Pore mutations in ammonium transporter AMT1 with increased electrogenic ammonium transport activity.

J Biol Chem

Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA.

Published: September 2009

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.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757203PMC
http://dx.doi.org/10.1074/jbc.M109.020842DOI Listing

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