Publications

• Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals.

  Furukawa J., Inoue K., Maeda J., Yasujima T., Ohta K., Kanai Y., Takada T., Matsuo H., Yuasa H.

  The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, thos ... >> More

  The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na(+) and H(+), but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes. << Less

  Sci. Rep. 5:15057-15057(2015) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy.

  Parker M.D., Hyde R.J., Yao S.Y., McRobert L., Cass C.E., Young J.D., McConkey G.A., Baldwin S.A.

  Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue prot ... >> More

  Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs. << Less

  Biochem. J. 349:67-75(2000) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation.

  Yao S.Y., Ng A.M., Vickers M.F., Sundaram M., Cass C.E., Baldwin S.A., Young J.D.

  The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenz ... >> More

  The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K(m) values 0.7-2.6 mm), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel. << Less

  J Biol Chem 277:24938-24948(2002) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

  El Bissati K., Downie M.J., Kim S.K., Horowitz M., Carter N., Ullman B., Ben Mamoun C.

  The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was un ... >> More

  The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine. << Less

  Mol. Biochem. Parasitol. 161:130-139(2008) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1).

  Yao S.Y., Ng A.M., Cass C.E., Baldwin S.A., Young J.D.

  The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inh ... >> More

  The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter. << Less

  J. Biol. Chem. 286:32552-32562(2011) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.