Thesis
Roots and gates : evolutionary origins and transport mechanisms of the Amt/Mep/Rh ammonium transport superfamily
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2026
- Thesis identifier
- T18093
- Person Identifier (Local)
- 202163726
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Ammonium transport across biological membranes is essential for life and is mediated by the Amt/Mep/Rh superfamily of membrane proteins, which are conserved across all domains of life. Despite a shared overall fold and key structural features, these proteins exhibit diverse physiological roles, functioning as high-affinity ammonium transporters, bidirectional detoxifiers, or non-transporting sensors. While individual subfamilies have been studied, the evolutionary and functional relationships among them remain incompletely understood. The overall goal of this project was to investigate this superfamily of transporters as a whole by generating a phylogenetic tree, developing a method of measuring the activity of proteins without the need to purify and reconstitute them and assess the function of a ubiquitously conserved feature, the Phe Gate. A root is proposed for the phylogenetic tree of ammonium transporters between those proteins that have a Glutamate residue at the entrance to an ammonium binding pocket and those that have Tyrosine residue. Because there were frequent pairings of archaeal and bacterial proteins, this tree gave evidence to the hypothesis that LUCA had ammonium transporters. This is at odds with previous research in predicting LUCA’s genome and constitutes an interesting find. The tree was also used to investigate other features of the protein’s family such as the occurrence of Amts fused to other enzymatic domains where it was found that in this tree, they only occur in proteins found in the Glu group. It was also found that the Twin His motif (HH) variation where the first His is changed for a Glu (EH), previously only seen in fungal proteins, is more common with an archaeal group sitting at the base of the fungal group containing a mixture of EH and HH proteins. The Phe Gate was found to be totally conserved. To circumvent the requirement for protein purification and reconstitution, a functional assay was developed by generating vesicles directly from E. coli cells overexpressing EcAmtB, with ammonium transport activity assessed using solid-supported membrane electrophysiology (SSME). This approach reliably detected transporter-mediated ammonium flux, demonstrating the assay’s viability for functional analysis in a nearnative membrane context. Despite its strict evolutionary conservation across the Amt/Mep/Rh superfamily, the functional role of the phenylalanine gate (Phe Gate) remains unresolved. Previous studies have proposed potential roles in substrate selectivity or facilitating deprotonation during transport. To investigate its function, Phe Gate variants of EcAmtB and NeRh50 were analysed using SSME. These variants exhibited a complete loss of transport activity without any alteration in potassium ion selectivity. These findings indicate that the Phe Gate is not merely a passive structural feature or selectivity filter but plays an essential mechanistic role in enabling ammonium transport. Overall, this work has increased our knowledge of the phylogenetic history of these proteins and sets the foundation for future work to be able to measure the activity of a greater diversity of ammonium transporters.
- Advisor / supervisor
- Hoskisson, Paul Y.
- Pritchard, Leighton
- Javelle, Arnaud
- Resource Type
- DOI
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PDF of thesis T18093 | 2026-09-22 | Public | Download |