Regulation of NH4  + Transport by Essential Cross Talk between AMT Monomers through the Carboxyl Tails  

Abstract

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.

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism inArabidopsis

“…It is thus conceivable that ammonium itself inhibits uptake at least during short-term exposure, possibly to prevent ammonium toxicity. The finding that mutants such as T460D as well as most other modifications of the conserved domain of the C terminus of AMT1 inactivate the transporter suggests that phosphorylation of T460 in AMT1;1 may lead to inhibition of uptake (Loqué et al, 2007;Neuhä user et al, 2007). This hypothesis is consistent with the observed downregulation of the transport by ammonium itself (Figure 6; Rawat et al, 1999).…”

“…Allosteric Inhibition of AMT1 by Phosphorylationregulation is mediated by a cytosolic C-terminal domain (Marini et al, 2000;Loqué et al, 2007;Neuhä user et al, 2007;Severi et al, 2007). The AMT/MEP C-terminal domain is highly conserved in >700 AMT homologs from cyanobacteria to higher plants with no obvious occurrence of cases lacking this domain (Loqué et al, 2007).…”

The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein

“…The first 22 residues of the hydrophilic C-terminal extension of Mep-Amt proteins are largely conserved and play an allosteric role in Mep-Amt activity control [31][32][33][34] . The conservation in the C terminus spreads beyond this domain considering several subgroups of fungal Mep-Amts (Supplementary Fig.…”

“…Structural data of bacterial Mep-Amt trimers indicate that the conserved domain of the C terminus of one monomer interacts with ILs of its own subunit and with the loops of the contiguous one 17,50 . The conserved portion of the C-terminal domain of Mep-Amt proteins is further reported to trans-activate the monomers via an allosteric switch [32][33][34] . Here, we show that the Mep2 C terminus contains at least three functional domains.…”

Molecular mechanisms of ammonium transport and accumulation in plants

“…This distinct cellular localization may explain its somewhat altered transcriptional regulation compared to other root AMTs. AtAMT1;2 is involved in ammonium transfer into the vascular tissue, since apoplasmic ammonium diffusion across the endodermis is restricted by the casparian strip [17]. When heterologously expressed in oocytes, AtAMT1;2 elicited large currents that were half maximal at $140 lM ammonium [17].…”