Abstract
A full-length cDNA for a membrane transporter was isolated from Brassica napus by its sequence homology to a previously cloned Arabidopsis low affinity nitrate transporter. The cDNA encodes a predicted protein of 589 amino acid residues with 12 putative transmembrane domains. The transporter belongs to a multigene family with members that have been identified in bacteria, fungi, plants, and animals and that are able to transport a range of different nitrogen-containing substrates, including amino acids, peptides, and nitrate. To identify the substrates of this plant gene, we have expressed the protein in Xenopus oocytes. The properties of the transporter are consistent with a proton cotransport mechanism for nitrate, and the voltage dependence of the Km for nitrate was determined. The Km for nitrate was shown to increase from 4 to 14 mM as the membrane voltage became more negative from -40 to -180 mV. Oocytes expressing the gene could accumulate internal nitrate to concentrations higher than those measured in water-injected controls. A range of different substrate molecules for the transporter was tested, but of these, histidine gave the largest currents, although the affinity was in the millimolar range. The pH dependence of the activity of the transporter was different for the substrates, with histidine transport favored at alkaline and nitrate at acid external pH. Kinetic analysis of the mechanism of histidine transport suggests a cotransport of protons and the neutral form of the amino acid, with the Km for histidine decreasing at more negative membrane voltages. This gene is the first member of this family of transporters for which the transport of two very different types of substrate, nitrate and histidine, has been demonstrated.
Highlights
A full-length cDNA for a membrane transporter was isolated from Brassica napus by its sequence homology to a previously cloned Arabidopsis low affinity nitrate transporter
We show that another member of this family, isolated from the plant Brassica napus, can transport both the amino acids and nitrate when expressed in Xenopus oocytes
(BnNRT1;2) can accumulate nitrate, but not chloride. These results show that this low affinity nitrate transporter is able to transport several different nitrogen-containing molecules; the transport requires high external concentration of the substrate, suggesting that if present in root cells, it is most likely to function in uptake of nitrate from the soil when it is available at high concentrations
Summary
(BnNRT1;2) can accumulate nitrate, but not chloride. These results show that this low affinity nitrate transporter is able to transport several different nitrogen-containing molecules; the transport requires high external concentration of the substrate, suggesting that if present in root cells, it is most likely to function in uptake of nitrate from the soil when it is available at high concentrations. The production of mRNA for the transporter is induced in roots by treatment with only low external concentrations of nitrate
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