Amino Acid Transport

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Model of amino acid symporter-mediated transport into plant cells: The H+-ATPase generates a substantial proton electrochemical potential difference (both ApH and AY) that can drive transport reactions three or four orders of magnitude away from equilibrium. A diagrammatic representation of the 11 transmembrane domains (TMDs) of NAT2/AAPl is also presented. demonstrated in isolated membrane vesicles by examining the effect of membrane electrical potential (AY) on ApH-dependent transport activity. Potassium gradients and valinomycin were used to manipulate AY. In the absence of compensating charge flow, a low rate of amino acid flux was observed. When the AY was clamped at zero, transport was stimulated fourfold, and when a negative AY was imposed, transport was stimulated sixfold. These data are consistent with an electrogenic transport system that results in primary charge separation as proton cotransport drives substrate accumulation (Li and Bush 1990). Similar results were reported for this porter expressed in Xenopus oocytes (Boorer et al. 1996). The stoichiometry of H"-amino acid cotransport was recently measured using electrophysiologic~ methods to measure conductance through another plant amino acid symporter (AAP5) expressed in Xenopus oocytes (Boorer and Fischer 1997). Boorer and Fischer (1997) used direct measurements of [3H]amino acid uptake and simultaneous measurements of inward current to show that the stoich~o~etry of the amino acid symporters is one w' per one amino acid. This held true for the basic, neutral, and acidic amino acids examined. Because the acidic amino acids also carried one positive charge, these results suggest they are transported in their neutral forms. The number of symporters that mediate amino acid transport into higher-plant cells has yet to be determined. <strong>Transport</strong> competition experiments with isolated membr~e vesicles provided evidence of at least four amino acid symporters: an acidic amino acid symporter, a basic amino acid symporter, and two symporters for the neutral amino acids (Li and Bush 1990, 1991). The neutral amino acid porters were resolved in the competition experiments because of their differential affinity for isoleucine, valine, threonine, and proline. A more thorough examination of the kinetics of inter-amino acid transport
competition among the neutral amino acids confirmed the initial conclusion. Li and Bush (1991) hypothesized that branching at the fharbon in isoleucine, valine, and threonine results in steric interactions that block their access to one of the neutral porters. More recently, several amino acid symporters have been cloned, and we now know that many amino acid symporters are found in the plant (see later discussion). The ability of various amino acid analogues to compete for transport into purified membrane vesicles was used to identify molecular determinants that are important for substrate recognition by the neutral amino acid symporters (Li and Bush 1992). D-ISOmers of alanine and isoleucine were not effective transport antagonists of the L-isomers, thereby implicating stereospecificity and suggesting that positional relations between the a-amino and carboxyl groups is an important parameter in substrate recognition. This conclusion was supported by the observation that p-alanine and analogues with methylation at the a-carbon, at the carboxyl group, or at the a-amino group, were not effective transport antagonists. In contrast, analogues with various substitutions at the distal end of the amino acid were less potent antagonists. More recently, el~trophysiological investigations of AAPS expressed in Xenopus oocytes also identified the a-amino and carboxyl groups, as well as the P-carbon, as important dete~inants in defining substrate specificity (Boorer and Fischer 1997). Thus, the binding site for the carboxyl end of the amino acid is a well-defined space that is characterized by compact, asymmetric positional relations and specific ligand interactions (Li and Bush 1992). The amino acid symporters are inhibited by carbonyl cyanide ~-chlorophenyl hydrazone (CCCP) and diethylpyrocarbonate (DEPC; Bush and Langston-~nkefer 1988; Li and Bush 1990). CCCP is a protonophore that inhibits amino acid transport by dissipating the proton-motive force that drives the symport reaction. DEPC is a small molecule that forms covalent bonds with the imidazole ring of histidine residues. The time-dependent inactivation of alanine transport in the presence and absence of amino acids showed that substrate binding protects the symporter from DEPC inactivation (Fig. 3). This observation is consistent with DEPC binding at (or at least confo~ationally linked to) the substrate-binding site of the carrier (Bush and Li 1991; see Bush 1993b, for discussion). Since histidine residues frequently participate in protonation reactions, this observation implicates a histidine residue in the reaction mechanism. Initial efforts to identify the amino acid syrnporters by using standard biochemical strategieswerelargelyunsuccessful.Althoughthiswasoftenquitefrustrating, it was not too surprising, given that these are very low-abundance, integral membrane prot6ins. Fortunately, a novel molecular approach was successfully used to clone several plant amino acid symporters and biochemical analysis has moved forward using these clones and a variety of expression systems. The first plant amino acid symporter cloned was identified using ~nctional complementation of yeast amino acid transport mutants (Frommer et al. 1993; Wsu et ai. 1993). ~acc~aro~~ces cerevisiae strains that are auxotrophic and transport-deficient for a specific amino acid were tr~sfo~ed with plant cDNA libraries that are constructed in yeast expression vectors. Those transformants that successfully expressed an appropriate plant amino acid symporter were easily identified by screening for growth on low concentrations of the limiting amino acid. Because these yeast strains are both auxotrophic and transport-deficient, a simple secondary screen in the absence of the limiting
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Amino Acid Transport

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