Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat.
In this study we examined the role of protein phosphorylation/dephosphorylation in the transport properties of the wheat (Triticum aestivum) root malate efflux transporter underlying Al resistance, TaALMT1. Pre-incubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurosporine) strongly inhibited both basal and Al(3+)-enhanced TaALMT1-mediated inward currents (malate efflux). Pre-incubation with phosphatase inhibitors (okadaic acid and cyclosporine A) resulted in a modest inhibition of the TaALMT1-mediated currents. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), enhanced TaALMT1-mediated inward currents. Since these observations suggest that TaALMT1 transport activity is regulated by PKC-mediated phosphorylation, we proceeded to modify candidate amino acids in the TaALMT1 protein in an effort to identify structural motifs underlying the process regulating phosphorylation. The transport properties of eight single point mutations (S56A, S183A, S324A, S337A, S351-352A, S384A, T323A and Y184F) generated in amino acid residues predicted to be phosphorylation sites and examined electrophysiologically. The basic transport properties of mutants S56A, S183A, S324A, S337A, S351-352A, T323A and Y184F were not altered relative to the wild-type TaALMT1. Likewise the sensitivity of these mutants to staurosporine resembled that observed for the wild-type transporter. However, the mutation S384A was noticeable, as in oocytes expressing this mutant protein TaALMT1-mediated basal and Al-enhanced currents were significantly inhibited, and the currents were insensitive to staurosporine or PMA. These findings indicate that S384 is an essential residue regulating TaALMT1 activity via direct protein phosphorylation, which precedes Al(3+) enhancement of transport activity.
Ligaba
Ayalew
A
Robert W. Holley Center for Agriculture and Health, USDA-ARS, Tower Road, Cornell University, Ithaca, NY 14853-2901, USA.
Kochian
Leon
L
Piñeros
Miguel
M
D000535:Aluminum / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008293:Malates / Q000378:metabolism*; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D014908:Triticum / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D008293:Malates; D027361:Organic Anion Transporters; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D010768:Phosphoserine; C016658:phorbolol myristate acetate; C030298:malic acid; D000535:Aluminum; D013755:Tetradecanoylphorbol Acetate