Position: Royal Society of Edinburgh Research Fellow
Division: Molecular Microbiology
Address: College of Life Sciences,
University of Dundee,
Dundee
Telephone: +44 1382 386203, int ext. 86203
Fax: +44 1382 388216
Email: a.javelle@dundee.ac.uk
Website: Javelle Group
Research in my laboratory is focused on membrane proteins involved in transport of ions across cellular membranes and in signalling processes relating to ion availability. Studies of membrane transporters/channels have had a great impact on our understanding human disease and drug design; indeed ion channels are, at present, the third biggest target classe in drug discovery and about 30% of current clinically marked drugs are targeted against these proteins.
The philosophy of my research, is to use simple and well studied bacterial model organisms, and a multidisciplinary approach to shed more light on two ubiquitous families of ion transporters both of which are conserved from bacteria to man: the ammonium transporter from the Amt/Rh family (TC 1.A.11) and the sulphate permease from the SulP/SLC26A family (TC 2.A.53).
The ammonia channel AmtB from E. coli as is the best system to investigate questions of structure, function and signal transduction relating to Amt proteins.
1 - Mechanistic studies of the ammonia channel AmtB from E. coli.
We have developed an in vivo transport assay using the ammonium analogue methylammonium, [14C]MA, as a tracer in intact cells and an in vitro assay by stopped flow analysis on proteoliposomes reconstituted with purified AmtB. The results of this analysis suggest that, contrary to the accepted idea that Amt proteins act as active NH4+/proton symporters, AmtB acts as a slowly-conducting channel for NH3. Furthermore, studies on competition between ammonium and methylammonium suggest that AmtB has a binding site for NH4+ on the periplasmic face. Interestingly, our data also suggest that ammonium assimilation by glutamine synthetase is coupled to the function of AmtB. These results have subsequently been supported by the resolution of the structure of AmtB. The structural features of AmtB indicate that NH4+ is recruited at the periplasmic side of the protein prior its deprotonation and the conduction of NH3. We have subsequently confirmed the general mechanism of transport and identified important residues in the recruitment, the deprotonation and the conduction mechanism using functional and structural analysis of mutants in combination with computational studies by molecular dynamic simulation.
2 – Posttranslational regulation of the ammonia channel AmtB from E. coli.
In bacteria the amtB gene is almost invariably transcriptionally linked to a second gene, glnK which encodes a signal transduction protein belonging to the PII family. Mike Merrick and co-workers have shown that the GlnK protein is sequestered to the membrane in an AmtB-dependent fashion, suggesting a new mechanism for controlling ammonium flux. By co-purification and western blot analysis we have confirmed that an AmtB-GlnK complex is formed at the membrane in response to small variations in the external ammonium concentration (between 5 and 50µM). The process occurs within seconds, blocks AmtB activity and is fully reversible. GC-MS analysis using [15N]NH4+ showed that variation in the intracellular glutamine pool in response to external ammonium fluctuation controls the process via the uridylylation/deuridylylation of GlnK. The process, which has since been shown to be ubiquitous in bacteria, provides a mechanism whereby not only is the activity of AmtB regulated in response to the cellular demand for ammonium but the cellular pool of GlnK is also modulated rapidly in response changes in the extracellular ammonium availability.
In animals, sulphate is extremely important for the process of sulfation. This is a critical step for the biotransformation, activity modulation or detoxification of xenobiotics, steroids, catecholamines, and bile acids in numerous organisms. Sulfation also serves a role in the biosynthetic pathway for the production of many biologically active substrates and structural components of membranes and tissues. In microorganisms and plants, sulphate is an essential precursor to the biosynthesis of sulphur-containing amino acids. Therefore sulphate transport across membranes is a fundamental and crucial biological process. The first sulphate transporter was identified in the filamentous fungus Neurospora crassa. Since then, it has been shown that these transporters, belonging to the SulP family (TC 2.A.53), are conserved from bacteria to man. The human genome encodes at least 10 SulP proteins, belonging to the solute carrier SLC26A family of various anions exchangers (SO42-, Cl-, HCO3-, I-…), amongst which 4 have been identified as being linked to known disease states. In bacteria, a second sulfate transport system is also present: the ABC-transporter from the SulT family (TC 3.A.1.6). Although SulP proteins are frequently encoded in bacterial genomes, little is known about their mechanism of action as well as their role in bacteria physiology. The goal of my studies is to use a multidisciplinary approach to shed more light on this family of putative anion transporters in bacteria and on their general mechanism of action.
