Ammonium is a nitrogen source supporting growth of yeast cells at an optimal rate. We recently reported the first characterization of an NH 4 ؉ transport protein (Mep1p) in Saccharomyces cerevisiae. Here we describe the characterization of two additional NH 4 ؉ transporters, Mep2p and Mep3p, both of which are highly similar to Mep1p. The Mep2 protein displays the highest affinity for NH 4 ؉ (K m , 1 to 2 M), followed closely by Mep1p (K m , 5 to 10 M) and finally by Mep3p, whose affinity is much lower (K m , ϳ1.4 to 2.1 mM). A strain lacking all three MEP genes cannot grow on media containing less than 5 mM NH 4 ؉ as the sole nitrogen source, while the presence of individual NH 4 ؉ transporters enables growth on these media. Yet, the three Mep proteins are not essential for growth on NH 4 ؉ at high concentrations (>20 mM). Feeding experiments further indicate that the Mep transporters are also required to retain NH 4 ؉ inside cells during growth on at least some nitrogen sources other than NH 4 ؉ . The MEP genes are subject to nitrogen control. In the presence of a good nitrogen source, all three MEP genes are repressed. On a poor nitrogen source, MEP2 expression is much higher than MEP1 and MEP3 expression. High-level MEP2 transcription requires at least one of the two GATA family factors Gln3p and Nil1p, which are involved in transcriptional activation of many other nitrogen-regulated genes. In contrast, expression of either MEP1 or MEP3 requires only Gln3p and is unexpectedly downregulated in a Nil1p-dependent manner. Analysis of databases suggests that families of NH 4 ؉ transporters exist in other organisms as well.

In Saccharomyces cerevisiae, the transport of ammonium across the plasma membrane for use as a nitrogen source is mediated by at least two functionally distinct transport systems whose respective encoding genes are called MEP1 and MEP2. Mutations in the MEP2 gene affect high affinity, low capacity ammonium transport while mutations in the MEP1 gene disrupt a lower affinity, higher capacity system. In this work, the MEP1 gene has been cloned and sequenced and its expression analyzed. The predicted amino acid sequence reveals a highly hydrophobic, 54 kDa protein with 10 or 11 putative membrane‐spanning regions. The predicted Mep1p protein shares high sequence similarity with several bacterial proteins of unknown function, notably the product of the nitrogen‐regulated nrgA gene of Bacillus subtilis, and with that of a partial cDNA sequence derived from Caenorhabditis elegans. The Mep1p and related proteins appear to define a new family of transmembrane proteins evolutionarily conserved in at least bacteria, fungi and animals. The MEP1 gene is most highly expressed when the cells are grown on low concentrations of ammonium or on ‘poor’ nitrogen sources like urea or proline. It is down‐regulated, on the other hand, when the concentration of ammonium is high or when other ‘good’ nitrogen sources like glutamine or asparagine are supplied in the culture medium. The overall properties of Mep1p indicate that it is a transporter of ammonium. Its main function appears to be to enable cells grown under nitrogen‐limiting conditions to incorporate ammonium present at relatively low concentrations in the growth medium.

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The kidney has an important role in the regulation of acid-base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH(3) diffusion coupled to H(+) secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading-a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg(-/-) acid-loaded mice show reduced apical permeability to NH(3) and impaired transepithelial NH(3) transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a ϳ35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH 4 ؉ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake. Ammonium transporters (AMTs)1 of the AMT/MEP/Rh protein family have been identified in all domains of life, including plants, bacteria, archea, yeast, and animals (1, 2). AMT/ MEP/Rh proteins are highly hydrophobic membrane proteins with a predicted molecular mass of ϳ45-55 kDa and 11 or 12 putative transmembrane spans. Initially AMT/MEP/Rh ammonium transporters from yeast and plants were identified molecularly by functional complementation of a yeast mutant defective in ammonium uptake (3-5). Later, homologs were isolated from bacteria (6) and animals (Caenorhabditis elegans), and phylogenetic analysis showed that mammalian Rh (rhesus) blood group polypeptides belong to the same superfamily (7). Heterologously expressed RhAG and a homolog from kidney (RhGK ϭ RhCG) were also shown to function as ammonium transporters (8, 9).Plants require transporters for NH 4 ϩ acquisition from a wide range of external concentrations and are able to concentrate and transiently accumulate NH 4 ϩ in the cytosol before being metabolized or further compartmentalized (10). Ammonium transport across root plasma membranes is biphasic, consisting of a high-affinity and a low-affinity nonsaturating component (11,12). The high-affinity transport system, which operates predominantly at low external ammonium concentrations, is energized by the membrane potential. In tomato, the NH 4 ϩ -uniporter LeAMT1;1 encodes a component of the high-affinity transport system that depends on the membrane potential (13). The molecular identity of the low-affinity transport system, however, is less clear. It contributes significantly to overall NH 4 ϩ uptake at higher external ammonium concentrations (Ͼ1 mM) and may have a distinct transport mechanism, because uncharged NH 3 or charged NH 4 ϩ may be the substrate (11, 12, 14,...