An extracellular hydrophilic carboxy-terminal domain regulates the activity of TaALMT1, the aluminum-activated malate transport protein of wheat

Abstract

Al3+-resistant cultivars of wheat (Triticum aestivum L.) release malate through the Al3+-activated anion transport protein Triticum aestivum aluminum-activated malate transporter 1 (TaALMT1). Expression of TaALMT1 in Xenopus oocytes and tobacco suspension cells enhances the basal transport activity (inward and outward currents present in the absence of external Al3+), and generates the same Al3+-activated currents (reflecting the Al3+-dependent transport function) as observed in wheat cells. We investigated the amino acid residues involved in this Al3+-dependent transport activity by generating a series of mutations to the TaALMT1 protein. We targeted the acidic residues on the hydrophilic C-terminal domain of TaALMT1 and changed them to uncharged residues by site-directed mutagenesis. These mutant proteins were expressed in Xenopus oocytes and their transport activity was measured before and after Al 3+ addition. Three mutations (E274Q, D275N and E284Q) abolished the Al3+-activated transport activity without affecting the basal transport activity. Truncation of the hydrophilic C-terminal domain abolished both basal and Al3+-activated transport activities. Al 3+-dependent transport activity was recovered by fusing the N-terminal region of TaALMT1 with the C-terminal region of AtALMT1, a homolog from Arabidopsis. These findings demonstrate that the extracellular C-terminal domain is required for both basal and Al3+-dependent TaALMT1 activity. Furthermore, we identified three acidic amino acids within this domain that are specifically required for the activation of transport function by external Al3+. © 2010 Blackwell Publishing Ltd.