A two-lane mechanism for selective biological ammonium transport
- University of Strathclyde, United Kingdom
- University of Dundee, United Kingdom
- Université Libre de Bruxelles, Belgium
Abstract
The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5 and Table 2.
Article and author information
Author details
Mélanie Boeckstaens
Gaëtan Dias Mirandela
Funding
Tenovus (S17-07)
- Arnaud Javelle
Scottish Universities Physics Alliance (NA)
- Ulrich Zachariae
Natural Environment Research Council (NE/M001415/1)
- Paul A Hoskisson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Williamson et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,944
- views
-
- 377
- downloads
-
- 24
- 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)
A two-lane mechanism for selective biological ammonium transport
eLife 9:e57183.
https://doi.org/10.7554/eLife.57183
Further reading
-
- Biochemistry and Chemical Biology
The Parkinson’s disease (PD)-linked protein Leucine-Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis–Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lies within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM and the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer–dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity, suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together, our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.
