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 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.
Zhou
J J
JJ
Biochemistry and Physiology Department, Integrated Approach to Crop Research (IACR)-Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.
Theodoulou
F L
FL
Muldin
I
I
Ingemarsson
B
B
Miller
A J
AJ
D000818:Animals; D029681:Arabidopsis Proteins; D001937:Brassica / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009566:Nitrates / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D018517:Plant Roots / Q000378:metabolism; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009566:Nitrates; D033902:Periplasmic Binding Proteins; D018075:RNA, Complementary; D012333:RNA, Messenger; C018963:histidine-binding protein; D006639:Histidine