Studies Of The Interaction Between The Ammonium Transport Protein AmtB And The Signal Transduction Protein GlnK

G. Coutts, M. Merrick

Dept of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK

In E. coli there are two members of the PII signal transduction protein family, GlnB and GlnK. GlnB facilitates the sensing of the intracellular nitrogen status and regulates the activities of the NtrBC system and adenyltransferase. While GlnK can partially substitute for some of the functions of GlnB, the primary function of GlnK is unknown. In both bacteria and archaea the glnK gene is highly conserved in an operon along with amtB, which encodes a high affinity ammonium transporter, and we have proposed that this conservation reflects the fact that GlnK and AmtB interact (Thomas et al. 2000a). If this is the case then we expect to find GlnK associated with the inner membrane in wild type or AglnB strains of E. coli but not in an A amtB strain. We have fractionated cells grown under nitrogen-limiting conditions and probed these fractions using Western blots with an antibody against PII (GlnK or GlnB) and we do indeed observe the predicted association.

To investigate the possible physiological role of the GlnK-AmtB association we examined the effects of ammonia shock on the interaction. It is known that ammonia shock results in the de-uridylation of GlnK in E. coli, and when strains were fractionated pre- and post-ammonia shock we observed a significant increase in the membrane-association of GlnK post-ammonia shock. We then used native PAGE to confirm that the non-uridylated form of GlnK has a markedly higher affinity for AmtB than uridylylated GlnK. A His-tagged form of AmtB was used to show that the level of AmtB in the membrane does not change post-ammonia shock.

The major part of AmtB that is located in the cytoplasm is the 32 amino acid C-terminal domain and this is therefore a potential site of interaction between AmtB and GlnK (Thomas et al. 2000b). Deletion of this region does not inactivate the transport activity of AmtB but does prevent GlnK association to the membrane. We therefore propose that the C-terminus is either required directly for interaction with GlnK or is necessary to allow AmtB to adopt a conformation that allows GlnK to interact.

In conclusion we propose the following model - that as a response to an increase in the intracellular nitrogen status the GlnK protein associates to AmtB thereby switching off ammonium transport, by post-translational control. The sequestration of GlnK to the membrane would also result in a rapid decrease in the intracellular pool of GlnK, which could in turn affect the activities of any cytoplasmic targets of GlnK.

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