Specificity and stoichiometry of the Arabidopsis H+/amino acid transporter AAP5.
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 microA 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 3H-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.
Boorer
K J
KJ
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA. kboorer@physiology.medsch.ucla.edu
Fischer
W N
WN
D026905:Amino Acid Transport Systems; D000818:Animals; D017920:Antiporters / Q000737:chemistry / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D001693:Biological Transport, Active; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014981:Xenopus
D026905:Amino Acid Transport Systems; D017920:Antiporters; D026901:Membrane Transport Proteins