CLONING AND FUNCTIONAL-CHARACTERIZATION OF A BRASSICA-NAPUS TRANSPORTER THAT IS ABLE TO TRANSPORT NITRATE AND HISTIDINE

A full-length cDNA for a membrane transporter was isolated from Brassi ca napus by its sequence homology to a previously cloned Arabidopsis l ow 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, incl uding amino acids, peptides, and nitrate. To identify the substrates o f this plant gene, we have expressed the protein in Xenopus oocytes, T he properties of the transporter are consistent with a proton cotransp ort mechanism for nitrate, and the voltage dependence of the K-m for n itrate was determined. The K-m for nitrate was shown to increase from 4 to 14 mM as the membrane voltage became more negative from -40 to -1 80 mV, Oocytes expressing the gene could accumulate internal nitrate t o concentrations higher than those measured in water-injected controls . A range of different substrate molecules for the transporter was tes ted, but of these, histidine gave the largest currents, although the a ffinity was in the millimolar range. The pH dependence of the activity of the transporter was different for the substrates, with histidine t ransport favored at alkaline and nitrate at acid external pH, Kinetic analysis of the mechanism of histidine transport suggests a cotranspor t of protons and the neutral form of the amino acid, with the K, for h istidine decreasing at more negative membrane voltages. This gene is t he first member of this family of transporters for which the transport of two very different types of substrate, nitrate and histidine, has been demonstrated.