An extracellular hydrophilic carboxy-terminal domain regulates the activity of TaALMT1, the aluminum-activated malate transport protein of wheat.
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 Al³+ 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. Al³+-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+.
Furuichi
Takuya
T
Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama 710-0046, Japan.
Sasaki
Takayuki
T
Tsuchiya
Yoshiyuki
Y
Ryan
Peter R
PR
Delhaize
Emmanuel
E
Yamamoto
Yoko
Y
D000535:Aluminum / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001692:Biological Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014908:Triticum / Q000235:genetics* / Q000378:metabolism
D027361:Organic Anion Transporters; D010940:Plant Proteins; D000535:Aluminum