The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in . The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.


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  1. Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. (1990). Basic local alignment search toolJ Mol Biol 215403410 [View Article][PubMed]. [Google Scholar]
  2. Avery S.V. (1995). Caesium accumulation by micro-organisms: uptake mechanisms, cation competition, compartmentalization and toxicityJ Ind Microbiol 147684 [View Article][PubMed]. [Google Scholar]
  3. Baxter I., Hosmani P.S., Rus A., Lahner B., Borevitz J.O., Muthukumar B., Mickelbart M.V., Schreiber L., Franke R.B., Salt D.E. (2009). Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis PLoS Genet 5e1000492 [View Article][PubMed]. [Google Scholar]
  4. Boyd J., Gradmann D., Boyd C.M. (2003). Transinhibition and voltage-gating in a fungal nitrate transporterJ Membr Biol 195109120 [View Article][PubMed]. [Google Scholar]
  5. Brownlee A.G., Arst H.N. Jr (1983). Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene clusterJ Bacteriol 15511381146[PubMed]. [Google Scholar]
  6. Cabrera E., González-Montelongo R., Giraldez T., de la Rosa D.A., Siverio J.M. (2014). Molecular components of nitrate and nitrite efflux in yeastEukaryot Cell 13267278 [View Article][PubMed]. [Google Scholar]
  7. Cove D.J. (1976). Chlorate toxicity in Aspergillus nidulans: the selection and characterisation of chlorate resistant mutantsHeredity (Edinb) 36191203 [View Article][PubMed]. [Google Scholar]
  8. Downey R.J., Gedeon C.A. (1994). Evidence for a H+ nitrate symporter in Aspergillus nidulans Microbios 783546[PubMed]. [Google Scholar]
  9. Enstone D.E., Peterson C.A., Ma F. (2002). Root endodermis and exodermis, structure, function, and responses to the environmentJ Plant Growth Regul 21335351 [View Article]. [Google Scholar]
  10. Forde B.G. (2000). Nitrate transporters in plants: structure, function and regulationBiochim Biophys Acta 1465219235 [View Article][PubMed]. [Google Scholar]
  11. Gao-Rubinelli F., Marzluf G.A. (2004). Identification and characterization of a nitrate transporter gene in Neurospora crassa Biochem Genet 422134 [View Article][PubMed]. [Google Scholar]
  12. Hampton C.R., Bowen H.C., Broadley M.R., Hammond J.P., Mead A., Payne K.A., Pritchard J., White P.J. (2004). Cesium toxicity in ArabidopsisPlant Physiol 13638243837 [View Article][PubMed]. [Google Scholar]
  13. Kinghorn J.R., Sloan J., Kana'n G.J.M., Dasilva E.R., Rouch D.A., Unkles S.E. (2005). Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporterGenetics 16913691377 [View Article][PubMed]. [Google Scholar]
  14. Kosola K.R., Bloom A.J. (1996). Chlorate as a transport analog for nitrate absorption by roots of tomatoPlant Physiol 11012931299[PubMed]. [Google Scholar]
  15. LaBrie S.T., Wilkinson J.Q., Crawford N.M. (1991). Effect of chlorate treatment on nitrate reductase and nitrite reductase gene expression in Arabidopsis thaliana Plant Physiol 97873879 [View Article][PubMed]. [Google Scholar]
  16. Léchenne B., Reichard U., Zaugg C., Fratti M., Kunert J., Boulat O., Monod M. (2007). Sulphite efflux pumps in Aspergillus fumigatus and dermatophytesMicrobiology 153905913 [View Article][PubMed]. [Google Scholar]
  17. McDonald D.W., Coddington A. (1974). Properties of the assimilatory nitrate reductase from Aspergillus nidulans Eur J Biochem 46169178 [View Article][PubMed]. [Google Scholar]
  18. Okamoto M., Vidmar J.J., Glass A.D.M. (2003). Regulation of NRT1 NRT2 gene families of Arabidopsis thaliana: responses to nitrate provisionPlant Cell Physiol 44304317 [View Article][PubMed]. [Google Scholar]
  19. Orsel M., Chopin F., Leleu O., Smith S.J., Krapp A., Daniel-Vedele F., Miller A.J. (2006). Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interactionPlant Physiol 14213041317 [View Article][PubMed]. [Google Scholar]
  20. Pao S.S., Paulsen I.T., Saier M.H. Jr (1998). Major facilitator superfamilyMicrobiol Mol Biol Rev 62134[PubMed]. [Google Scholar]
  21. Parker J.L., Newstead S. (2014). Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1Nature 5076872 [View Article][PubMed]. [Google Scholar]
  22. Siddiqi M.Y., King B.J., Glass A.D.M. (1992). Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activityPlant Physiol 100644650 [View Article][PubMed]. [Google Scholar]
  23. Sun J., Bankston J.R., Payandeh J., Hinds T.R., Zagotta W.N., Zheng N. (2014). Crystal structure of the plant dual-affinity nitrate transporter NRT1.1Nature 5077377 [View Article][PubMed]. [Google Scholar]
  24. Trueman L.J., Richardson A., Forde B.G. (1996). Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii Aspergillus nidulans Gene 175223231 [View Article][PubMed]. [Google Scholar]
  25. Unkles S.E., Hawker K.L., Grieve C., Campbell E.I., Montague P., Kinghorn J.R. (1991). crnA encodes a nitrate transporter in Aspergillus nidulans Proc Natl Acad Sci U S A 88204208 [View Article][PubMed]. [Google Scholar]
  26. Unkles S.E., Zhou D., Siddiqi M.Y., Kinghorn J.R., Glass A.D.M. (2001). Apparent genetic redundancy facilitates ecological plasticity for nitrate transportEMBO J 2062466255 [View Article][PubMed]. [Google Scholar]
  27. Unkles S.E., Rouch D.A., Wang Y., Siddiqi M.Y., Glass A.D.M., Kinghorn J.R. (2004). Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporterProc Natl Acad Sci U S A 1011754917554 [View Article][PubMed]. [Google Scholar]
  28. Unkles S.E., Symington V.F., Kotur Z., Wang Y., Siddiqi M.Y., Kinghorn J.R., Glass A.D.M. (2011). Physiological and biochemical characterization of AnNitA, the Aspergillus nidulans high-affinity nitrite transporterEukaryot Cell 1017241732 [View Article][PubMed]. [Google Scholar]
  29. Unkles S.E., Karabika E., Symington V.F., Cecile J.L., Rouch D.A., Akhtar N., Cromer B.A., Kinghorn J.R. (2012). Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifsBiochem J 4473542 [View Article][PubMed]. [Google Scholar]
  30. Wang Y., Li W., Siddiqi Y., Kinghorn J.R., Unkles S.E., Glass A.D.M. (2007). Evidence for post-translational regulation of NrtA, the Aspergillus nidulans high-affinity nitrate transporterNew Phytol 175699706 [View Article][PubMed]. [Google Scholar]
  31. Wang Y., Li W., Siddiqi Y., Symington V.F., Kinghorn J.R., Unkles S.E., Glass A.D.M. (2008). Nitrite transport is mediated by the nitrite-specific high-affinity NitA transporter and by nitrate transporters NrtANrtB in Aspergillus nidulans. Fungal Genet Biol 4594102 [View Article][PubMed]. [Google Scholar]
  32. White P.J., Broadley M.R. (2000). Mechanisms of caesium uptake by plantsNew Phytol 147241256 [View Article]. [Google Scholar]
  33. Yan H., Huang W., Yan C., Gong X., Jiang S., Zhao Y., Wang J., Shi Y. (2013). Structure and mechanism of a nitrate transporterCell Reports 3716723 [View Article][PubMed]. [Google Scholar]
  34. Zheng H., Wisedchaisri G., Gonen T. (2013). Crystal structure of a nitrate/nitrite exchangerNature 497647651 [View Article][PubMed]. [Google Scholar]
  35. Zhou J.J., Trueman L.J., Boorer K.J., Theodoulou F.L., Forde B.G., Miller A.J. (2000). A high affinity fungal nitrate carrier with two transport mechanismsJ Biol Chem 2753989439899 [View Article][PubMed]. [Google Scholar]

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