Direct observation of electrogenic NH ₄ ⁺ transport in ammonium transport (Amt) proteins

Abstract

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH 4 + scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH 4 + /NH 3 transport is used instead in acid-base and pH homeostasis in kidney or NH 4 + /NH 3 (and eventually CO 2 ) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.ammonium transport proteins | Amt/Rh family | cation transport

Mechanism of NH 4 + Recruitment and NH 3 Transport in Rh Proteins

“…We have rather proposed that NH 4 + transfers a proton to the signature histidine dyad, followed by diffusion of NH 3 down the hydrophobic pore, and the transport of the excess proton through a water wire filling this pore (Wang et al, 2012). The overall process results in the net transport of NH 4 + , in agreement with the bulk of functional studies on Amt proteins (Ninnemann et al, 1994;Siewe et al, 1996;Marini et al, 1997;Ludewig et al, 2002;Wacker et al, 2014), and supported by other free energy calculations on Amt-1 (Ullmann et al, 2012).…”

“…However, functional studies paint a more complex picture. The measurement of electric current provides strong evidence that Amt proteins transport NH 4 + (Ludewig et al, 2002;Wacker et al, 2014), although the closely related prokaryotic homolog AmtB from E. coli was reported to transport NH 3 (Khademi et al, 2004;Javelle et al, 2005), a view that was later challenged (Javelle et al, 2007). The debate is more open in the case of Rh proteins, which were reported to transport NH 3 (Ludewig, 2004;Ripoche et al, 2004;MouroChanteloup et al, 2010) or NH 4 + (Hemker et al, 2003;Nakhoul et al, 2005Nakhoul et al, , 2010 or both (Bakouh et al, 2004;Benjelloun et al, 2005).…”

A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform

“…Based on electrophoretic analyses and docking calculations, we propose that trimeric Bot1 is the predominant conformation in planta or that mono-, di-, and trimeric forms are in a dynamic equilibrium. It is common for transporters to form multimeric complexes, e.g., the structures of functional dimers of chloride channels (Lim et al, 2013;Stölting et al, 2014) and trimers of ammonium transport proteins (Khademi and Stroud, 2006;Wacker et al, 2014) have been described.…”

Atmospheric nitrogen assimilation in Ustilago maydis.

“…These are trimeric integral membrane complexes through which ammonium (NH ) molecules are electrogenicly transported unambiguously as determined in two Archaeoglobus fulgidus ammonium transporters utilizing solid-supported membrane (SSM)-based electrophysiology (Wacker et al, 2014). This same work determined a pH dependence on activity such that pH below 5.0 resulted in a drop-off of ammonium transport activity as well as high specificity for ammonium.…”

“…In various filamentous pathogens, the filamentation response tends to be reduced or inhibited as a function of lowering pH (Wacker et al, 2014). This trend could reflect the impact of pH on the ability of active transporters to function in a more hydronium- Ammonium binds, and then is deprotonated.…”