Thesis

Mechanistic characterisation of the Rh50 ammonium transporter protein from Nitrosomonas europaea

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2023
Thesis identifier
  • T16485
Person Identifier (Local)
  • 201986815
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • The exchange of ammonium across cellular membranes is a fundamental process in all domains of life which is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. The functional diversity of Amt/Mep and Rh has been known for decades: bacteria, fungi, and plants use Amt/Mep proteins to scavenge ammonium for biosynthetic assimilation. Mammals on the other hand use the Rh proteins for ammonium detoxification in erythrocytes, kidney, and liver tissues. Crucially Rh malfunction is associated with various pathologies, including hereditary anaemias, overhydrated stomatocytosis, and early-onset depressive disorders. However, their mechanism and the substrate translocated (NH4 + /NH3) remain elusive. Recently, our group proposed a new model for the mechanism of electrogenic ammonium transport in AmtB protein from E. coli, where NH4 + undergoes deprotonation, allowing NH3 and H+ to follow two separate pathways, and join in the cytoplasm. Despite their fundamentally divergent physiological functions, the Amt/Mep/Rh proteins are structurally very similar, raising the possibility of a conserved transport mechanism across the family. In this context, we investigated the mechanism of ammonium translocation through rhesus protein (Rh50) from Nitrosomonas europaea, as a first step towards expanding the Rh protein research and understanding of their mechanism. An in vitro assay based on Solid Supported Membrane Electrophysiology (SSME) was developed and the electrogenic activity in Rh50 was confirmed (Chapter 3). We provided a detailed characterisation of the activity, selectivity, and kinetics of WT Rh50 (Chapter 4). Further characterisation of WT Rh50 and Rh50 variants led to the proposal of the coexistence of two mechanisms in the protein: mechanism I wherein NH4 + is deprotonated and H+ and NH3 are carried separately across the membrane, and mechanism II, where NH4 + is directly translocated through by-passing deprotonation (Chapter 4). Through mutagenesis studies, we also revealed valuable information on bidirectionality of the protein which we propose to be directly linked with a hydration pattern of the protein (Chapter 5). The body of this work provides a basis for future Rh protein research which will enable us to better understand how Rh mutations lead to pathologies in humans.
Advisor / supervisor
  • Hoskisson, Paul A.
  • Javelle, Arnaud
Resource Type
Note
  • This thesis was previously held under Moratorium from 9th February 2023 until 9th February 2025.
DOI
Date Created
  • 2022

Relations

Items

Thumbnail Title Date Uploaded Visibility Actions
file details File 2023-05-18 Private
file details PDF of thesis T16485 2023-05-22 Public Download