The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic ''gate'' formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH4 ؉ deprotonation takes place at S1 before NH3 conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.

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.

SummaryExternal hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1 , GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum . We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1 , AMT2 , AMT3 , GDHA and GLNA . In response to exogenously supplied ammonium or glutamine, AMT1 , AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum . Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH 4 + + + + . AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

The Amt proteins are high affinity ammonium transporters that are conserved in all domains of life. In bacteria and archaea the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P II signal transduction protein family, proteins that regulate many facets of nitrogen metabolism. We have now shown that Escherichia coli AmtB is inactivated by formation of a membrane-bound complex with GlnK. Complex formation is reversible and occurs within seconds in response to micromolar changes in the extracellular ammonium concentration. Regulation is mediated by the uridylylation/deuridylylation of GlnK in direct response to fluctuations in the intracellular glutamine pool. Furthermore under physiological conditions AmtB activity is required for GlnK deuridylylation. Hence the transporter is an integral part of the signal transduction cascade, and AmtB can be formally considered to act as an ammonium sensor. This system provides an exquisitely sensitive mechanism to control ammonium flux into the cell, and the conservation of glnK linkage to amtB suggests that this regulatory mechanism may occur throughout prokaryotes.

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The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.

SummaryThe SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM-dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C 4-dicarboxylic acids and that its substrate specificity differs from the well-characterized succinate transporter, DctA. Accordingly ychM was re-designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C4-dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.