Abstract
The ammonium flux across prokaryotic, plant, and animal membranes is regulated by structurally related ammonium transporters (AMT) and/or related Rhesus (Rh) glycoproteins. Several plant AMT homologs, such as AtAMT1;2 from Arabidopsis, elicit ionic, ammonium-dependent currents when expressed in oocytes. By contrast, functional evidence for the transport of NH3 and the lack of coupled ionic currents has been provided for many Rh proteins. Furthermore, despite high resolution structures the transported substrate in many bacterial homologs, such as AmtB from Escherichia coli, is still unclear. In a heterologous genetic screen in yeast, AtAMT1;2 mutants with reduced transport activity were identified based on the resistance of yeast to the toxic transport analog methylamine. When expressed in oocytes, the reduced transport capacity was confirmed for either of the mutants Q67K, M72I,and W145S. Structural alignments suggest that these mutations were dispersed at subunit contact sites of trimeric AMTs, without direct contact to the pore lumen. Surprisingly, and in contrast to the wild type AtAMT1;2 transporter, ionic currents were not associated with the substrate transport in these mutants. Whether these data suggest that the wild type AtAMT1;2 functions as H(+)/NH3 co-transporter, as well as how the strict substrate coupling with protons is lost by the mutations, is discussed.
Highlights
The substrate transported by ammonium transporters (AMT)/Rh proteins (NH3/NH4ϩ/NH3ϩHϩ) is strongly disputed
Two other mutants (G357V and L399R, isolated in duplicate) improved the growth of the wild type yeast on MeA above the level of the empty plasmid transformed controls. These plasmids failed to support the growth of the triple-⌬mep strain on low ammonium (Fig. 1A). These latter two mutations and the two mutations V179L and G291S occurred in residues that were conserved among most AMT/Rh proteins, but the three other mutations (Q67K, M72I, and W145S) were confined to stretches that were exclusively conserved among plant AMT1 sequences, but not in AmtB, AtAMT2, and Rh sequences
The mutations in the residues Gln-67, Met-72, and Trp-145 were characterized in more detail. The function of these mutants in yeast was consistent with the idea that ammonium and methylamine were transported at a reduced rate or that less AMT transporter protein was expressed at the plasma membrane of yeast
Summary
The substrate transported by AMT/Rh proteins (NH3/NH4ϩ/NH3ϩHϩ) is strongly disputed. Results: In the net NH4ϩ transporter AtAMT1;2 mutations led to a change in coupling of NH3 and Hϩ transport. By contrast to the uncertain functional mechanism in EcAmtB, direct electrogenic transport and membrane potential-driven accumulation of ionic NH4ϩ and methylammonium (MeAϩ) have been measured for several plant AMTs [20, 21]. The data are discussed in light of the proposed transport mechanisms for AMTs
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