Pore mutations in ammonium transporter AMT1 with increased electrogenic ammonium transport activity.
AMT/Mep ammonium transporters mediate high affinity ammonium/ammonia uptake in bacteria, fungi, and plants. The Arabidopsis AMT1 proteins mediate uptake of the ionic form of ammonium. AMT transport activity is controlled allosterically via a highly conserved cytosolic C terminus that interacts with neighboring subunits in a trimer. The C terminus is thus capable of modulating the conductivity of the pore. To gain insight into the underlying mechanism, pore mutants suppressing the inhibitory effect of mutations in the C-terminal trans-activation domain were characterized. AMT1;1 carrying the mutation Q57H in transmembrane helix I (TMH I) showed increased ammonium uptake but reduced capacity to take up methylammonium. To explore whether the transport mechanism was altered, the AMT1;1-Q57H mutant was expressed in Xenopus oocytes and analyzed electrophysiologically. AMT1;1-Q57H was characterized by increased ammonium-induced and reduced methylammonium-induced currents. AMT1;1-Q57H possesses a 100x lower affinity for ammonium (K(m)) and a 10-fold higher V(max) as compared with the wild type form. To test whether the trans-regulatory mechanism is conserved in archaeal homologs, AfAmt-2 from Archaeoglobus fulgidus was expressed in yeast. The transport function of AfAmt-2 also depends on trans-activation by the C terminus, and mutations in pore-residues corresponding to Q57H of AMT1;1 suppress nonfunctional AfAmt-2 mutants lacking the activating C terminus. Altogether, our data suggest that bacterial and plant AMTs use a conserved allosteric mechanism to control ammonium flux, potentially using a gating mechanism that limits flux to protect against ammonium toxicity.
Loqué
Dominique
D
Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA.
Mora
Silvia I
SI
Andrade
Susana L A
SL
Pantoja
Omar
O
Frommer
Wolf B
WB
D000818:Animals; D019611:Archaeoglobus fulgidus / Q000378:metabolism; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D005816:Genetic Complementation Test; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D015533:Transcriptional Activation; D014981:Xenopus
D027682:Cation Transport Proteins; D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; C088952:ammonium transporters, plant