary and structure-function analyses led to the concept that selective channel-like gates may contribute to substrate specificity (Krypotou et al. 2015).

The generalized transport reaction catalyzed by NCS1 family permeases is:

Nucleobase or Vitamin (out) + H+ (out) → Nucleobase or Vitamin (in) + H+ (in)

This family belongs to the APC Superfamily.



Belenky, P.A., T.G. Moga, and C. Brenner. (2008). Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1. J. Biol. Chem. 283: 8075-8079.

Danielsen, S., M. Kilstrup, K. Barilla, B. Jochimsen, and J. Neuhard. (1992). Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase. Mol. Microbiol. 6: 1335-1344.

De Koning, H. and G. Diallinas. (2000). Nucleobase transporters. Molec. Memb. Biol. 75: 75-94.

Enjo, F., K. Nosaka, M. Ogata, A. Iwashima, and H. Nishimura. (1997). Isolation and characterization of a thiamin transport gene, THI10, from Saccharomyces cerevisiae. J. Biol. Chem. 272: 19165-19170.

Gabriel, F., A. Sabra, S. El-Kirat-Chatel, S. Pujol, V. Fitton-Ouhabi, D. Brèthes, K. Dementhon, I. Accoceberry, and T. Noël. (2014). Deletion of the uracil permease gene confers cross-resistance to 5-fluorouracil and azoles in Candida lusitaniae and highlights antagonistic interaction between fluorinated nucleotides and fluconazole. Antimicrob. Agents Chemother. 58: 4476-4485.

Goudela, S., H. Tsilivi, and G. Diallinas. (2006). Comparative kinetic analysis of AzgA and Fcy21p, prototypes of the two major fungal hypoxanthine-adenine-guanine transporter families. Mol. Membr. Biol. 23: 291-303.

Kazmier K., Sharma S., Islam SM., Roux B. and Mchaourab HS. (2014). Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1. Proc Natl Acad Sci U S A. 111(41):14752-7.

Krypotou E., Kosti V., Amillis S., Myrianthopoulos V., Mikros E. and Diallinas G. (2012). Modeling, substrate docking, and mutational analysis identify residues essential for the function and specificity of a eukaryotic purine-cytosine NCS1 transporter. J Biol Chem. 287(44):36792-803.

Krypotou, E., T. Evangelidis, J. Bobonis, A.A. Pittis, T. Gabaldón, C. Scazzocchio, E. Mikros, and G. Diallinas. (2015). Origin, diversification and substrate specificity in the family of NCS1/FUR transporters. Mol. Microbiol. 96: 927-950.

Ma, P., S.G. Patching, E. Ivanova, J.M. Baldwin, D. Sharples, S.A. Baldwin, and P.J. Henderson. (2016). The allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity. Microbiology. [Epub: Ahead of Print]

Moraes, T.F. and R.A. Reithmeier. (2012). Membrane transport metabolons. Biochim. Biophys. Acta. 1818: 2687-2706.

Papadaki, G.F., S. Amillis, and G. Diallinas. (2017). Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions. Genetics 207: 1387-1400.

Pinson, B., C. Napias, J. Chevallier, P.J.A. Van den Broek, and D. Brèthes. (1997). Characterization of the Saccharomyces cerevisiae cytosine transporter using energizable plasma membrane vesicles. J. Biol. Chem. 272: 28918-28924.

Pyrris, Y., G.F. Papadaki, E. Mikros, and G. Diallinas. (2024). The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit. Microb Cell 11: 1-15.

Rapp, M., J. Schein, K.A. Hunt, V. Nalam, G.S. Mourad, and N.P. Schultes. (2016). The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility. Protoplasma 253: 611-623.

Rodionov, D.A., A.G. Vitreschak, A.A. Mironov, and M.S. Gelfand. (2002). Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. J. Biol. Chem. 277: 48949-48959.

Rodionov, D.A., C. Yang, X. Li, I.A. Rodionova, Y. Wang, A.Y. Obraztsova, O.P. Zagnitko, R. Overbeek, M.F. Romine, S. Reed, J.K. Fredrickson, K.H. Nealson, and A.L. Osterman. (2010). Genomic encyclopedia of sugar utilization pathways in the Shewanella genus. BMC Genomics 11: 494.

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.

Rodriguez, C., J.C. Bloch, and M.R. Chevallier. (1995). The immunodetected yeast purine-cytosine permease is not N-linked glycosylated, nor are glycosylation sequences required to have a functional permease. Yeast 11: 15-23.

Saier, M.H., Jr., B.H. Eng, S. Fard, J. Garg, D.A. Haggerty, W.J. Hutchinson, D.L. Jack, E.C. Lai, H.J. Liu, D.P. Nusinew, A.M. Omar, S.S. Pao, I.T. Paulsen, J.A. Quan, M. Sliwinski, T.-T. Tseng, S. Wachi, and G.B. Young. (1999). Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim. Biophys. Acta 1422: 1-56.

Schein, J.R., K.A. Hunt, J.A. Minton, N.P. Schultes, and G.S. Mourad. (2013). The nucleobase cation symporter 1 of Chlamydomonas reinhardtii and that of the evolutionarily distant Arabidopsis thaliana display parallel function and establish a plant-specific solute transport profile. Plant Physiol. Biochem 70: 52-60.

Schultz, A.C., P. Nygaard, and H.H. Saxild. (2001). Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J. Bacteriol. 183: 3293-3302.

Smits, P.H.M., M. De Haan, C. Maat, and L.A. Grivell. (1994). The complete sequence of a 33 kb fragment on the right arm of chromosome II from Saccharomyces cerevisiae reveals 16 open reading frames, including ten new open reading frames, five previously identified genes and a homologue of the SCO1 gene. Yeast 10(Suppl A): S75-S80.

Stolz, J. and M. Vielreicher. (2003). Tpn1p, the plasma membrane vitamin B6 transporter of Saccharomyces cerevisiae. J. Biol. Chem. 278: 18990-18996.

Suzuki, S., and P.J. Henderson. (2006). The hydantoin transport protein from Microbacterium liquefaciens. J. Bacteriol. 188: 3329-3336.

Vlanti, A. and G. Diallinas. (2008). The Aspergillus nidulans FcyB cytosine-purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis. Mol. Microbiol. 68: 959-977.

Wagner, R., J. de Montigny, P. de Wergifosse, J.L. Souciet, and S. Potier. (1998). The ORF YBL042 of Saccharomyces cerevisiae encodes a uridine permease. FEMS Microbiol. Lett. 159: 69-75.

Weyand, S., T. Shimamura, S. Yajima, S. Suzuki, O. Mirza, K. Krusong, E.P. Carpenter, N.G. Rutherford, J.M. Hadden, J. O'Reilly, P. Ma, M. Saidijam, S.G. Patching, R.J. Hope, H.T. Norbertczak, P.C. Roach, S. Iwata, P.J. Henderson, and A.D. Cameron. (2008). Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322: 709-713.

Witz, S., B. Jung, S. Fürst, and T. Möhlmann. (2012). De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis. Plant Cell 24: 1549-1559.

Witz, S., P. Panwar, M. Schober, J. Deppe, F.A. Pasha, M.J. Lemieux, and T. Möhlmann. (2014). Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis. PLoS One 9: e91343.

Yoo, H.S., T.S. Cunningham, and T.G. Cooper. (1992). The allantoin and uracil permease gene sequences of Saccharomyces cerevisiae are nearly identical. Yeast 8: 997-1006.

Zhang, J., K.M. Smith, T. Tackaberry, X. Sun, P. Carpenter, M.D. Slugoski, M.J. Robins, L.P. Nielsen, I. Nowak, S.A. Baldwin, J.D. Young, and C.E. Cass. (2006). Characterization of the transport mechanism and permeant binding profile of the uridine permease Fui1p of Saccharomyces cerevisiae. J. Biol. Chem. 281: 28210-28221.


TC#NameOrganismal TypeExample
2.A.39.1.1Cytosine permease Bacteria CodB of E. coli (P0AA82)

The putative hydroxymethylpyrimidine transporter, CytX (Rodionov et al. 2002)


CytX of Pseudomonas putida (B1JAG2)


The putative hydroxymethylpyrimidine porter, CytX (Rodionov et al., 2002

Proteobacteria, Fimicutes, Archaea

CytX of Neisseria meningitidis (A1KWE6)


Putative nucleobase transporter


Putative nucleobase transporter of Alicyclobacillus acidocaldarius (F8IKT4)

2.A.39.1.5Putative purine-cytosine permease yxlABacilliyxlA of Bacillus subtilis





TC#NameOrganismal TypeExample
2.A.39.2.1Cytosine-purine permease Yeast Fcy2 of Saccharomyces cerevisiae (P17064)
2.A.39.2.2The vitamin B6:H+ symporter, Tpn1 (pyridoxine, pyridoxal and pyridoxamine are substrates) (Stolz and Vielreicher, 2003)YeastTpn1 of Saccharomyces cerevisiae (P53099)
2.A.39.2.3The hypoxanthine/adenine/guanine (purine) transporter, Fcy21 (Goudela et al., 2006)YeastFcy21 of Candida albicans (Q708J7)

The cytosine-purine-scavenging protein, FcyB (low capacity, high affinity; 45% identical and maybe orthologous to the yeast purine-cytosine protein (2.A.39.2.3) (Vlanti and Diallinas 2008).  Substrate docking and mutational analyses have revealed residues essential for specificity and function (Krypotou et al. 2012). The substrate specificities and phylogenies of members of the NCS1 family have been reported (Krypotou et al. 2015).


FycB of Aspergillus nidulans (B1PXD0)


TC#NameOrganismal TypeExample
2.A.39.3.1Allantoin permease Yeast Dal4 of Saccharomyces cerevisiae

Uracil uptake porter, FurD of 544 aas (Krypotou et al. 2015).


FurD of Emericella nidulans (Aspergillus nidulans)


Purine/uracil uptake porter of 599 aas and 12 TMSs, NCS1 or PLUTO.  Nucleobase:proton symporter that facilitates the uptake of nucleobases in the cells. Can transport adenine, guanine and uracil (Witz et al. 2014). Contributes to uracil import into plastids for plastidic uracil salvage which is essential for plant growth and development (Witz et al. 2012).

PLUTO of Arabidopsis thaliana (Mouse-ear cress)


NCS1 of 540 aas and 12 TMSs.  Transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. The closely related Zea mays NCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in these NCS1 profiles are due to a limited number of amino acid differences (Rapp et al. 2016).

NCS1 of Setaria viridis (Green bristlegrass) (Setaria italica subsp. viridis)


Uracil-specific permease of 581 aas and 11 or 12 TMSs. FUR4.  Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungal agents when they are used simultaneously with 5-fluorouracil although the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. Subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Thus, intracellular fluorinated nucleotides play a role in azole resistance, either by preventing azoles from targeting the catalytic site of lanosterol 14-alpha-demethylase or by acting as a molecular switch for the triggering of efflux transport (Gabriel et al. 2014; ).

FUR4 of Clavispora lusitaniae (Candida lusitaniae)


NCS1 of 528 aas and 12 TMSs.  Transporter of adenine, guanine, uracil and allantoin (Schein et al. 2013).

NCS1 of Chlamydomonas reinhardtii (Chlamydomonas smithii)


Uptake porter for allantoin, uric acid (urate) and uracil and related analyogues, FurE of 527 aas and 12 TMSs.  Evidence has been presented that both the C- and/or N-terminal domains are involved in intramolecular dynamics critical for substrate selection (Papadaki et al. 2017).  The last two TMSs in the APC-type FurE transporter act as an intramolecular chaperone, essential for concentrative ER-exit (Pyrris et al. 2024).

FurE of Emericella nidulans (Aspergillus nidulans)

2.A.39.3.2Uracil/uridine permease Yeast Fur4 of Saccharomyces cerevisiae
2.A.39.3.3Uridine (nucleoside; fluorouridine; not uracil or allantoin) permease, Fui1 (Uridine:H+ (1:1) symporter) (inhibited by analogues with modifications at positions C5') (Zhang et al., 2006) Yeast Fui1 (YBL042; YBC2) of Saccharomyces cerevisiae

Allantoin permease, PucI, with 12 TMSs and both N- and C-termini on the inside. PucI transports allantoin with a Km of 24 mμM, but recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides (Ma et al. 2016).


PucI (YwoE) of Bacillus subtilis

2.A.39.3.5The probable hydantoin permease, HyuP (most similar to the hydantoin transporter of Microbacterium liquefaciens; Suzuki and Henderson, 2006) BacteriaHyuP of Arthrobacter aurescens (Q9F467)

The benzyl-hydantoin:cation symporter-1, Mhp1 (84% identical to 2.A.39.3.5).  The 3-d structures in the open and closed states (2.85 Å resolution) are known (Weyand et al., 2008). Models of the ion-coupled coonformational cycle have been proposed (Kazmier et al. 2014).


Mhp1 of Microbacterium liguefaciens
(2JLN_A) (210060745)

2.A.39.3.7Uracil permeaseYeastFur4 of Schizosaccharomyces pombe

Allantoin permease; encoded in an operon with allantoinase and other degradative enzymes (Moraes and Reithmeier 2012).


YbbW of Escherichia coli


NCS-1 homologue of unknown function and of 652 aas with 14 TMSs in a 2 + 2 + 2... arrangment.

Red Algae

NCS-1 homologue of Galdieria sulphuraria


TC#NameOrganismal TypeExample
2.A.39.4.1Thiamine permease, Thi10 Yeast Thi10 of Saccharomyces cerevisiae
2.A.39.4.2The nicotinamide riboside transporter, Nrt1 (Belenky et al., 2008)YeastNrt1 of Saccharomyces cerevisiae (Q08485)

TC#NameOrganismal TypeExample

The putative mannitol porter, MtlP (Rodionov et al. 2010).


MtlP of Shewanella frigidimarina (Q082R8)