Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor.

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of a putative partner to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. Finally, we show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans.

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32.

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.

Ammonia excretion in Caenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1.

The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmol gFW(-1) day(-1)) and very little urea (0.21±0.004 µmol gFW(-1) day(-1)). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H(+)-ATPase (subunit A) and Na(+)/K(+)-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H(+)-ATPase, carbonic anhydrase, Na(+)/K(+)-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmol l(-1) NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na(+)/K(+)-ATPase also increased significantly in response to 1 mmol l(-1) NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins.

Coexistence of Ammonium Transporter and Channel Mechanisms in Amt-Mep-Rh Twin-His Variants Impairs the Filamentation Signaling Capacity of Fungal Mep2 Transceptors.

Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family's characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors. IMPORTANCE Fungal infections represent a significant threat to human health and cause huge damage to crop yields worldwide. The dimorphic switch between yeast and filamentous growth is associated with the virulence of pathogenic fungi. Of note, fungal Mep2 proteins of the conserved Amt-Mep-Rh family play a transceptor role in the induction of filamentation; however, the signaling mechanism remains largely unknown. Amt-Mep-Rh proteins ensure the specific scavenging of NH4+ through a mechanism relying on substrate deprotonation, thereby preventing competition and translocation of similar-sized K+. Our multidisciplinary approaches using E. coli AmtB, Saccharomyces cerevisiae, and Candida albicans Mep2 show that double variation of the family-defining Twin-His motif triggers a mechanistic switch from a specific transporter to an unspecific ion channel with both mechanisms still coexisting in single variants. Moreover, we show that this mechanistic alteration is associated with loss of signaling ability of Mep2, supporting a transport mechanism-driven process in filamentation induction.

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

Human apolipoprotein L1 interferes with mitochondrial function in Saccharomyces cerevisiae.

To the best of our knowledge, the vertebrate apolipoprotein L (APOL) family has not previously been ascribed to any definite pathophysiological function, although the conserved BH3 protein domain suggests a role in programmed cell death or an interference with mitochondrial processes. In the present study, the human APOL1 was expressed in the yeast Saccharomyces cerevisiae in order to determine the molecular action of APOL1. APOL1 inhibited cell proliferation in a non­fermentable carbon source, such as glycerol, while it had no effect on proliferation in fermentable carbon sources, such as galactose. APOL1, expressed in yeast, is localized in the mitochondrial fraction, as determined via western blotting. APOL1 induced a loss of mitochondrial function, demonstrated by a loss of respiratory index, and mitochondrial membrane potential. Green fluorescent protein tagging of mitochondrial protein revealed that APOL1 was associated with abnormal mitochondrial and lysosomal morphologies, observed by a loss of the normal mitochondrial tubular network. Thus, the results of the present study suggest that APOL1 could be a physiological regulator of mitochondrial function.

A two-lane mechanism for selective biological ammonium transport.

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.

Pib2-Dependent Feedback Control of the TORC1 Signaling Network by the Npr1 Kinase.

To adjust cell growth and metabolism according to environmental conditions, the conserved TORC1 signaling network controls autophagy, protein synthesis, and turnover. Here, we dissected the signals controlling phosphorylation and activity of the TORC1-effector kinase Npr1, involved in tuning the plasma membrane permeability to nitrogen sources. By evaluating a role of pH as a signal, we show that, although a transient cytosolic acidification accompanies nitrogen source entry and is correlated to a rapid TORC1-dependent phosphorylation of Npr1, a pH drop is not a prerequisite for TORC1 activation. We show that the Gtr1/Gtr2 and Pib2 regulators of TORC1 both independently and differently contribute to regulate Npr1 phosphorylation and activity. Finally, our data reveal that Npr1 mediates nitrogen-dependent phosphorylation of Pib2, as well as a Pib2-dependent inhibition of TORC1. This work highlights a feedback control loop likely enabling efficient downregulation and faster re-activation of TORC1 in response to a novel stimulating signal.

Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily.

Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins, and despite their significance in physiological processes, the nature of substrate translocation (NH3/NH4+) by the distinct members of this family is still a matter of controversy. Using Saccharomyces cerevisiae cells expressing representative AMT-Mep-Rh ammonium carriers and taking advantage of the natural chemical-physical property of the N isotopic signature linked to NH4+/NH3 conversion, this study shows that only cells expressing AMT-Mep-Rh proteins were depleted in 15N relative to 14N when compared to the external ammonium source. We observed 15N depletion over a wide range of external pH, indicating its independence of NH3 formation in solution. On the basis of inhibitor studies, ammonium transport by nonspecific cation channels did not show isotope fractionation but competition with K+. We propose that kinetic N isotope fractionation is a common feature of AMT-Mep-Rh-type proteins, which favor 14N over 15N, owing to the dissociation of NH4+ into NH3 + H+ in the protein, leading to 15N depletion in the cell and allowing NH3 passage or NH3/H+ cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH4+/K+ ratio, allowing organisms to specifically scavenge NH4+. We show that 15N isotope fractionation may be used in vivo not only to determine the molecular species being transported by ammonium transport proteins, but also to track ammonium toxicity and associated amino acids excretion.

[From the discovery of microbial Mep-Amt ammonium transporters to human Rhesus factors]. / De la découverte des transporteurs d'ammonium Mep-Amt microbiens aux facteurs Rhésus humains.

Ammonium, ubiquitous on Earth, plays major and distinct roles in most organisms. While it can be a nitrogen source for many microorganisms and plants, it is a cytotoxic metabolic product actively detoxified by the liver in animals. Furthermore, in the latter, ammonium synthesis in the kidney is involved in acid/base homeostasis. Ammonium transport is ensured by a family of proteins, called Mep-Amt-Rh. This family is conserved in all domains of life and comprises the human Rh factors, notably known in transfusional medicine. While the study of bacterial, fungal and vegetal Mep-Amt transporters reveals a fine-tuned and rapid regulation of these proteins in function of environmental changes, the regulation of animal Rh proteins has been poorly addressed. This review notably highlights the importance of the yeast model in the study of the regulation of these proteins as well as in the functional characterization of Mep-Amt-Rh members of diverse origins.

The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner.

The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast.

The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein.

The TORC1 complex controls cell growth upon integrating nutritional signals including amino-acid availability. TORC1 notably adapts the plasma membrane protein content by regulating arrestin-mediated endocytosis of amino-acid transporters. Here we demonstrate that TORC1 further fine tunes the inherent activity of the ammonium transport protein, Mep2, a yeast homologue of mammalian Rhesus factors, independently of arrestin-mediated endocytosis. The TORC1 effector kinase Npr1 and the upstream TORC1 regulator Npr2 control Mep2 transport activity by phospho-silencing a carboxy-terminal autoinhibitory domain. Under poor nitrogen supply, Npr1 enables Mep2 S457 phosphorylation and thus ammonium transport activity. Supplementation of the preferred nitrogen source glutamine leads to Mep2 inactivation and instant S457 dephosphorylation via plasma membrane Psr1 and Psr2 redundant phosphatases. This study underscores that TORC1 also adjusts nutrient permeability to regulate cell growth in a fast and flexible response to environmental perturbation, establishing a hierarchy in the transporters to be degraded, inactivated or maintained active at the plasma membrane.

SNPs altering ammonium transport activity of human Rhesus factors characterized by a yeast-based functional assay.

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCG(R202C) may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.

Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation.

Ammonium transport proteins of the Mep/Amt/Rh family include microbial and plant Mep/Amt members, crucial for ammonium scavenging, and animal Rhesus factors likely involved in ammonium disposal. Recent structural information on two bacterial Mep/Amt proteins has revealed the presence, in the hydrophobic conducting pore, of a pair of preserved histidines proposed to play an important role in substrate conductance, by participating either in NH(4)(+) deprotonation or in shaping the pore. Here we highlight the existence of two functional Mep/Amt subfamilies distinguishable according to whether the first of these histidines is conserved, as in yeast ScMep2, or replaced by glutamate, as in ScMep1. Replacement of the native histidine of ScMep2 with glutamate leads to conversion from ScMep2 to ScMep1-like properties. This includes a two-unit upshift of the optimal pH for transport and an increase of the transport rate, consistent with alleviation of an energy-limiting step. Similar effects are observed when the same substitution is introduced into the Escherichia coli AmtB protein. In contrast to ScMep1, ScMep2 is proposed to play an additional signaling role in the induction of filamentous growth, a dimorphic change often associated with virulence in pathogenic fungi. We show here that the histidine to glutamate substitution in ScMep2 leads to uncoupling of the transport and sensor functions, suggesting that a ScMep2-specific transport mechanism might be responsible for filamentation. Our overall data suggest the existence of two functional groups of Mep/Amt-type proteins with different transport mechanisms and distinct impacts on cell physiology and signaling.

The yeast ammonium transport protein Mep2 and its positive regulator, the Npr1 kinase, play an important role in normal and pseudohyphal growth on various nitrogen media through retrieval of excreted ammonium.

Three ammonium transport systems of the Mep/Amt/Rh superfamily contribute to ammonium uptake for use as a nitrogen source in Saccharomyces cerevisiae. A specific sensor role has further been proposed for Mep2 in the stimulation of pseudohyphal development during ammonium limitation. Optimal ammonium transport by the Mep proteins requires the Npr1 kinase, a potential target of the target-of-rapamycin signalling pathway. We show here that the growth impairment of cells lacking Npr1 on many nitrogen sources is shared by cells deprived of the three Mep proteins and is a consequence of deficient ammonium retrieval. Expression of a newly isolated Npr1-independent and hyperactive Mep2 in cells lacking Npr1 and/or the Mep proteins restores growth on low ammonium but also on other nitrogen sources. This hyperactive Mep2 variant efficiently counteracts ammonium excretion. Hence, ammonium uptake activity plays an important role in compensating for leakage of catabolic ammonium. Our data also reveal that the requirement of Npr1 for ammonium-induced pseudohyphal growth is an indirect consequence of its necessity for Mep2-mediated ammonium transport. Finally, we show that Mep2 participates, through ammonium leakage compensation, in pseudohyphal growth induced by amino acid starvation. This argues further in favour of tight coupling of Mep2 transport and sensor functions.

Transduction of the nitrogen signal activating Gln3-mediated transcription is independent of Npr1 kinase and Rsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae.

Nitrogen Catabolite Repression (NCR) allows the adaptation of yeast cells to the quality of nitrogen supply by inhibiting the transcription of genes encoding proteins involved in transport and degradation of nonpreferred nitrogen sources. In cells using ammonium or glutamine, the GATA transcription factor Gln3 is sequestered in the cytoplasm by Ure2 whereas it enters the nucleus after a shift to a nonpreferred nitrogen source like proline or upon addition of rapamycin, the TOR complex inhibitor. Recently, the Npr1 kinase and the Rsp5, Bul1/2 ubiquitin ligase complex were reported to have antagonistic roles in the nuclear import and Gln3-mediated activation. The Npr1 kinase controls the activity of various permeases including transporters for nitrogen sources that stimulate NCR such as the Mep ammonium transport systems. Combining data from growth tests, Northern blot analysis and Gln3 immunolocalization, we show that the Npr1 kinase is not a direct negative regulator of Gln3-dependent transcription. The derepression of Gln3-activated genes in ammonium-grown npr1 cells results from the reduced uptake of the nitrogen-repressing compound because NCR could be restored in npr1 cells by repairing ammonium-uptake defects through different means. Finally, we show that the impairment of the ubiquitin ligase complex does not prevent induction of NCR genes under nonpreferred nitrogen conditions. The apparent Rsp5-, Bul1/2-dependent Gln3 activation keeps to the cellular status, as it is only observed in cells having left the balanced phase of exponential growth.

Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters.

Ammonium transport proteins belonging to the Mep/Amt/Rh family are spread throughout all domains of life. A conserved aspartate residue plays a key role in the function of Escherichia coli AmtB. Here, we show that the analogous aspartate residue is critical for the transport function of eukaryotic family members as distant as the yeast transporter/sensor Mep2 and the human RhAG and RhCG proteins. In yeast Mep2, replacement of aspartate(186) with asparagine produced an inactive transporter localized at the cell surface, whilst replacement with alanine was accompanied by stacking of the protein in the endoplasmic reticulum. Introduction of an acidic residue, glutamate, produced a partially active protein. A carboxyl group at position 186 of Mep2 therefore appears mandatory for function. Kinetic analysis shows the Mep2(D186E) variant to be particularly affected at the level of substrate affinity, suggesting an involvement of aspartate(186) in ammonium recognition. Our data also put forward that ammonium recognition and/or transport by Mep2 is required for the sensor role played in the development of pseudohyphal growth. Finally, replacement of the conserved aspartate with asparagine in human RhAG and RhCG proteins resulted in the loss of bi-directional transport function. Hence, this aspartate residue might play a preserved functional role in Mep/Amt/Rh proteins.