In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase

Arnaud Javelle, Gavin Thomas, Anne-Marie Marini, Reinhard Krämer, Mike Merrick

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)215-222
Number of pages8
JournalBiochemical journal
Volume390
Issue number1
DOIs
Publication statusPublished - 15 Aug 2005

Keywords

  • adenosine triphosphate
  • biological transport, active
  • cation transport proteins
  • Escherichia coli
  • Escherichia coli proteins
  • glutamate-ammonia ligase
  • kinetics
  • membrane potentials
  • methylamines
  • time factors

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