Rhesus factors and ammonium: a function in efflux?
© BioMed Central Ltd 2001
Published: 9 March 2001
Completion of fungal, plant and human genomes paved the way to the identification of erythrocytic rhesus proteins and their kidney homologs as ammonium transporters.
Ammonium is the preferred nitrogen source of bacteria and fungi, and plants acquire nitrogen from the soil in the form of ammonium . In animals and humans, assimilated forms of nitrogen - amino acids - are much preferred for nutrition, and, in the case of ammonotelic animals, ammonium is used for the excretion of nitrogen instead. In the human kidney, ammonium is produced, reabsorbed and excreted as a means to maintain pH balance and to get rid of surplus inorganic nitrogen. Whether ammonium transport also has a role in the pH regulation of other organs is not known and the molecular mechanisms were not, up to now, understood.
Properties and role of ammonium
In aqueous solution, ammonium (NH4+) is in equilibrium with ammonia (NH3) with a pKa of 9.25. Thus, at typical cytosolic pH, approximately 99% is present in the cationic form. Ammonia can pass through lipid bilayers along its concentration gradient, whereas ammonium is orders of magnitude less permeant. A transmembrane electrical and pH gradient will therefore affect NH4+/NH3, equilibrium. Plants and some microorganisms acidify their external environment and utilize membrane-bound ammonium transport proteins, so internal ammonia concentration may be more than 100 times enriched compared to the external space. Besides pH, membrane potential and transporters, enzymatic reactions that generate or remove ammonium are also important in maintaining its concentration. For example, in microorganisms and plants, ammonium is rapidly and efficiently assimilated by enzymes such as glutamine synthetase and glutamate dehydrogenase. As high levels of ammonium can be cytotoxic, cytoplasmic ammonium has to be kept low. To maintain desired cytoplasmic concentrations and to promote nitrogen assimilation and growth, microorganisms and plants have evolved highly specific uptake and, because of passive leakage of ammonia out of the cells, re-uptake mechanisms. The mechanism of transport for import and export in different species - whether the transporters use ammonium or ammonia as a substrate and whether they couple the transport to protons or other ions - remains to be fully elucidated.
Transporters for ammonium
Ammonium transport has been studied for many decades in microorganisms, but the molecular structure of the transporters and the transport mechanism remained obscure. Genetic studies in Escherichia coli identified mutants defective in ammonium transport, but it was not possible to identify a bona fide gene encoding a polytopic membrane protein . In the late 1970s, using a thorough screen for yeast mutants resistant to methylammonium (a toxic analog of ammonium carried by most ammonium transporters), a strain defective in ammonium uptake was identified . Through complementation, ammonium transporter genes were identified, and the respective proteins from both yeast (Mep) and plants (AMT) were characterized [4,5]. The genes from the two organisms are closely related and encode plasma membrane proteins that have an external amino terminus, an internal carboxyl terminus and 11 transmembrane spans . Yeast was then used as a tool to study the biochemical properties of both transporters. Transport was saturable with high affinities and was selective for ammonium and methylammonium . It was also energy-dependent and was able to concentrate ammonium several hundred-fold inside the cells.
Selectivity of ammonium transporters
Many homologs of the Mep and AMT ammonium transporters have been identified in yeast and plants. Saccharomyces cerevisiae has three paralogous Mep proteins with different affinities and capacities , and the higher plant Arabidopsis thaliana has six AMT paralogs [1,7]. Genetic evidence indicates there are regulatory interactions between the Mep proteins, which may point to an interaction as an oligomeric complex . Furthermore, it is interesting to note that Mep2 is glycosylated and has been implicated in ammonium sensing during pseudohyphal growth .
The identification of yeast and plant transporters also led to the characterization of bacterial ammonium transporters because the genomes of archaebacteria and eubacteria contained homologous sequences. A bacterial homolog, like yeast Mep and plant AMTs, behaves strictly like an ammonium transporter [12,13]. The presence of homologs in all these species made it probable that related proteins might also be present in animals and humans. Indeed, they are, and the fascinating surprise was that the mammalian homologs, although distantly related, had previously been described but no transport function had been assigned: they were identified as the rhesus (Rh) blood group antigens . Over stretches of approximately 200 amino acids, identity between Mep and AMT and Rh proteins can be as high as 25%.
The rhesus blood group antigens
Interestingly, Rh antigens had long been recognized for their role in transfusion-incompatible immune reactions and hemolytic disease of newborn babies. Although they have been biochemically characterized, a clear physiological role could not be assigned . The Rh complex is expressed in erythrocytes and, perhaps similar to Mep proteins, is a heterotetramer consisting of two RhAG glycoproteins (also known as Rh50 for their electrophoretic mobility in SDS gels), which are glycosylated in topologically similar positions to Mep2, and two highly homologous non-glycosylated RhCE or RhD subunits (also referred to as Rh30). Each monomer contains 12 putative transmembrane domains, and both amino and carboxyl termini are cytosolic. Bacterial ammonium transporters have a similar structure . The CE/D subunits of the complex present the epitopes of the Rh antigens D or E/e and C/c on their external loops. The RhAG subunits are essential for expression of the whole complex, whereas the combination with RhCE and RhD varies. Two different genetic defects can lead to the rare Rhnull disease, which results in changed erythrocyte morphology and physiology: either RhAG is mutated and thus does not translocate the Rh complex to the cell surface, or RhD is deleted and RhCE is simultaneously mutated .
The homology of Rh proteins with Mep and AMT proteins prompted Marini et al.  to investigate whether human RhAG expressed alone can mediate ammonium transport in ammonium-uptake deficient yeast. RhAG conferred growth on ammonium as sole nitrogen source. Surprisingly, and in contrast to the Mep proteins in yeast, RhAG also conferred resistance to very high concentrations of toxic methylammonium, indicating it has a role in export. RhAG promoted efflux of ammonium when cultured on an amino acid carbon source, the catabolism of which leads to ammonium production. These experiments suggest the efflux is mediated either by uniport of ammonia or by an antiport mechanism (Figure 1c,d). As the plasma membrane of erythrocytes seems to be permeable to NH3 but not to NH4+  and the pH of the cytosol is higher, facilitated diffusion of the uncharged molecule down its concentration gradient may be the actual mechanism. Such a mechanism would explain the coincidental uptake and efflux capabilities of RhAG.
Recently, two homologous glycoproteins were identified from mouse and human: RhBG, which is predominantly expressed in kidney, liver and skin  and RhCG (also known as RhGK), which is predominantly expressed in kidney and testis [17,20].
A role for RhBG and RhCG in kidney physiology
Ammonium excretion by the kidney is tightly coupled to the maintenance of systemic acid-base balance . Ammonium is not primarily a constituent of the ultrafiltrate; it is generated by metabolism of glutamine in proximal epithelial cells and is released into the tubular fluid. Interestingly, RhBG localizes to proximal tubules , and ammonium is concentrated several-fold at the end of the proximal tubule. A significant proportion of ammonium is reabsorbed in the thick ascending limb of the loop of Henle, the apical membrane of which is highly permeable to NH4+ but not to NH3 . Lumen-positive voltage drives paracellular absorption of NH4+. The final nephron segment, the collecting duct, seems impermeable to NH4+, but permeable to NH3. Protons are secreted into the lumen and ammonia released from cortical cells will be protonated to form ammonium, trapped in the lumen and excreted. RhCG, which is also able to efflux ammonium , localizes to the collecting duct .
Functional and evolutionary relationships
Taken together, the results suggest that different transporters from the same superfamily do transport the same substances, but use different transport mechanisms. This may not be too surprising, since different functional relationships are found in diverse classes of membrane transporters, such as glucose transporters of the major facilitator superfamily (for example, the yeast protein ScHXT1 compared with the Arabidopsis protein AtSTP1) and amino acid transporters of the amino acid/polyamine/choline (APC) family (for example, the human protein HsCAT1 compared with the Arabidopsis protein AtCAT1). In both cases, the plant protein probably mediates proton co-transport, whereas the other mediates uniport.
The absence of Rh-like proteins in higher plants suggests that either some of the AMT proteins also use a different coupling mechanism or that other, sequence-unrelated proteins are responsible for ammonium/ammonia export. In contrast to urea-excreting mammals, fish excrete mainly ammonium, through the gills. We would therefore predict that Rh-like proteins (such as AAF63256, also known as DrRh1) may be highly expressed in the gills and may be responsible for ammonium/ammonia excretion in fish. Knock-out mutants in plants, mice and fish will be excellent tools for dissecting the individual roles of AMT, Mep and Rh-like proteins in the different genomes and their relative contribution to net ammonium flux compared with other transporters that can mediate ammonium transport (such as potassium transporters, Na+/H+ antiporters or Na+/K+/Cl- cotransporters). Genome-wide analysis will also help to unravel the evolution of the mechanism of ammonium/ammonia transport.
We are grateful for support by the GABI genome project funded by the BMBF.
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