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

Abstract

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.

Fig. 1. Functional AMT-Mep–type proteins deplete cells in ¹⁵N.

Yeast growth (top) and natural N isotopic abundance (bottom) in yeast strains grown for 48 hours (A and B) or 20 hours (C) at pH 4.3 in the presence of 13 mM K⁺ and 76 mM ammonium (A) or 1 mM ammonium (B and C). Wild type, Σ1278b; wild type (ura3), 23344c; wild type (ura3) + pFL38, wild type (ura3) + empty plasmid; triple mepΔ, 31019b; triple mepΔ + pFL38, triple mepΔ + empty plasmid; ScMep1, triple mepΔ + ScMep1; ScMep2, triple mepΔ + ScMep2; ScMep2^H194E, triple mepΔ + ScMep2^H194E; ScMep3, triple mepΔ + ScMep3 yeast strains. Data are presented as means ± SE (n ≥ 3). Letters represent significant differences among yeast strains for a given ammonium concentration (P ≤ 0.05). *Significant differences between ammonium concentrations for a given yeast strain. δ¹⁵N of (NH₄)₂SO₄, 0.0 to 0.5 mUr. a.u., arbitrary units.

Fig. 2. Cellular δ¹⁵N derived from Mep-, TIP-, and AKT-type transporters show differential pH dependence.

Cell growth (OD₆₀₀; A and B), N content (%; C and D), and cellular N isotopic composition (δ¹⁵N; E and F). in Mep-containing cells and triple-mepΔ and yeast cells expressing other ammonium transporters. Yeast cells were grown at pH 4.3 in the presence of 13 mM K⁺ and 76 mM ammonium. Yeast strains used in this study include wild-type (Σ1278b), triple mepΔ (31019b), wild-type (ura3) (23344c), triple mepΔ + ScMep2, and triple mepΔ + ScMep2^H194E. Other putative carriers of NH₄⁺ (such as AtAKT1 from A. thaliana, triple mepΔ + AtAKT1) or NH₃ (such as TaTIP2;1 from T. aestivum, triple mepΔ + TaTIP2;1) that are not members of the AMT-Mep-Rh family were also tested and considered controls for different ammonium transport mechanisms. TaTIP2;1 from T. aestivum–containing cells and triple-mepΔ cells were grown on galactose (3%) at pH above 6.5 (B and D). Equations for regression curves displayed in (A): wild type: y = 0.21x − 14.69, R² = 0.5755; wild type (ura3): y = 0.43x − 13.70, R² = 0.8480; triple mepΔ (on glucose; pH 2.5 to 6.5): y = −0.2x − 1.87, R² = 0.7711; triple mepΔ + ScMep2: y = 0.52x − 9.98, R² = 0.5694; triple mepΔ + ScMep2^H194E: y = 0.79x − 13.38, R² = 0.6476; triple mepΔ + TaTIP2;1: y = −2.42x + 5.99, R² = 0.8135; triple mepΔ (on galactose; pH 6.5 to 8): y = −0.04x − 0.70, R² = 0.0531; triple mepΔ + AtAKT1: y = −0.21x − 0.61, R² = 0.7516. δ¹⁵N of (NH₄)₂SO₄, −1.0 to 0.5 mUr. Data are presented as means ± SE (A to D; n = 3).

Fig. 3. Mep-type proteins mediate intracellular alkalinization upon ammonium uptake.

Stopped-flow fluorescence signals obtained for intact triple-mepΔ (31019b) cells transformed with YCpMep1, YCpMep2, or YCpMep3 were compared with the negative control (31019b) and wild-type strain (Σ1278b). (A) Intracellular alkalinization was indicated by an increase in the fluorescence intensity resulting from the addition of 25 mM NH₄Cl (pH 8.0) to the external medium at t₀ = 23°C. (B) Arrhenius plot displaying the ln(k) of the changes in intracellular pH resulting from the transport of NH₃ as a function of the inverse of the temperature. Data are presented as means ± SE (n = 3).

Fig. 4. Functional AMT-Rh protein superfamily depletes cells in ¹⁵N.

Isotopic N signature of triple-mepΔ (31019b) cells transformed with different AMT-Rh subfamily transporters from protista, eubacteria, plants, and animals. Yeast growth (top) and natural N isotope abundance (bottom) of different yeast strains grown at pH 4.3 in the presence of 13 mM K⁺ and 76 mM NH₄⁺ (A) or 0.5 mM NH₄⁺ (B). *TaTIP2;1-containing cells were grown at pH 7.0. AMT-Rh carrier-containing strains grown in the presence of 76 mM NH₄⁺ were compared with their respective negative control strains (31019b + pDR196 and 31019b + p426MET25) and other putative carriers of NH₄⁺ (such as AtAKT1 from A. thaliana) or NH₃ (such as TaTIP2;1), which are not members of the AMT-Mep-Rh family, as controls. Control strains did not show growth in the presence of 0.5 mM NH₄⁺. EcAmtB (AMT from Escherichia coli), triple mepΔ + EcAmtB; Cr-AMT1;1 (Amt1;1 from Chlamydomonas reinhardtii), triple mepΔ + CrAmt1;1; Cr-AMT1;2 (AMT1;2 from C. reinhardtii), triple mepΔ + CrAMT1;2; At-AMT1;1 (AMT1;1 from A. thaliana), triple mepΔ + AtAMT1;1; At-AMT1;2 (AMT1;2 from A. thaliana), triple mepΔ + AtAMT1;2; At-AMT1;3 (AMT1;3 from A. thaliana), triple mepΔ + AtAMT1;3; At-AMT2;1 (AMT2;1 from A. thaliana), triple mepΔ + AtAMT2;1; Hs-RhCG (RhCG from Homo sapiens), triple mepΔ + HsRhCG. Data are presented as means ± SE (n ≥ 3). Letters represent significant differences (P ≤ 0.05). δ¹⁵N of (NH₄)₂SO₄, −0.8 to 0.1 mUr. nd, not determined.

Fig. 5. Inhibition of NSC1-type channels does not affect cellular ¹⁵N depletion.

Inhibition of NSC1-type channels with hygromycin B in wild-type (Σ1278b) and triple-mepΔ (31019b) cells grown in the presence of 13 mM K⁺ and 76 mM ammonium. (A) Yeast growth (OD₆₀₀), (B) natural N isotopic abundance (in milliUreys), and (C) total N content (in micromoles) in wild-type and triple-mepΔ yeast grown under control conditions (black bars) or in the presence of 300 μM hygromycin B, the NSCC type 1 inhibitor (white bars), at the end of the growth period. Yeast cells were grown for 48 hours at 185 rpm, 30°C, and pH 4.3. Data are presented as means ± SE (n = 3). δ¹⁵N of (NH₄)₂SO₄, 0.0 mUr.

Fig. 6. External ammonium/potassium ratio affects cellular δ¹⁵N.

Tracking δ¹⁵N values in the presence of different external ammonium and potassium concentrations in S. cerevisiae (A to D) wild-type (Σ1278b; black) and (E to H) triple-mepΔ (31019b; red) strains. (A and E) Yeast growth (OD₆₀₀). (B and F) Yeast δ¹⁵N (in milliUreys). (C and G) Amino acid concentrations detected in the media after yeast growth (μmol g⁻¹ DW). (D and H) Yeast N content (%). Yeast cells were grown for 48 hours at 185 rpm, 30°C, and pH 4.3. Data are presented as means ± SE (n = 3). δ¹⁵N of (NH₄)₂SO₄, 0.0 to 0.044 mUr. DW, dry weight.