Ammonium Permease Research Articles

The Mep2p ammonium permease controls nitrogen starvation‐induced filamentous growth in Candida albicans

Nitrogen starvation is one of the signals that induce Candida albicans, the major fungal pathogen of humans, to switch from yeast to filamentous growth. In response to nitrogen starvation, C. albicans expresses the MEP1 and MEP2 genes, which encode two ammonium permeases that enable growth when limiting concentrations of ammonium are the only available nitrogen source. In addition to its role as an ammonium transporter, Mep2p, but not Mep1p, also has a central function in the induction of filamentous growth on a solid surface under limiting nitrogen conditions. When ammonium is absent or present at low concentrations, Mep2p activates both the Cph1p-dependent mitogen-activated protein (MAP) kinase pathway and the cAMP-dependent signalling pathway in a Ras1p-dependent fashion via its C-terminal cytoplasmic tail, which is essential for signalling but dispensable for ammonium transport. In contrast, under ammonium-replete conditions that require transporter-mediated uptake Mep2p is engaged in ammonium transport and signalling is blocked such that C. albicans continues to grow in the budding yeast form. Mep2p is a less efficient ammonium transporter than Mep1p and is expressed at much higher levels, a distinguishing feature that is important for its signalling function. At sufficiently high concentrations, ammonium represses filamentous growth even when the signalling pathways are artificially activated. Therefore, C. albicans has established a regulatory circuit in which a preferred nitrogen source, ammonium, also serves as an inhibitor of morphogenesis that is taken up into the cell by the same transporter that mediates the induction of filamentous growth in response to nitrogen starvation.

Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae controls a variety of properties that depend on the nutrient composition of the medium. High activity of the pathway occurs in the presence of rapidly fermented sugars like glucose or sucrose, but only as long as growth is maintained. Growth arrest of fermenting cells or growth on a respiratory carbon source, like glycerol or ethanol, is associated with low activity of the PKA pathway. We have studied how different nutrients trigger rapid activation of the pathway. Glucose and sucrose activate cAMP synthesis through a G-protein-coupled receptor system, consisting of the GPCR Gpr1, the Galpha protein Gpa2 and its RGS protein Rgs2. Glucose is also sensed intracellularly through its phosphorylation. Specific mutations in Gpr1 abolish glucose but not sucrose signalling. Activation of the PKA pathway by addition of a nitrogen source or phosphate to nitrogen- or phosphate-starved cells, respectively, is not mediated by an increase in cAMP. Activation by amino acids is triggered by the general amino acid permease Gap1, which functions as a transporter/receptor. Short truncation of the C-terminus results in constitutively activating alleles. Activation by ammonium uses the ammonium permeases Mep1 and Mep2 as receptor. Specific point mutations in Mep2 uncouple signalling from transport. Activation by phosphate is triggered a.o. by the Pho84 phosphate permease. Several mutations in Pho84 separating transport and signalling or triggering constitutive activation have been obtained.

Nitrogen catabolite repression in Saccharomyces cerevisiae during wine fermentations.

We carried out fermentations with several nitrogen sources in different concentrations and studied nitrogen regulation by following the transcriptional profile of the general amino-acid permease (GAP1) and the ammonium permeases (MEP1, MEP2, MEP3). In wine fermentations the cells evolve from a nitrogen-repressed situation at the beginning of the process to a nitrogen-derepressed situation as the nitrogen is consumed. These nitrogen-repressed/derepressed conditions determined the different patterns of ammonium and amino-acid consumption. Arginine and alanine were hardly used under the repressed conditions, while the uptake of branched-chain and aromatic amino acids increased.

Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism.

Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.

Transcriptional effects of the signal transduction protein P II ( glnB gene product) on NtcA-dependent genes in Synechococcus sp. PCC 7942

P II proteins signal the cellular nitrogen status in numerous bacteria, and in cyanobacteria P II is subjected to serine phosphorylation when the cells experience a high C to N balance. In the unicellular cyanobacterium Synechococcus sp. PCC 7942, the P II protein ( glnB gene product) is known to mediate the ammonium-dependent inhibition of nitrate and nitrite uptake. The analysis of gene expression through RNA/DNA hybridization indicated that a P II-null mutant was also impaired in the induction of NtcA-dependent, nitrogen assimilation genes amt1 (ammonium permease), glnA (glutamine synthetase) and nir (nitrite reductase), as well as of the N-control gene ntcA, mainly under nitrogen deprivation. This gene expression phenotype of the glnB mutant could be complemented by wild-type P II protein or by modified P II proteins that cannot be phosphorylated and mimic either the phosphorylated (GlnB S49D and GlnB S49E) or unphosphorylated (GlnB S49A) form of P II. However, strains carrying the GlnB S49D and GlnB S49E mutant proteins exhibited higher levels of expression of nitrogen-regulated genes than the strains carrying the wild-type P II or the GlnB S49A protein.

Amino acids control ammonia pulses in yeast colonies

Individual yeast colonies produce pulses of volatile ammonia separated by phases of medium acidification. Colonies of Saccharomyces cerevisiae mutant defective in the general amino acid permease, Gap1p, exhibit decreased ammonia production. Mutations in the S. cerevisiae amino acid sensor SPS completely abolish the colony ammonia pulses. In contrast, the ammonia pulse production is independent of external concentrations of ammonium and of its uptake by the ammonium permeases Mep1p, Mep2p, and Mep3p. It is concluded that in S. cerevisiae colonies, the extracellular amino acids, but not the extracellular ammonium, serve as a source for volatile ammonia production. These phenomena are not restricted to S. cerevisiae, since we observe that extracellular levels of 8 out of the 20 tested amino acids are necessary for ammonia pulses produced by Candida mogii colonies.

Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA

The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family). Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters. To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized. Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity. Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue. A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A. nidulans ammonium transporter, MepA. These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.

Isolation and characterization of two ammonium permease genes, meaA and mepA, from Aspergillus nidulans.

Ammonium and the analogue methylammonium are taken into the cell by active transport systems which constitute a family of transmembrane proteins that have been identified in fungi, bacteria, plants, and animals. Two genes from Aspergillus nidulans, mepA and meaA, which encode ammonium transporters with different affinities have been characterized. The MepA transporter exhibits the highest affinity for methylammonium (Km, 44.3 microM); in comparison, the Km for MeaA is 3.04 mM. By use of targeted gene replacement strategies, meaA and mepA deletion mutants were created. Deletion of both meaA and mepA resulted in the inability of the strain to grow on ammonium concentrations of less than 10 mM. The single meaA deletion mutant exhibited reduced growth at the same concentrations, whereas the mepA deletion mutant displayed wild-type growth. Interestingly, multiple copies of mepA were found to complement the methylammonium resistance phenotype conferred by the deletion of meaA. The expression profiles for mepA and meaA differed; the mepA transcript was detected only in nitrogen-starved cultures, whereas meaA was expressed under both ammonium-sufficient and nitrogen starvation conditions. Together, these results indicate that MeaA constitutes the major ammonium transport activity and is required for the optimal growth of A. nidulans on ammonium as the sole nitrogen source and that MepA probably functions in scavenging low concentrations of ammonium under nitrogen starvation conditions.

Sensing nitrogen limitation in Corynebacterium glutamicum: the role of glnK and glnD.

A novel nitrogen control system regulating the transcription of genes expressed in response to nitrogen starvation in Corynebacterium glutamicum was identified by us recently. In this communication, we also show that the nitrogen regulation cascade in C. glutamicum functions by a new mechanism, although components highly similar to sensor and signal transmitter proteins of Escherichia coli are used, namely uridylyltransferase and a PII-type GlnK protein. The genes encoding these key components of the nitrogen regulation cascade, glnD and glnK, are organized in an operon together with amtB, which codes for an ammonium permease. Using a combination of site-directed mutagenesis, RNA hybridization experiments, reporter gene assays, transport measurements and non-denaturing gel electrophoresis followed by immunodetection, we showed that GlnK is essential for nitrogen control and that signal transduction is transmitted by uridylylation of this protein. As a consequence of the latter, a glnD deletion strain lacking uridylyltransferase is impaired in its response to nitrogen shortage. The glnD mutant revealed a decreased growth rate in the presence of limiting amounts of ammonium or urea; additionally, changes in its protein profile were observed, as shown by in vivo labelling and two-dimensional PAGE. In contrast to E. coli, expression of glnD is upregulated upon nitrogen limitation in C. glutamicum. This indicates that the glnD gene product is probably not the primary sensor of nitrogen status in C. glutamicum as shown for enterobacteria. In accordance with this hypothesis, we found a deregulated nitrogen control as a result of the overexpression of glnD. Furthermore, quantification of cytoplasmic amino acid pools excluded the possibility that a fall in glutamine concentration is perceived as the signal for nitrogen starvation by C. glutamicum, as is found in enterobacteria. Direct measurements of the intracellular ammonium pool indicated that the concentration of this compound might indicate the cellular nitrogen status. Deduced from glnK and glnD expression patterns and the genetic organization of these genes, this regulatory mechanism is also present in Corynebacterium diphtheriae, the causative agent of diphtheria.

Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane.

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)). Transport and accumulation of the ammonium analog [(14)C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+). In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A Deltavph1 mutant of S. cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2). By inference, their natural substrate is NH(3), a gas. We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).

Repression of GCN4 mRNA Translation by Nitrogen Starvation in Saccharomyces cerevisiae

Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

Uncoupling transport and sensing of the ammonium permease Mep2

Uncoupling transport and sensing of the ammonium permease Mep2

Biochemical and genetic analysis of a Chlamydomonas reinhardtii mutant devoid of chloroplastic glutamine synthetase activity

A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity, and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants, GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently, indicating that resistance was not due to an altered form of GS2.

Nitrogen catabolite repression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Dal80, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence 5'GATAA 3'. Gln3 and Gat1 act positively on gene expression whereas Dal80 and Deh1 act negatively. Expression of nitrogen catabolite pathway genes known to be regulated by these four regulators are glutamine, glutamate, proline, urea, arginine. GABA, and allantonie. In addition, the expression of the genes encoding the general amino acid permease and the ammonium permease are also regulated by these four regulatory proteins. Another group of genes whose expression is also regulated by Gln3, Gat1, Dal80, and Deh1 are some proteases, CPS1, PRB1, LAP1, and PEP4, responsible for the degradation of proteins into amino acids thereby providing a nitrogen source to the cell. In this review, all known promoter sequences related to expression of nitrogen catabolite pathways are discussed as well as other regulatory proteins. Overview of metabolic pathways and promotors are presented.

Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains.

Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the Galpha subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of Deltamep2/Deltamep2 and Deltagpa2/Deltagpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in Deltamep1/Deltamep1 Deltamep2/Deltamep2 and Deltamep1/Deltamep1 Deltagpa2/Deltagpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a Deltamep1/Deltamep1 Deltamep2/Deltamep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth.

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.

Intracellular asparagine pool as a factor in control of ammonium uptake by Saccharomyces cerevisiae

During growth of Saccharomyces cerevisiae in defined media containing 1 m m ammonium and a single amino acid at 2 m m , asparagine, and to a lesser extent aspartic acid and glutamine, significantly delayed ammonium uptake. Addition of asparagine or glutamine to the medium during growth on ammonium caused an immediate cessation of ammonium uptake. Measurement of the pool sizes indicated that ammonium uptake responded to the internal concentration of asparagine rather than that of glutamine and, because of the speed of the response the most likely mechanism was inhibition of the ammonium permease system.

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent Km values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 μM and 2.0±1.6 μM respectively, and the Vmax was 47±2.6 nmol min-1 (mg protein)-1 for MNF 2030 and 101±5.1 nmol min-1 (mg protein)-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.

The Influence of Ammonium Permease Activity and Carbon Source on the Uptake of Ammonium from Simple Defined Media by Saccharomyces cerevisiae

When growing under defined conditions, cells of the yeast Saccharomyces cerevisiae absorbed ammonium more rapidly with glucose as carbon source than with maltose. Ammonium pool sizes and permease activity were also higher in the glucose-grown cells and the relationship implies that firstly, the sugar is a primary determinant of ammonium permease activity and, secondly, the activity of the permease largely determines both the rate of ammonium uptake and ammonium pool size in the first part of the fermentation.

The Ammonium Permease of Rhizobium leguminosarum MNF3841

SUMMARY: An ammonium permease was derepressed when Rhizobium leguminosarum was grown in chemostat culture under conditions of nitrogen limitation. The ammonium permease was characterized by direct measurements with an ion-specific ammonium electrode. Cells grown under ammonia, nitrate, glutamate or methylamine limitation had permease activity, while those grown with excess (10 mM) ammonium chloride or glutamate did not. On transfer from N-limited to N-excess conditions, the permease disappeared rapidly, and all activity was lost within 18 h. Uptake by the permease was sensitive to azide, carbonyl cyanide m-chlorophenylhydrazone. 2,4-dinitrophenol, nigericin and valinomycin. The apparent Km for NH+4 was 0-015 mM; ammonium uptake had a narrow maximum around pH 7.5. The internal ammonia concentration of N-limited cells was 0-4 mM, with up to 60-fold gradients of NH+4 forming across the membrane within 30 min. Hydrazine and hydroxylamine strongly inhibited ammonium uptake, with methylamine, glutamine, aspartate and glycine less effective as inhibitors. Isolated pea bacteroids capable of transporting succinate did not possess the ammonium permease.