Amt Proteins Research Articles

Phosphorylation and Ankyrin-G Binding of the C-terminal Domain Regulate Targeting and Function of the Ammonium Transporter RhBG

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

The 1.3-A resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins.

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 A. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally.

The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion.

The Amt/Mep ammonium channels are trimers in which each monomer contains a long, narrow, hydrophobic pore. Whether the substrate conducted by these pores is NH(3) or NH(4)(+) remains controversial. Substitution of leucine for the highly conserved tryptophan 148 residue at the external opening to Escherichia coli AmtB pores allowed us to address this issue. A strain carrying AmtB(W148L) accumulates much larger amounts of both [(14)C]methylammonium and [(14)C]methylglutamine in a washed cell assay than a strain carrying wild-type AmtB. Accumulation of methylammonium occurs within seconds and appears to reflect channel conductance, whereas accumulation of methylglutamine, which depends on the ATP-dependent activity of glutamine synthetase, increases for many minutes. Concentration of methylammonium was most easily studied in strains that lack glutamine synthetase. It is eliminated by the protonophore carbonyl cyanide m-chlorophenyl hydrazone and is approximately 10-fold higher in the strain carrying AmtB(W148L) than wild-type AmtB. The results indicate that AmtB allows accumulation of CH(3)NH(3)(+) ion in response to the electrical potential across the membrane and that the rate of flux through AmtB(W148L) is approximately 10 times faster than through wild-type AmtB. We infer that both mutant and wild-type proteins also carry NH(4)(+). Contrary to our previous views, we assess that E. coli AmtB does not differ from plant Amt proteins in this regard; both carry ions. We address the role of W148 in decreasing the activity and increasing the selectivity of AmtB and the implications of our findings with respect to the function of Rh proteins, the only known homologues of Amt/Mep proteins.

Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteria

Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family. In this study we have characterized in vitro complex formation between the AmtB and P(II) proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-P(II) complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg(2+) and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P(II) complex defines a new regulatory role for Amt proteins in Prokaryotes.

Evolution and functional characterization of the RH50 gene from the ammonia-oxidizing bacterium Nitrosomonas europaea.

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an RH50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium Nitrosomonas europaea. Here we have used a phylogenetic approach to study the evolution of the N. europaea RH50 gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the N. europaea genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50(Ne) protein and the corresponding knockout mutant, we determined that NeRh50 can mediate ammonium uptake. The RH50(Ne) gene may thus have replaced functionally the AMT gene, which is missing in the genome of N. europaea and may be regarded as a case of nonorthologous gene displacement.

The NtcA-Regulated amtB Gene Is Necessary for Full Methylammonium Uptake Activity in the Cyanobacterium Synechococcus elongatus

The Amt proteins constitute a ubiquitous family of transmembrane ammonia channels that permit the net uptake of ammonium by cells. In many organisms, there is more than one amt gene, and these genes are subjected to nitrogen control. The mature Amt protein is a homo- or heterooligomer of three Amt subunits. We previously characterized an amt1 gene in the unicellular cyanobacterium Synechococcus elongatus strain PCC 7942. In this work, we describe the presence in this organism of a second amt gene, amtB, which encodes a protein more similar to the bacterial AmtB proteins than to any other characterized cyanobacterial Amt protein. The expression of amtB took place in response to nitrogen step-down, required the NtcA transcription factor, and occurred parallel to the expression of amt1. However, the transcript levels of amtB measured after 2 h of nitrogen deprivation were about 100-fold lower than those of amt1. An S. elongatus amtB insertional mutant exhibited an activity for uptake of [14C]methylammonium that was about 55% of that observed in the wild type, but inactivation of amtB had no noticeable effect on the uptake of ammonium when it was supplied at a concentration of 100 microM or more. Because an S. elongatus amt1 mutant is essentially devoid of [14C]methylammonium uptake activity, the mature Amt transporter is functional in the absence of AmtB subunits but not in the absence of Amt1 subunits. However, the S. elongatus amtB mutant could not concentrate [14C]methylammonium within the cells to the same extent as the wild type. Therefore, AmtB is necessary for full methylammonium uptake activity in S. elongatus.

Ammonium channel expression is essential for brain development and function in the larva of Ciona intestinalis

Ammonium uptake into the cell is known to be mediated by ammonium transport (Amt) proteins, which are present in all domains of life. The physiological role of Amt proteins remains elusive; indeed, loss-of-function experiments suggested that Amt proteins do not play an essential role in bacteria, yeast, and plants. Here we show that the reverse holds true in the tunicate Ciona intestinalis. The genome of C. intestinalis contains two AMT genes, Ci-AMT1a and Ci-AMT1b, which we show derive from an ascidian-specific gene duplication. We analyzed Ci-AMT expression during embryo development. Notably, Ci-AMT1a is expressed in the larval brain in a small number of cells defining a previously unseen V-shaped territory; these cells connect the brain cavity to the external environment. We show that the knockdown of Ci-AMT1a impairs the formation of the brain cavity and consequently the function of the otolith, the gravity-sensing organ contained in it. We speculate that the normal mechanical functioning (flotation and free movement) of the otolith may require a close regulation of ammonium salt(s) concentration in the brain cavity, because ammonium is known to affect both fluid density and viscosity; the cells forming the V territory may act as a conduit in achieving such a regulation.

The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel.

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.5 A. This structure of PII in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GlnK. GlnK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation.

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.

The Amt/Mep/Rh family of ammonium transport proteins (Review)

The Amt/Mep/Rh family of integral membrane proteins comprises ammonium transporters of bacteria, archaea and eukarya, as well as the Rhesus proteins found in animals. They play a central role in the uptake of reduced nitrogen for biosynthetic purposes, in energy metabolism, or in renal excretion. Recent structural information on two prokaryotic Amt proteins has significantly contributed to our understanding of this class, but basic questions concerning the transport mechanism and the nature of the transported substrate, NH3 or [NH4(+)], remain to be answered. Here we review functional and structural studies on Amt proteins and discuss the bioenergetic issues raised by the various mechanistic proposals present in the literature.

The conserved carboxy-terminal region of the ammonia channel AmtB plays a critical role in channel function

The ammonium transport (Amt) proteins are a highly conserved family of integral membrane proteins found in eubacteria, archaea, fungi and plants. Genetic, biochemical and bioinformatic analyses suggest that they have a common tertiary structure comprising eleven trans-membrane helices with an N-out, C-in topology. The cytoplasmic C-terminus is variable in length but includes a core region of some 22 residues with considerable sequence conservation. Previous studies have indicated that this C-terminus is not absolutely required for Amt activity but that mutations that alter C-terminal residues can have very marked effects. Using the Escherichia coli AmtB protein as a model system for Amt proteins, we have carried out an extensive site-directed mutagenesis study to investigate the possible role of this region of the protein. Our data indicate that nearly all mutations fall into two phenotypic classes that are best explained in terms of two distinct effects of the C-terminal region on AmtB activity. Residues within the C-terminus play a significant role in normal AmtB function and the C-terminal region might also mediate co-operativity between the three subunits of AmtB.

An Unusual Twin-His Arrangement in the Pore of Ammonia Channels Is Essential for Substrate Conductance

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.

Molecular Dynamics Simulations on the Escherichia coli Ammonia Channel Protein AmtB: Mechanism of Ammonia/Ammonium Transport

Molecular dynamics (MD) simulations have been performed at the atomic level to study the ammonium/ammonia transport across the Escherichia coli AmtB membrane protein. Although ammonia primarily exists in the form of NH(4)(+) in aqueous solution, the recent X-ray structure determination of AmtB reveals that the ammonium/ammonia transporter proteins are ammonia-conducting channels rather than ammonium ion transporters [Khademi, S.; et al. Science 2004, 305, 1587; Zheng, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17090]. Our simulations showed that the entrance of NH(4)(+) into the periplasmic recruitment vestibule requires only 3.1 kcal/mol of energy. This is consistent with the X-ray crystal structure, where one NH(4)(+) is captured in the binding vestibule. In this vestibule, NH(4)(+) loses one water of hydration, but the loss is compensated by a hydrogen bond, first with the backbone carbonyl oxygen of Phe161 then with the hydroxyl group of Ser219, as well as the stabilizing pi-cation interactions with the aromatic rings of Trp148 and Phe107 in the AmtB protein. In the end of this recruitment vestibule, the phenyl ring of Phe107 dynamically switches to an open state. This is correlated with a slight rotation and shifting of the indole ring of Trp148, which eventually creates a slot for the initially buried carboxylate group of Asp160 to become exposed to the bulk solvent. A hydrogen bond wire between NH(4)(+) and the carboxylate group of Asp160 via two water molecules was observed. Thus, Asp160 is most likely the proton acceptor from NH(4)(+). This explains the high conservation of Asp160 in Amt proteins and why the D160A mutant would completely quench the activity of AmtB [Javelle, A.; et al. J. Biol. Chem. 2004, 279, 8530; Marini, A. M.; et al. Curr. Genet. 2006, 49, 364]. Once NH(4)(+) deprotonates, the phenyl ring of Phe215 rotates to open, and the subsequent passage of NH(3) through the channel is straightforward.

Mechanisms of ammonium transport, accumulation, and retention in ooyctes and yeast cells expressing Arabidopsis AtAMT1;1

Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Δ ψ-driven transport of NH 4 + through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture.

The ammonia channel protein AmtB from Escherichia coli is a polytopic membrane protein with a cleavable signal peptide

The Escherichia coli ammonia channel protein, AmtB, is a homotrimeric polytopic inner membrane protein in which each subunit has 11 transmembrane helices. We have shown that the structural gene amtB encodes a preprotein with a signal peptide that is cleaved off to produce a topology with the N-terminus in the periplasm and the C-terminus in the cytoplasm. Deletion of the signal peptide coding region results in significantly lower levels of AmtB accumulation in the membrane but modification of the signal peptidase cleavage site, leading to aberrant cleavage, does not prevent trimer formation and does not inactivate the protein. The presence of a signal peptide is apparently not a conserved feature of all prokaryotic Amt proteins. Comparison of predicted AmtB sequences suggests that while Amt proteins in Gram-negative organisms utilize a signal peptide, the homologous proteins in Gram-positive organisms do not.

The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins

The Escherichia coli ammonium transport protein (AmtB) has become the model system of choice for analysis of the process of ammonium uptake by the ubiquitous Amt family of inner membrane proteins. Over the past 6 years we have developed a range of genetic and biochemical tools in this system. These have allowed structure/function analysis to develop rapidly, offering insight initially into the membrane topology of the protein and most recently leading to the solution of high-resolution 3D structures. Genetic analysis has revealed a novel regulatory mechanism that is apparently conserved in prokaryotic Amt proteins and genetic approaches are also now being used to dissect structure/function relationships in Amt proteins. The now well-recognised homology between the Amt proteins, found in archaea, eubacteria, fungi and plants, and the Rhesus proteins, found characteristically in animals, also means that studies on E. coli AmtB can potentially shed light on structure/function relationships in the clinically important Rh proteins.

Biological gas channels for NH 3 and CO 2: evidence that Rh (Rhesus) proteins are CO 2 channels

Physiological evidence from our laboratory indicates that Amt/Mep proteins are gas channels for NH 3, the first biological gas channels to be described. This view has now been confirmed by structural evidence and is displacing the previous belief that Amt/Mep proteins were active transporters for the NH 4 + ion. Still disputed is the physiological substrate for Rh proteins, the only known homologues of Amt/Mep proteins. Many think they are mammalian ammonium (NH 4 + or NH 3) transporters. Following Monod's famous dictum, “Anything found to be true of E. coli must also be true of elephants” [Perspect. Biol. Med. 47(1) (2004) 47], we explored the substrate for Rh proteins in the unicellular green alga Chlamydomonas reinhardtii. C. reinhardtii is one of the simplest organisms to have Rh proteins and it also has Amt proteins. Physiological studies in this microbe indicate that the substrate for Rh proteins is CO 2 and confirm that the substrate for Amt proteins is NH 3. Both are readily hydrated gases. Knowing that transport of CO 2 is the ancestral function of Rh proteins supports the inference from hematological research that a newly evolving role of the human Rh30 proteins, RhCcEe and RhD, is to help maintain the flexible, flattened shape of the red cell.

Rh proteins vs Amt proteins: an organismal and phylogenetic perspective on CO 2 and NH 3 gas channels

Rh (Rhesus) proteins are homologues of ammonium transport (Amt) proteins. Physiological and structural evidence shows that Amt proteins are gas channels for NH 3, but the substrate of Rh proteins, be it CO 2 as shown in green alga, or NH 3/NH 4 + as shown in mammalian cells, remains disputed. We assembled a large dataset generated of Rh and Amt to explore how Rh originated from and evolved independently of Amt relatives. Analysis of this rich data implies that Rh was split from Amt first to emerge in archaeal species. The Rh ancestor underwent divergence and duplication along speciation, leading to neofunctionalization and subfunctionalization of the Rh family. The characteristic organismal distribution of Rh vs. Amt reflects their early separation and subsequent independent evolution: they coexist in microbes and invertebrates but do not in fungi, vascular plants or vertebrates. Rh gene-duplication was prominent in vertebrates: while epithelial RhBG/RhCG displayed strong purifying selection, erythroid Rh30 and RhAG experienced different episodes of positive selection in each of which adaptive evolution occurred at certain time points and in a few codon sites. Mammalian Rh30 and RhAG were subject to particularly strong positive selection in some codon sites in the lineage from rodents to human. The grounds of this adaptive evolution may be driven by the necessity to increase the surface/volume ratio of biconcave erythrocytes for facilitative gas diffusion. Altogether, these results are consistent with Rh proteins not being the orthologue of Amt proteins but having gained the function for CO 2/HCO 3 – transport, with important roles in systemic pH regulation.

Evolutionary conservation and diversification of Rh family genes and proteins.

Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, NH(3)/NH(4)(+), whereas others think that Rh proteins transport CO(2) and Amt proteins NH(3). In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution.

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.