In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase.
The Escherichia coli AmtB protein is member of the ubiquitous Amt family of ammonium transporters. Using a variety of [14C]methylammonium-uptake assays in wild-type E. coli, together with amtB and glutamine synthetase (glnA) mutants, we have shown that the filtration method traditionally used to measure [14C]methylammonium uptake actually measures intracellular accumulation of methylglutamine and that the kinetic data deduced from such experiments refer to the activity of glutamine synthetase and not to AmtB. Furthermore, the marked difference between the K(m) values of glutamine synthetase calculated in vitro and those calculated in vivo from our data suggest that ammonium assimilation by glutamine synthetase is coupled to the function of AmtB. The use of a modified assay technique allows us to measure AmtB activity in vivo. In this way, we have examined the role that AmtB plays in ammonium/methylammonium transport, in the light of conflicting proposals with regard to both the mode of action of Amt proteins and their substrate, i.e. ammonia or ammonium. Our in vivo data suggest that AmtB acts as a slowly conducting channel for NH3 that is neither dependent on the membrane potential nor on ATP. Furthermore, studies on competition between ammonium and methylammonium suggest that AmtB has a binding site for NH4+ on the periplasmic face.
Javelle
Arnaud
A
Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk NR4 7UH, UK.
Thomas
Gavin
G
Marini
Anne-Marie
AM
Krämer
Reinhard
R
Merrick
Mike
M
D000255:Adenosine Triphosphate / Q000502:physiology; D001693:Biological Transport, Active; D027682:Cation Transport Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008744:Methylamines / Q000378:metabolism; D013997:Time Factors
C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008744:Methylamines; D000255:Adenosine Triphosphate; C027451:methylamine; D005974:Glutamate-Ammonia Ligase