RESUMEN
Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K+ ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether the base (NH3) or NH3 coupled with an H+ are transported. Here, using expression of two plant AMTs (AtAMT1;2 and AMT2) in budding yeast, we found that systematic replacements in the conserved twin-histidine motif, a hallmark of most AMT/Mep/Rh, alter substrate recognition, transport capacities, N isotope selection, and selectivity against K+ AMT-specific differences were found for histidine variants. Variants that completely lost ammonium N isotope selection, a feature likely associated with NH4+ deprotonation during passage, substantially transported K+ in addition to NH4+ Of note, the twin-histidine motif was not essential for ammonium transport. However, it conferred key AMT features, such as high substrate affinity and selectivity against alkali cations via an NH4+ deprotonation mechanism. Our findings indicate that the twin-His motif is the core structure responsible for substrate deprotonation and isotopic preferences in AMT pores and that decreased deprotonation capacity is associated with reduced selectivity against K+ We conclude that optimization for ammonium transport in plant AMT represents a compromise between substrate deprotonation for optimal selectivity and high substrate affinity and transport rates.
