Mutational Analysis of the Candida albicans Ammonium Permease Mep2p Reveals Residues Required for Ammonium Transport and Signaling

Strains and growth conditions. C. albicans strains used in this study are listed in Table 1. All strains were stored as frozen stocks with 15% glycerol at −80°C and subcultured on SD agar plates (6.7 g yeast nitrogen base without amino acids [BIO 101, Vista, CA], 20 g glucose, 15 g agar per liter) at 30°C. For routine growth of the strains, YPD liquid medium (20 g peptone, 10 g yeast extract, 20 g glucose per liter) was used. To support growth of ura3 strains, 100 μg/ml uridine was added to the media. To observe MEP2 expression, strains were grown overnight in liquid SD-Pro medium, which contains 0.1% proline instead of ammonium, diluted 50-fold, and grown for 6 hours at 30°C in SD medium containing 100 μM ammonium (SLAD) or 100 μM proline (SLPD). To study filament formation, the strains were grown as single colonies for 6 days at 37°C on SD-2% agar plates containing 100 μM ammonium, proline, or urea as described in the text.

Plasmid construction.Plasmids pMEP2K1 and pMEP2ΔC2, containing the full-length MEP2 and MEP2^ΔC440 alleles, respectively, have been described previously (1). Additional C-terminally truncated MEP2 alleles were generated by amplifying the MEP2 gene from pMEP2K1 with primer MEP3 and one of the primers MEP81 to MEP87 or MEP59, which introduce a stop codon at the desired position (all primers used in this study are listed in Table 2). The truncated MEP2 alleles were substituted for full-length MEP2 in pMEP2K1, resulting in plasmids pMEP2ΔC6 to pMEP2ΔC13, which contain the MEP2^ΔC439 to MEP2^ΔC432 alleles, respectively.

Plasmid pMEP21H2 contains a hybrid MEP2^1-418-MEP1^417−534 allele (1). Truncated derivatives in which a stop codon was inserted behind codon 438 or 433 of MEP1 were generated by amplification of part of the hybrid gene with the primer pair MEP39p-MEP23 or MEP39p-MEP91 and its substitution for the corresponding region in pMEP21H2, resulting in plasmid pMEP21H4 or pMEP21H6, respectively.

MEP2 ^ΔC435 alleles in which individual codons were replaced by the corresponding MEP1 codons were generated by amplifying the MEP2 gene from pMEP2ΔC10 with primer MEP3 and one of the primers MEP95 to MEP102, which introduce the Y433F, A428D, L427E, T425V, L423A, M422E, E420G, and D419N exchanges, and then substituted for the wild-type MEP2 allele in pMEP2K1 to result in plasmids pMEP2ΔC10M1 to pMEP2ΔC10M8, respectively.

MEP2 alleles with single amino acid substitutions were generated in the following way. For pMEP2D180L, the N-terminal part of MEP2 plus upstream sequences was amplified from pMEP2K1 with the primers MEP3 and MEP38p and the remaining part of the MEP2 coding region was amplified with primers MEP39p and MEP32. The two fragments were digested with KpnI and BamHI, respectively, and ligated together into the vector pBluescript. Plasmids pMEP2D180N, pMEP2Y122A, pMEP2F126A, pMEP2W165A, pMEP2W167A, pMEP2H188A, pMEP2S243A, and pMEP2H342A were generated in an analogous fashion, using the primer pairs MEP3-MEP67p and MEP39p-MEP32, MEP3-MEP68p and MEP70p-MEP32, MEP3-MEP69p and MEP70p-MEP32, MEP3-MEP73p and MEP74p-MEP32, MEP3-MEP73p and MEP80p-MEP32, MEP3-MEP71p and MEP72p-MEP32, MEP3-MEP75p and MEP76p-MEP32, and MEP3-MEP77p and MEP78p-MEP32, respectively. A PstI-SacI fragment from pMEP2K1 containing the C-terminal part of MEP2, the ACT1 transcription termination sequence, the URA3 marker, and MEP2 downstream sequences was then ligated between the same sites of the plasmids listed above to generate plasmids pMEP2K4 to pMEP2K12, containing the MEP2^Y122A, MEP2^F126A, MEP2^W165A, MEP2^W167A, MEP2^D180L, MEP2^D180N, MEP2^H188A, MEP2^S243A, and MEP2^H342A alleles, respectively. pMEP2K16 containing the F239A mutation was obtained by ligating the PstI-SacI fragment from plasmid pMEP2K1 into PstI/SacI-digested pMEP2G16 (see below). Plasmids containing green fluorescent protein (GFP)-tagged versions of the mutated MEP2 alleles were created by substituting the KpnI-BamHI fragments from plasmids pMEP2D180L, pMEP2D180N, pMEP2Y122A, pMEP2F126A, pMEP2W165A, pMEP2W167A, pMEP2H188A, pMEP2S243A, and pMEP2H342A for the corresponding fragment in the previously described plasmid pMEP2G2 (1), resulting in plasmids pMEP2G3 and pMEP2G8 to pMEP2G15, respectively. To obtain MEP2G16, the N-terminal and C-terminal parts of MEP2 were amplified with the primer pairs MEP3-MEP94p and MEP76p-MEP32. The PCR products were digested at an internal NsiI site and at the introduced BamHI site, respectively, and ligated together into NsiI/BamHI-digested pMEP2G2.

C. albicans transformation. C. albicans strains were transformed by electroporation (22) with gel-purified inserts from the plasmids described above. Uridine-prototrophic transformants were selected on SD agar plates. Single-copy integration of all constructs into one of the genomic MEP2 alleles, which can be distinguished by an EcoRI restriction site polymorphism, was confirmed by Southern hybridization.

Isolation of genomic DNA and Southern hybridization.Genomic DNA from C. albicans strains was isolated as described previously (29). Ten micrograms of DNA was digested with EcoRI, separated in a 1% agarose gel and, after ethidium bromide staining, transferred by vacuum blotting onto a nylon membrane and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence-labeled probes was performed with the Amersham ECL direct nucleic acid labeling and detection system (GE Healthcare, Braunschweig, Germany) according to the instructions of the manufacturer.

Fluorescence microscopy and flow cytometry.Fluorescence microscopy was performed with a Zeiss LSM 510 inverted confocal laser scanning microscope equipped with a Zeiss Axiovert 100 microscope. Imaging scans were acquired with an argon laser with a wavelength of 488 nm and corresponding filter settings for GFP and parallel transmission images. The cells were observed with a ×63 oil immersion objective. Fluorescence-activated cell sorter analysis was performed with a FACSCalibur flow cytometry system equipped with an argon laser emitting at 488 nm (Becton Dickinson, Heidelberg, Germany). Fluorescence was measured on the FL1 fluorescence channel equipped with a 530-nm-band-pass filter. Twenty thousand cells were analyzed per sample and were counted at a low flow rate. Fluorescence and forward scatter data were collected by using logarithmic amplifiers. The mean fluorescence values were determined with CellQuest Pro (Becton Dickinson) software.

Ammonium removal assays.Ammonium removal from the medium was assayed as described previously (25, 27). Briefly, strains were grown to late log phase in SD-Pro medium, and the cultures were diluted to an optical density at 600 nm of 1.0 in the same medium plus 250 μM ammonium sulfate. At the indicated times, a portion of the culture was taken, the cells were removed by centrifugation, and the ammonium concentration in the culture supernatant was determined using a glutamate dehydrogenase-linked assay in which NH₄⁺ plus NADH plus H⁺ plus α-ketoglutarate is converted to glutamate plus NAD⁺ plus H₂O. An increase in the NADH concentration (measured by determining the absorbance at 340 nm 1 min after the addition of NADH) indicates ammonium removal by the cells.

Identification of the minimal region in the C-terminal cytoplasmic tail of Mep2p that is required for the induction of filamentous growth.Previous experiments demonstrated that deletion of the last 40 amino acids of Mep2p did not impair its ability to induce filamentous growth of C. albicans in response to nitrogen limitation. Cells expressing the MEP2^ΔC440 allele even had a hyperfilamentous phenotype, because mRNA levels of the truncated allele were strongly increased compared with those of the wild-type allele (1). In contrast, deletion of 17 additional amino acids abolished the capacity of MEP2 to induce filamentous growth, although the MEP2^ΔC423 allele was expressed at equally high levels and ammonium uptake by the truncated Mep2p protein was not affected. This result indicated that at least some residues in the region between amino acids 424 and 440 of Mep2p are part of a signaling domain that is specifically required for morphogenesis but not ammonium transport (Fig. 1A). To define the minimal region that is essential for the induction of filamentous growth, we generated progressive truncations starting from amino acid 440 of Mep2p. The truncated alleles were expressed in mep1Δ mep2Δ double mutants, and the ability of the strains to undergo filamentous growth on SLAD plates was evaluated. As expected, all tested truncated alleles rescued the growth defect of the double mutants under limiting ammonium conditions, and strains expressing the MEP2^ΔC439 to MEP2^ΔC433 alleles also exhibited the hyperfilamentous phenotype (Fig. 1B, panels a to c, and data not shown). In contrast, strains expressing the MEP2^ΔC432 allele were unable to undergo filamentous growth (Fig. 1B, panel d), indicating that the tyrosine at position 433 is the last residue that is essential for signaling by Mep2p.

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FIG. 1.

Effects of deletions and mutations in the C-terminal cytoplasmic tail of Mep2p on its ability to induce filamentous growth. (A) Alignment of the region between amino acids 419 and 440 of Mep2p with the corresponding region in Mep1p. Identical residues are marked by stars below the alignment. The tyrosine at position 433 of Mep2p is labeled by an arrowhead. The methionine, leucine, and alanine at positions 422, 427, and 428, respectively, whose substitution by glutamine or aspartate occurring at these positions in Mep1p abolished filamentous growth, are highlighted in bold. (B) Colony phenotypes of mep1Δ mep2Δ double mutants expressing the indicated MEP2 and MEP2-MEP1 hybrid alleles. Photos were taken after 6 days of growth on SLAD plates at 37°C. The two independently constructed series of strains (Table 1) behaved identically, and only one of them is shown in each case. Strains MEP12M6A and -B, in which only the URA3 gene, but not MEP2, was reintegrated, were used as controls (mep1Δ mep2Δ).

Interestingly, Mep2p is highly similar in this region to Mep1p, which does not normally induce filamentous growth in C. albicans (Fig. 1A). It was previously shown that substitution of the Mep1p C-terminal tail for the C terminus of Mep2p abolished filamentous growth (1). To exclude the possibility that the C terminus of Mep1p, which is longer than that of Mep2p, contains an inhibitory domain, we created new hybrid alleles in which the last 22 and 17 codons of the hyperactive MEP2^ΔC440 and MEP2^ΔC435 alleles, respectively, were replaced by the corresponding sequence of MEP1. Strains expressing the hybrid MEP2^1-418-MEP1^417-438 and MEP2^1-418-MEP1^417-433 alleles did not undergo filamentous growth (Fig. 1B, panels e and f), demonstrating that residues in the region between amino acids 419 and 433 of Mep2p, in which it differs from Mep1p, must be essential for signaling. To investigate which of these eight amino acids are required for the signaling function of Mep2p, each of them was exchanged individually with the corresponding Mep1p amino acid in the MEP2^ΔC435 allele. The amino acid substitutions D419N, E420G, L423A, T425V, and Y433F did not affect the ability of the MEP2^ΔC435 allele to induce filamentous growth (Fig. 1B, panels g, h, i, l, m, and p). In contrast, strains expressing the MEP2^ΔC435 alleles with the M422E, L427E, and A428D mutations did not undergo filamentous growth or very weakly formed filaments on SLAD plates (Fig. 1B, panels k, n, and o). Therefore, the presence of a negatively charged amino acid (Glu or Asp) instead of the hydrophobic amino acids Met, Leu, and Ala at positions 422, 427, and 428, respectively, strongly reduces or abolishes the signaling activity of Mep2p, explaining the signaling defect of the Mep2p-Mep1p hybrid proteins.

Expression of mutated Mep2p proteins.It is thought that ammonium binding or transport by the Mep2p proteins of S. cerevisiae and C. albicans influences the signaling activity of the proteins (1, 25). Although there is experimental evidence that ammonium transport is required for signaling by Mep2p in S. cerevisiae (3, 26, 32, 39), an alternative possibility that was suggested for C. albicans (1) is that Mep2p can induce filamentous growth only when it is not engaged in ammonium transport (i.e., when ammonium is absent or present only at low concentrations) and that transport activity inhibits signaling. To test the latter hypothesis, we attempted to generate transport-deficient Mep2p derivatives. The conserved aspartate at position 160 of Escherichia coli AmtB (in the mature protein after removal of the signal sequence) has been suggested to be an initial ammonium binding site (36). We therefore mutated D180 of Mep2p, which corresponds to D160 of AmtB, to either leucine or asparagine (also see Discussion). The resolution of the AmtB crystal structure revealed additional highly conserved residues that might stabilize ammonium in an outer vestibule of the protein (F103, F107, W148, and S219) as well as two pore-lining histidines (H168 and H318) that are believed to make contacts with ammonium during transport (19, 21). Since mutation of these residues might abolish ammonium transport, we changed the corresponding amino acids in Mep2p (Y122, F126, W167, H188, S243, and H342), as well as W165, which is located near W167, to alanine. The mutated alleles were introduced into mep1Δ mep2Δ double mutants, and their capacity to restore growth of the mutants on plates containing limiting ammonium concentrations was assessed. As shown in Fig. 2, strains expressing the MEP2^W165A, MEP2^W167A, and MEP2^S243A alleles grew as well as control strains containing a wild-type MEP2 allele, indicating that the mutated proteins were still able to transport ammonium. Growth was also restored by the MEP2^Y122A and MEP2^F126A alleles, albeit at reduced levels compared with the wild-type MEP2 allele. In contrast, the D180L, D180N, H188A, and H342A mutations abolished growth of strains expressing the corresponding MEP2 alleles, indicating that these mutations rendered the ammonium permease nonfunctional. To test if the mutated Mep2p proteins were correctly expressed in the cell membrane, we introduced C-terminally GFP-tagged versions of all alleles into mep1Δ mep2Δ double mutants and observed expression of the fusion proteins by fluorescence microscopy. Mep2p-GFP fusion proteins containing the Y122A, F126A, W165A, W167A, and S243A mutations were all correctly localized at the cell periphery (Fig. 3A). However, while cells expressing GFP-tagged MEP2^W165A and MEP2^S243A alleles exhibited similar fluorescence to that of cells expressing a GFP-tagged wild-type MEP2 gene, the Y122A, F126A, and W167A mutations resulted in reduced fluorescence of the corresponding transformants. In contrast, GFP-tagged Mep2p proteins containing the D180L, D180N, H188A, and H342A mutations could not be detected in the cells by fluorescence microscopy (data not shown). Therefore, the latter mutations, which abolished growth at low ammonium concentrations, did not specifically affect ammonium transport but rather impaired Mep2p expression or protein stability. To compare the expression levels of the mutated Mep2p proteins, the strains carrying the GFP-tagged alleles were analyzed by flow cytometry. As shown in Fig. 3B, cells containing the MEP2^D180L, MEP2^D180N, and MEP2^H342A alleles exhibited only background fluorescence, confirming that these proteins were not expressed at detectable levels. The fluorescence of cells containing the MEP2^H188A allele was marginally higher (3.5-fold) than that of control cells without a GFP-tagged MEP2 gene. The W165A and S243A mutations resulted in only slightly reduced fluorescence compared with that of the wild-type control (53% and 62% of the wild-type level, respectively). In contrast, and in line with the microscopic observations, the fluorescence of the cells expressing the MEP2^Y122A, MEP2^F126A, and MEP2^W167A alleles was more strongly reduced (20%, 23%, and 21% of the wild-type level, respectively). Similar results were obtained when MEP2 expression was induced in medium containing proline instead of ammonium as the limiting nitrogen source, in which a functional Mep2p protein is not required for growth (Fig. 3C).

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FIG. 2.

Growth of mep1Δ mep2Δ double mutants expressing wild-type or mutated MEP2 alleles, as indicated, on plates containing a limiting concentration (1 mM) of ammonium. The plates were incubated for 4 days at 30°C. The two independently constructed series of strains behaved identically, and only one of them is shown. Strains MEP12M6A and -B, in which only the URA3 gene , but not MEP2, was reintegrated, were used as controls.

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FIG. 3.

Expression of GFP-tagged Mep2p proteins. (A) mep1Δ mep2Δ double mutants carrying wild-type or mutated MEP2 alleles were grown for 6 h at 30°C in liquid SLAD medium and observed by fluorescence microscopy to confirm proper localization of GFP-tagged Mep2p. Similar results were obtained when the cells were grown in SLPD medium containing proline instead of ammonium (data not shown). (B) Fluorescence of strains grown in SLAD medium was quantified by flow cytometry. The bars represent the means and standard deviations from five (control, wild type, and Y122A, F126A, W165A, W167A, and S243A mutants) or two (D180L, D180N, H188A, and H342A mutants) experiments performed with each of two independently constructed series of strains. The mep1Δ mep2Δ double mutants MEP12M6A and -B served as controls. (C) Fluorescence of strains grown in SLPD medium was quantified by flow cytometry. The bars represent the means and standard deviations from two independent experiments.

Ammonium transport and signaling activity of mutated Mep2p proteins.To assess the effects of the various amino acid substitutions on the signaling activity of Mep2p, the ability of strains expressing the corresponding MEP2 alleles to undergo filamentous growth in response to nitrogen limitation was tested. As expected, MEP2 alleles containing the D180L, D180N, H188A, and H342A mutations, whose encoded proteins were not detectably expressed in the cell membrane, did not restore filamentous growth of mep1Δ mep2Δ double mutants on plates containing limiting concentrations of different nitrogen sources (data not shown). In contrast, strains expressing the MEP2^W165A and MEP2^S243A alleles exhibited wild-type filament formation on SLAD plates (Fig. 4). Filamentous growth was slightly reduced in strains expressing the MEP2^Y122A allele compared with that of the wild-type control, which could be explained by the fivefold reduced expression levels of the mutated Mep2p protein (see above). However, filament formation on SLAD plates was completely abolished in strains containing the MEP2^F126A and MEP2^W167A alleles (Fig. 4), although the corresponding Mep2p proteins were expressed at similar levels to that of Mep2p containing the Y122A substitution (Fig. 3), indicating that the F126A and W167A mutations impaired the signaling activity of Mep2p.

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FIG. 4.

Filament formation of strains expressing the indicated MEP2 alleles in a mep1Δ mep2Δ background. Individual colonies were photographed after 6 days of growth at 37°C on SLAD plates. The two independently constructed series of strains behaved identically, and only one of them is shown. Strains MEP12M6A and -B (mep1Δ mep2Δ) were used as controls. All pictures are shown at the same magnification.

To investigate if the signaling activities of mutated Mep2p proteins correlated with their ability to transport ammonium, we compared ammonium uptake by strains expressing wild-type, MEP2^Y122A, MEP2^F126A, MEP2^W167A, and MEP2^S243A alleles in an ammonium removal assay (see Materials and Methods). As shown in Fig. 5A, the S243A mutation, which did not impair growth and filament formation on SLAD plates, had no detectable effect on ammonium uptake. In contrast, ammonium uptake was reduced to different degrees in cells expressing the MEP2^Y122A, MEP2^F126A, and MEP2^W167A alleles compared with that of cells expressing a wild-type MEP2 allele. Interestingly, the W167A mutation, which abolished nitrogen starvation-induced filament formation, had a weaker effect on ammonium uptake than the Y122A substitution, which still allowed filamentous growth. Therefore, the signaling activity of the mutated Mep2p proteins was not directly correlated with their ammonium transport activity. The ammonium uptake capacity of cells expressing the mutated MEP2 alleles corresponded well with their ability to grow in liquid medium (Fig. 5B) as well as on solid medium (Fig. 4) containing limiting ammonium concentrations. The F126A mutation, which had the severest effect on ammonium uptake, also reduced growth of the cells most strongly. The Y122A mutation, which had an intermediate effect on ammonium transport, also resulted in a significant growth reduction. In contrast, the minor effect of the W167A mutation on ammonium uptake did not translate into a detectable growth defect of the cells.

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FIG. 5.

(A) Ammonium uptake by mep1Δ mep2Δ double mutants expressing wild-type or mutated MEP2 alleles. Ammonium uptake was measured as described in Materials and Methods. Two independently constructed strains were used in each case. Strains MEP12M6A and -B (mep1Δ mep2Δ mutants) served as controls. (B) Growth of the same strains in liquid medium under limiting ammonium conditions. Precultures of the strains grown in SD-Pro medium were diluted 10⁻² in SD medium containing 1 mM ammonium as the sole nitrogen source, and growth was monitored by measuring the optical densities (OD₆₀₀) of the cultures over time.

In order to test the filament formation capacity of cells expressing mutated MEP2 alleles independently of their ability to grow under nitrogen-limiting conditions, we assessed filamentous growth of the strains on plates containing limiting concentrations of other nitrogen sources, i.e., urea or the amino acid proline. Under these conditions, ammonium permeases are not required for growth, but Mep2p is still necessary for the induction of filament formation (1). The effects of the various mutations on filamentous growth on plates containing 100 μM urea corresponded to those seen on SLAD plates, i.e., the Y122A substitution resulted in a slight reduction of filament formation, whereas the F126A and W167A mutations abolished filamentous growth (Fig. 6). Interestingly, on plates containing 100 μM proline, strains expressing the MEP2^F126A allele produced filamentous colonies, whereas the W167A mutation also abolished filament formation under these conditions. Proline is known to induce filament formation in C. albicans (23), although at the limiting concentrations used in our assays Mep2p is nevertheless required for filamentous growth. It therefore seems that Mep2p containing the F126A mutation can still support filament formation when additional signaling pathways are activated.

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FIG. 6.

Filament formation of mep1Δ mep2Δ double mutants (left two panels) and mep2Δ single mutants (right panels) expressing wild-type or mutated MEP2 alleles on agar plates containing 100 μM of the indicated nitrogen sources. Individual colonies were photographed after 6 days of growth at 37°C. The mep1Δ mep2Δ double mutants MEP12M6A and -B and the mep2Δ single mutants MEP2M5A and -B were used as controls. The two independently constructed series of strains behaved identically, and only one of them is shown.

In a complementary approach to study the effects of MEP2 mutations on signaling separately from their effects on growth of the cells, we introduced the MEP2^Y122A, MEP2^F126A, and MEP2^W167A alleles into mep2Δ single mutants, which can grow normally on SLAD plates because they express Mep1p. As shown in Fig. 6 (right panels), the filament formation behaviors of these strains corresponded to those of the mep1Δ mep2Δ double mutants expressing the same alleles, i.e., the Y122A mutation resulted in reduced filamentous growth and the F126A and W167A mutations abolished filament formation.

An F239A mutation abolishes ammonium uptake and induction of filamentous growth by Mep2p.Very recently, Javelle et al. demonstrated that mutation of the conserved phenylalanine at amino acid position 215 in AmtB of E. coli abolished ammonium transport (14). F215, together with F107, is thought to form an extracytosolic gate to the conductance channel in AmtB, and they concluded that F215 is essential for the deprotonation of ammonium to allow transport of ammonia into the cell. In order to obtain a similarly transport-deficient Mep2p protein in C. albicans, we replaced the corresponding residue, F239, with an alanine. Indeed, expression of the MEP2^F239A allele did not restore growth of mep1Δ mep2Δ double mutants on SLAD plates, indicating that the F239A mutation abolished ammonium uptake by Mep2p (Fig. 7A). Fluorescence microscopy of cells expressing a GFP-tagged Mep2p^F239A protein demonstrated that the mutated protein was properly localized (Fig. 7B), and quantification of the cellular fluorescence by flow cytometry showed that the expression levels of the mutated protein reached approximately 35% of those of wild-type Mep2p in cells grown in SLAD medium and approximately 50% of those of wild-type Mep2p in cells grown in SLPD (data not shown). We then investigated if the transport-deficient Mep2p protein was able to induce filament formation. As shown in Fig. 7C, mep1Δ mep2Δ double mutants expressing the MEP2^F239A allele were unable to undergo filamentous growth on agar plates containing limiting concentrations of different nitrogen sources (left three panels), and the mutated allele also did not restore filamentous growth of mep2Δ single mutants on SLAD plates (right panels). Therefore, F239 is essential for both ammonium transport and induction of filamentous growth by Mep2p in C. albicans.

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FIG. 7.

(A) An F239A mutation abolishes ammonium uptake by Mep2p. mep1Δ mep2Δ double mutants expressing wild-type MEP2 or the MEP2^F239A allele were grown for 4 days at 30°C on plates containing 2 mM ammonium. (B) The F239A mutation does not affect localization of Mep2p. mep1Δ mep2Δ double mutants carrying GFP-tagged wild-type or MEP2^F239A alleles were grown for 6 h at 30°C in liquid SLAD medium and observed by fluorescence microscopy. (C) The F239A mutation in Mep2p prevents the induction of filamentous growth in response to nitrogen limitation. mep1Δ mep2Δ double mutants (left three panels) and mep2Δ single mutants (right panels) expressing wild-type MEP2 or the MEP2^F239A allele were grown for 6 days at 37°C on agar plates containing 100 μM of the indicated nitrogen sources, and individual representative colonies were photographed. The two independently constructed series of strains behaved identically in all experiments, and only one of them is shown in panels A to C. The mep1Δ mep2Δ double mutants MEP12M6A and -B and the mep2Δ single mutants MEP2M5A and -B were used as controls.