Ammonium transport across plant plasma membranes is facilitated by AMT/Rh-type ammonium transporters (AMTs), which also have homologs in most organisms. In the roots of the plant Arabidopsis (Arabidopsis thaliana), AMTs have been identified that function directly in the high-affinity NH4  + acquisition from soil. Here, we show that AtAMT1;2 has a distinct role, as it is located in the plasma membrane of the root endodermis. AtAMT1;2 functions as a comparatively low-affinity NH4  + transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH4  + transport activity. Coexpressing these mutants along with wild-type AtAMT1;2 substantially reduced the activity of the wild-type transporter. A molecular model of AtAMT1;2 provides a plausible explanation for the dominant inhibition, as the C terminus of one monomer directly contacts the neighboring subunit. It is suggested that part of the cytoplasmic C terminus of a single monomer can gate the AMT trimer. This regulatory mechanism for rapid and efficient inactivation of NH4  + transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium.

Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP-like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP-like mutants conducted urea but not NH 3 , and a residue without direct contact to the pore lumen was critical for conduction in the mutants.

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4 + and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4 + and MeA+ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH4 +, or cotransports NH3/H+, RhCG mediates electroneutral NH3 transport.

AMT (ammonium transporter)/Rh (Rhesus) ammonium transporters/channels are identified in all domains of life and fulfil contrasting functions related either to ammonium acquisition or excretion. Based on functional and crystallographic high-resolution structural data, it was recently proposed that the bacterial AmtB (ammonium transporter B) is a gas channel for NH3 [Khademi, O'Connell, III, Remis, Robles-Colmenares, Miercke and Stroud (2004) Science 305, 1587-1594; Zheng, Kostrewa, Berneche, Winkler and Li (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 17090-17095]. Key residues, proposed to be crucial for NH3 conduction, and the hydrophobic, but obstructed, pore were conserved in a homology model of LeAMT1;1 from tomato. Transport by LeAMT1;1 was affected by mutations of residues that were predicted to constitute the aromatic recruitment site for NH4+ at the external pore entrance. Despite the structural similarities, LeAMT1;1 was shown to transport only the ion; each transported 14C-methylammonium molecule carried a single positive elementary charge. Similarly, NH4+ (or H+/NH3) was transported, but NH3 conduction was excluded. It is concluded that related proteins and a similar molecular architecture can apparently support contrasting transport mechanisms.

AtAMT1;1 was the founding member of the family of AMT/Rh ammonium transporters and accounts for about one third of the total ammonium absorption in the roots of the model plant Arabidopsis. Recent evidence suggested that at least some AMT/Rh proteins are NH3 gas channels. In order to evaluate the transported form of ammonium in AtAMT1;1, the protein was functionally expressed in Xenopus oocytes. AtAMT1;1 elicited NH4+ and methylammonium (MeA+) inward currents that saturated in a voltage-dependent manner with a half maximal concentration of 2.7 +/- 1.6 microM for NH4+ and 5.0 +/- 0.7 microM for the transport analogue methylammonium. AtAMT1;1 was plasma membrane localized and expressed in the root cortex and epidermis, including root hairs. The AtAMT1;1-GFP fusion construct under control of its endogenous promoter revealed additional localization of the protein in the pericycle, in the leaf epidermis, and in mesophyll cells. The functional data and its localization suggest that AtAMT1;1 participates in concentrative NH4+ acquisition in roots, in long-distance transport to the shoots, and in re-uptake of apoplastic NH4+ that derives from photorespiration in shoots.