KINETICS AND SPECIFICITY OF A H-ACID TRANSPORTER FROM ARABIDOPSIS-THALIANA( AMINO)

The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiolo gical, radiotracer flux, and electron microscopic methods to character ize the biophysical properties, kinetics, and specificity of the trans porter. Uptake of alanine was H+-dependent increasing from 14 pmol/ooc yte/h at 0.032 mu M H+ to 370 pmol/oocyte/h at 10 mu M H+. AAP1 was el ectrogenic; there was an amino acid-induced depolarization of the oocy te plasma membrane and net inward currents through the transporter due to the transport of amino acids. AAP1 transported a wide spectrum of amino acids favoring neutral amino acids with short side chains. The m aximal current (i(max)) for alanine, proline, glutamine, histidine, an d glutamate was voltage-and [H+](o)-dependent. Similarly, the i(max)(H ) was voltage-and [amino acid](o)-dependent. The i(max) for both H+ an d amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transp orter. The K-0.5 of the transporter for amino acids decreased as [H+]( o) increased and was lower at negative membrane potentials. The K-0.5 for H+ was relatively voltage-independent and decreased as [amino acid ](o) increased. This positive cooperativity suggests that the transpor ter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were >1, suggesting that the transporter has more than one binding site for both H+ and amino acid, Freeze-fracture elec tron microscopy was used to estimate the number of transporters expres sed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 i ncreased similar to 5-fold above water-injected controls and correspon ded to a turnover number 350 to 800 s(-1).