Ammonium Transporters: A molecular dual carriageway

In order to enter a cell, an ammonium ion must first dissociate to form an ammonia molecule and a hydrogen ion (a proton), which then pass through the cell membrane separately and recombine inside.
  1. William J Allen
  2. Ian Collinson  Is a corresponding author
  1. School of Biochemistry, University of Bristol, United Kingdom

Ammonium is an important metabolite for all forms of life. It acts as a major source of nitrogen for plants, fungi and bacteria, while in animals, it is used to maintain the pH balance inside cells (and is often produced as a toxic by-product of cellular processes). Each cell is surrounded by a membrane that is impermeable to charged particles like ammonium, so the uptake or release of such molecules requires specialized transporter proteins embedded in the membrane.

Following the pioneering work of biochemists and biophysicists, notably Peter Mitchell and Ronald Kaback, and a wealth of atomic structures revealed by X-ray crystallography and electron cryo-microscopy, the dynamic and biochemical actions of many biological transporters are well understood. So much so, it is rare nowadays to encounter a surprising new mechanism underlying solute transport. Now, in eLife, Ulrich Zachariae, Arnaud Javelle and colleagues at the University of Dundee, University of Strathclyde and Université Libre de Bruxelles, report just such a new mechanism for proton-driven ammonium transport (Williamson et al., 2020).

Even though the first atomic structure of an ammonium transporter was published 16 years ago, exactly how ammonium is transported could not be agreed on (Khademi et al., 2004). To cross the cell membrane, ammonium must pass through the centre of the transporter protein, which is very hydrophobic and therefore unfavourable to the passage of charged molecules. This led to the proposal that ammonium (with the chemical formula NH4+) dissociates into ammonia (NH3) and a positively charged hydrogen ion (H+), with only uncharged NH3 traversing the membrane (Khademi et al., 2004). The importance of this deprotonationevent is supported by other experimental evidence (Ariz et al., 2018). Yet, other studies have shown that for many ammonium transporters, movement of NH3 across the membrane is electrogenic, i.e., accompanied by a flow of positive charge generating an electric current (Ludewig et al., 2002).

The team, which includes Gordon Williamson, Giulia Tamburrino, Adriana Bizior and Mélanie Boeckstaensas joint first authors, reconcile these observations into a single, convincing model by showing that ammonium is first deprotonated on one side of the membrane, and that NH3 and H+ both travel across separately, whereupon they reunite to reform ammonium (Figure 1). Williamson et al. used detailed computer simulations to generate predictions for how transport is achieved, which were then validated in functional and electrophysiological analyses. As such, this work is an excellent example of how integrating computational and empirical observations can produce insights that would otherwise be very difficult to attain.

The mechanism of ammonium transporters.

Ammonium is an important metabolite for many organisms. Until now, it was unclear how exactly ammonium is transported in and out of cells. Williamson et al. demonstrated that ammonium (NH4+) dissociates into ammonia gas (NH3) and a positively charged hydrogen (H+), which then traverse the cell membrane separately and re-join at the other end. (A) The ammonium transporter (teal) spans the whole membrane (grey). The two ends are open to the water-filled environment (cyan), but the only open passage through the centre is strongly water-repellent (pink). The entrance to the water channel has a pocket that specifically recognizes ammonium. (B) Ammonium binds into the pocket near the amino acid Asp160, which is essential to trigger the opening of the channel. The environment inside the pocket also allows ammonium to separate into NH3 and H+. NH3 can now cross the membrane through the hydrophobic channel (pink), and H+ crosses separately via the bridge formed of two copies of the amino acid histidine (His168 and His318). (C) Once inside the cell, NH3 and H+ recombine to make ammonium (NH4+).

A critical feature of any metabolite transporter is that it does not accidentally transport other molecules. This is a particular danger for potassium ions (K+), which are a similar size and shape to ammonium, but also for protons, which must cross the membrane as part of the transport reaction. The concentrations of these ions are tightly regulated inside cells, so even small leaks of either would be detrimental to most cells. The results of Williamson et al. reveal just how this selectivity is achieved. For example, the water-filled channel through which H+ moves is only formed when ammonium binds at the entrance to the transporter. The formation of the channel depends on a single amino acid, which is present in all known ammonium transporters: even a conservative amino acid substitution at this site completely stops the transporter from working (Figure 1A).

To prevent any potassium from leaking through, the fully formed water channel is blocked at its centre by two copies of the amino acid histidine (Figure 1B). Histidine readily takes up and releases protons at physiological (slightly basic) pH values (pH 7.4), so H+ can hop across the histidine bridge with relative ease. But the twin histidines block anything larger from getting through. If both of them are removed, the transporter becomes an open channel, letting both protonated ammonium and potassium flow through freely (Williamson et al., 2020).

Ammonium transporters share a similar three-dimensional structure, even in organisms with distant evolutionary origins. But it remains to be seen how widespread this specific transport mechanism is: do most ammonium transporters work in the same way, or are there some that transport NH3 while leaving the proton behind? And what structural features determine this? Also, further research is needed to determine how exactly the channel forms, how the proton is removed from the ammonium, and how the speed at which ammonium crosses the membrane is regulated. The breakthrough by Williamson et al. opens the door to these questions that could lead towards a complete understanding of a fundamental biological process widespread throughout nature.

Ammonium transporters are relevant to numerous, disparate scientific fields. For example, the central role ammonium plays in the global nitrogen cycle makes them important both for improving crop yields and for the breakdown of pollutants (Li et al., 2017; Fowler et al., 2013). Human ammonium transporters are essential for kidney function, and mutations have been implicated in various diseases (Kurtz, 2018; Huang and Ye, 2010). For those interested in other membrane transport processes, the idea of using selective protonation or deprotonation of small, ionisable solutes, or even the amino acid side chains of larger protein molecules, is compelling (Jiang et al., 2015; Wiechert and Beitz, 2017).

References

Article and author information

Author details

  1. William J Allen

    William J Allen is in the School of Biochemistry, University of Bristol, United Kingdom

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9513-4786
  2. Ian Collinson

    Ian Collinson is in the School of Biochemistry, University of Bristol, United Kingdom

    For correspondence
    ian.collinson@bristol.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3931-0503

Publication history

  1. Version of Record published: August 25, 2020 (version 1)

Copyright

© 2020, Allen and Collinson

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,791
    views
  • 220
    downloads
  • 1
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. William J Allen
  2. Ian Collinson
(2020)
Ammonium Transporters: A molecular dual carriageway
eLife 9:e61148.
https://doi.org/10.7554/eLife.61148
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Natalia Dolgova, Eva-Maria E Uhlemann ... Oleg Y Dmitriev
    Research Article

    Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Isabelle Petit-Hartlein, Annelise Vermot ... Franck Fieschi
    Research Article

    NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.