Specificity and Stoichiometry of the ArabidopsisH+/Amino Acid Transporter AAP5

Abstract

The H؉ -dependent AAP5 amino acid transporter from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used radiotracer flux and electrophysiology methods to investigate its substrate specificity and stoichiometry. Inward currents of up to 9 A were induced by a broad spectrum of amino acids, including anionic, cationic, and neutral amino acids. The apparent affinity of AAP5 for amino acids was influenced by the position of side chain branches, bulky ring structures, and charged groups. The maximal current was dependent on amino acid charge, but was relatively independent of amino acid structure. A detailed kinetic analysis of AAP5 using lysine, alanine, glutamate, and histidine revealed H ؉ -dependent differences in the apparent affinity constants for each substrate. The differences were correlated to the effect of H ؉ concentration on the net charge of each amino acid and suggested that AAP5 transports only the neutral species of histidine and glutamate. Stoichiometry experiments, whereby the uptake of 3 H-labeled amino acid and net inward charge were simultaneously measured in voltage-clamped oocytes, showed that the charge:amino acid stoichiometry was 2:1 for lysine and 1:1 for alanine, glutamate, and histidine. The results confirm that histidine is transported in its neutral form and show that the positive charge on lysine contributes to the magnitude of its inward current. Thus, the transport stoichiometry of AAP5 is 1 H ؉ :1 amino acid irrespective of the net charge on the transported substrate. Structural features of amino acid molecules that are involved in substrate recognition by AAP5 are discussed.Transport of amino acids across the plasma membrane of higher plants is mediated by proton-coupled transport proteins that utilize the electrochemical gradient for H ϩ to drive the uphill transport of amino acids (reviewed in Refs. 1-4). Kinetic analysis of amino acid uptake into plasma membrane vesicles isolated from sugar beet leaves suggests the presence of four H ϩ -coupled amino acid transport systems (5-7), and at least 10 H ϩ /amino acid transporters have been isolated by complementing yeast amino acid transport mutants with plant cDNA libraries (8 -12). These transporters have very broad and overlapping specificities. However, each exhibits a preference for amino acids possessing a particular molecular geometry or charge. Analysis of the substrate specificity of amino acid transporters in yeast cells and plasma membrane vesicles is traditionally accomplished by measuring the inhibition of amino acid transport activity by various substrates. Competition experiments yield information on substrates that interact with amino acid transporters, but do not allow a distinction between substrates that are transported and those that act as inhibitors.We previously analyzed the specificity and kinetic properties of the Arabidopsis AAP1 H ϩ /amino acid transporter by expressing the cloned gene in Xenopus oocytes and measuring substrate-induced currents using electrophysiology methods (13). AAP1 transpo...

The Effect of a Genetically Reduced Plasma Membrane Protonmotive Force on Vegetative Growth of Arabidopsis

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“…H + /amino acid cotransporters have substrate specificities based on side chain charges (Fischer et al, 1995), but they respond to the membrane potential because they transport one proton per one amino acid irrespective of their overall net charge (Boorer and Fischer, 1997). Alternatively, if a particular amino acid is taken up with more than one proton, which was proposed in the case of Val uptake, wildtype and mutant plants will respond differently to such an amino acid (Borstlap and Schuurmans, 2000).…”

High Affinity Amino Acid Transporters Specifically Expressed in Xylem Parenchyma and Developing Seeds of Arabidopsis

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Genes and Proteins for Solute Transport and Sensing

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