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	Fig 1. The ammonium transport protein Mep2 is regulated by its C-terminal extremity and the Npr1 kinase.(A) Topology model of the Mep2 protein with the location of the mutated residues and amino-acid deletions used in this study. In blue: the N4, D186, H199, G349 and S426; in magenta: the conserved histidine-twin H194 and H348; in orange: the phosphorylation site S457. The enhancer, linker and autoinhibitory domains are labelled in green, grey and red, respectively. (B) Model of Mep2 regulation by the TORC1 effector kinase Npr1, modified from [39]. The transport activity of the hydrophobic core of yeast Mep2 is fine-tuned by a mechanism involving the modulation of the spatial organization of different regions in the CTD. Left panel: When the Npr1 kinase is active, it enables S457 phosphorylation and silencing of the C-terminal autoinhibitory domain (amino-acids 450–485) of Mep2. The enhancer C-terminal domain, limited to residues 428–441, is free to activate the transport protein. Right panel: When Npr1 is inactive, Mep2 is rapidly dephosphorylated by the Psr1/2 phosphatases. The non-phosphorylated autoinhibitory domain prevents the enhancer domain to activate the Mep2 protein. (C) Homozygous diploid triple-mepΔ (ZAM38) and triple-mepΔ npr1-1 (ZMB058) cells were transformed with the pFL38 empty plasmid (-) or with YCpMep2, YCpMep2CΔ428–431, YCpMep2CΔ428–449, YCpMep2CΔ434–449, YCpMep2CΔ434–485, YCpMep2CΔ442–449, YCpMep2CΔ442–485, YCpMep2CΔ450–485 or YCpMep2CΔ469–485. Ammonium removal rates of cells growing in SHPD (0.1% proline) liquid medium. At time 0, 500 μM ammonium was added and its removal from the medium was monitored for 1 h. Ammonium remaining in the medium is expressed as percentage of the initial concentration. Averages and standard deviations are reported (n = 3).
	Fig 2. Transport activity of Mep2 CTD variants correlates with pseudohyphal growth efficiency.Homozygous diploid triple-mepΔ (ZAM38) and triple-mepΔ npr1-1 (ZMB058) cells were transformed with the pFL38 empty plasmid (-) or with YCpMep2, YCpMep2S426stop, YCpMep2CΔ428–431, YCpMep2CΔ428–449, YCpMep2CΔ434–449, YCpMep2CΔ434–485, YCpMep2CΔ442–449, YCpMep2CΔ442–485, YCpMep2CΔ450–485 or YCpMep2CΔ469–485. (A) Growth tests of low-density cell suspensions streaked on SLAD (100 μM ammonium) and SHAD (1 mM ammonium) media at day 3 (3d) and 7 (7d), at 29°C. (B) Pseudohyphal growth tests of high-density cell suspensions dropped on SLAD and SHAD media at day 7. (C) Immunodetection of Mep2 from membrane-enriched cell extracts treated with N-glycosidase F. Cells were grown in buffered minimal medium containing 0.1% proline. Pma1 was detected as a loading control.
	Fig 3. The S457D phosphomimetic mutation does not lock Mep2 in a conformation allowing constitutive filamentation.(A-B) Homozygous diploid triple-mepΔ (ZAM38) and mep2Δ (ZAB2) cells were transformed with the pFL38 empty plasmid (-) or with YCpMep2, YCpMep2D186N, YCpMep2S457D or YCpMep2D186N, S457D. (A) Growth tests of low-density cell suspensions on SLAD and SHAD media at day 3 (3d) and 7 (7d), at 29°C. (B) Pseudohyphal growth tests of high-density cell suspensions dropped on SLAD and SHAD media at day 7. (C) Immunodetection of Mep2 from membrane-enriched extracts, treated with N-glycosidase F, of haploid triple-mepΔ cells (31019b) transformed with YCpMep2, YCpMep2D186N, YCpMep2S457D or YCpMep2D186N, S457D plasmids and grown in buffered minimal medium containing 0.1% proline. Pma1 was detected as a loading control. (D-E) Homozygous diploid triple-mepΔ (ZAM38) and triple-mepΔ psr1Δ psr2Δ (ZAB1) cells transformed with the pFL38 empty plasmid (-) or with YCpMep2 or YCpMep2S457D. (D) Growth tests of low-density cell suspensions on SLAD and SHAD media at day 3 (3d) and 7 (7d). The homozygous diploid triple-mepΔ growth tests showed in panel A served as common control for tests A and D. (E) Pseudohyphal growth tests of high-density cell suspensions dropped on SLAD and SHAD media at day 7.
	Fig 4. The Mep2 C-terminal tail can be dispensable for filamentation induction.(A-B) Homozygous diploid triple-mepΔ (ZAM38) and triple-mepΔ npr1-1 (ZMB058) cells were transformed with the pFL38 empty plasmid (-) or with YCpMep2, YCpMep2S426stop, YCpMep2H199Y, YCpMep2H199Y, S426stop, YCpMep2G349C, YCpMep2G349C, S426stop, YCpMep2H194E, YCpMep2H348A or YEpMep1. (A) Pseudohyphal growth tests of high-density cell suspensions dropped on SLAD and SHAD media at day 7. (B) Growth tests of low-density cell suspensions on SLAD and SHAD media at day 3 (3d) and 7 (7d), at 29°C. (C-E) Ammonium removal rates of homozygous diploid triple-mepΔ (ZAM38) cells growing in SHPD (0.1% proline) liquid medium. At time 0, 500 μM ammonium was added and its removal from the medium was monitored for 1 h. Ammonium remaining in the medium is expressed as percentage of the initial concentration. Averages and standard deviations are reported (n = 3). (C) Cells transformed with the pFL38 empty plasmid or with YCpMep2, YCpMep2H199Y, YCpMep2H199Y, S426stop. (D) Cells transformed with the pFL38 empty plasmid or with YCpMep2, YCpMep2H348A, YCpMep2G349C, YCpMep2G349C, S426stop, YCpMep2S426stop. (E) Cells transformed with the pFL38 empty plasmid or with YCpMep2, or YCpMep2H194E.
	Fig 5. Visualization of interatomic interactions implicating H199 and G349 residues in the ScMep2 crystal structure.A close-up (A), a global trimeric (C) and a monomer (D) view of the ScMep2 structure (PDB id 5AEX, chain A). ScMep2 protein is illustrated in grey ribbons with TM5 in blue, TM10 in yellow and TM11 in turquoise, and with the C-terminal coil in red. The H199 side chain is depicted in stick representation with carbon atoms in purple and the α carbon atom of G349 is drawn as a purple sphere. Side chains found in interaction with H199 are shown in stick representation with carbon atoms in green. The histidine-twin H194-H348 is also depicted with carbon atoms in orange. The nitrogen and oxygen atoms are colored in blue and red, respectively. In (A), different type of non-covalent interactions are illustrated by yellow dashed bonds as π-π stack interactions and van der Waals interactions. The distances associated with non-covalent interactions are indicated, as well as the distance between Cα of G349 and ring of H199. Residues drawn are labelled. The non-covalent interactions were defined with the ARPEGGIO program and the images were created using a combination of MolScript and Raster3D programs. (B) WebLogo plots of transmembrane helices TM5 and TM10 in Mep1-like vs Mep2-like sequences. The logo consists of amino acid stack for each position in the aligned sequence dataset. The height of the stack indicates the sequence conservation at that position (maximum = 4 bits), while the height of the amino acid one-letter symbol within the stack shows the relative frequency of the amino acid at that position. The color scheme is aromatic amino acids (F, H, W, Y) in green, polar (N, Q, S, T) in purple, negatively charged (D, E) in red, positively charged (K, R) in blue, aliphatic (A, I, L, M, V) in black, cysteine (C) in brown, proline (P) in grey and glycine (G) in orange. Sequence numbering is according ScMep1 or ScMep2 sequences.
	Fig 6. Substrate translocation via Mep1 and Mep2 has specific impacts on cytosolic pH.Cells transformed with pYES-pHluorin were grown in buffered minimal medium (pH = 6.1) containing 0.1% proline and evolution of pHc, corrected for negative control variations (delta-pH), was followed after addition of ammonium 20 mM (grey triangle) (A) and/or 2 mM (black square) (A-D). (A) Triple-mepΔ (31019b) cells. (B) Wild-type (23344c) cells. (C) mep2Δ mep3Δ (31018b) cells, expressing chromosomal MEP1. (D) mep1Δ mep3Δ (31022a) cells, expressing chromosomal MEP2. (A-D) Averages and standard deviations are reported (n = 3). (E) Combined representation of delta-pH evolution from (B-D) after addition of 2 mM ammonium. Wild-type (black circle), mep2Δ mep3Δ (red circle) and mep1Δ mep3Δ (green circle) cells. Averages are reported (n = 3).
	Fig 7. Substrate translocation via Mep2H194E induces a pH decrease.Haploid triple-mepΔ cells (31019b) were transformed with pMep2-pHluorin (A), pMep2D186N-pHluorin (B) and pMep2H194E-pHluorin (C) and grown in a buffered minimal medium (pH = 6.1) containing 0.1% proline. Evolution of pH, corrected for negative control variations (delta-pH), was followed after addition of ammonium 2 mM (black square) and 0.5 mM (open square). The cells were also visualized by fluorescence microscopy (panel 3). Scale bar, 5 μm.
	Fig 8. Functional characterization of Mep1 and Mep2 in Xenopus oocytes.(A) Current / Voltage plot showing currents induced by 3 mM ammonium in dependence of the membrane potential. Mep1 is shown as closed black circles. Mep2, Mep2H194E, Mep2S457D and Mep2H194E S457D are shown as open, circles, squares, triangles and diamonds, respectively. All currents were recorded at pH 5.5 from the same batch of oocytes. Data is given as means (n≥4) ± SD. (B) Currents induced by 3 mM NH4Cl at a membrane potential of -100 mV. Data is shown as means ± SE (n≥4) and significant differences at P<0.01 according to Tukey’s test are indicated by different letters. (C) Concentration-dependence of the ammonium currents of Mep1 at pH 5.5. Net currents at -100 mV are given in dependence of the ammonium concentration. Data is given as means ± SD (n = 5). (D) Mep2-GFP, Mep2N4Q-GFP, Mep2H194E-GFP and Mep2S457D-GFP fusion protein localization at the plasma membrane of oocytes. Control shows non-injected (NI) oocytes. Dark pictures show GFP fluorescence in the membrane, bright pictures show bright-field (BF) images of the oocyte. Shown is one representative picture of n = 30. Scale bar, 200 μm. (E) Influx of 15N-labelled ammonium into oocytes expressing Mep1, Mep2, and Mep2S457D. Water injected oocytes were used as negative control. Oocytes were incubated for 20 min in media containing 3 mM NH4Cl at pH 5.5. Data is given as means ± SE (n≥8) and significant differences at P<0.01 according to Tukey’s test are indicated by different letters.
	Fig 9. The H188E pore mutation abolishes the signaling ability of C. albicans Mep2 expressed in S. cerevisiae.Homozygous diploid triple-mepΔ (ZAM38) cells were transformed with the pFL38 empty plasmid (-) or with the centromeric plasmids, YCpMep2, YCpCaMep2, YCpCaMep2H188E, or with the episomal plasmids, YEpCaMep2 or YEpCaMep2H188E. (A) Growth tests of low-density cell suspensions on SLAD and SHAD media at day 3 (3d) and 7 (7d) at 29°C. (B) Pseudohyphal growth tests of high-density cell suspensions dropped on SLAD and SHAD media at day 7 at 29°C.

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