Figure 7. Suppressor mutants restore transport and fluorescence response.
(A) Growth of the yeast Δmep1,2,3 strain transformed with suppressor mutants and grown on solid media containing the indicated concentrations of NH4Cl, (NH4)2SO4 (as anion control) or 1 mM arginine (growth control) as the sole nitrogen source for 3 days. Note that yeast expressing AmTrac-T464D-A141E grew poorly at high concentrations of ammonium, suggesting high capacity transport activity leading to ammonium toxicity. (B) Lateral view and (C) cytoplasmic side view of AfAmt1 according to the crystal structure (Andrade et al., 2005). The corresponding residues in AfAmt1 that suppress the T464D mutation in AmTrac are indicated by spheres. TMH-V is shown as red helix, TMH-VI in blue. The connecting L5–6 is labeled. Note that residues corresponding to cis-suppressing mutations reside in the internal pore region. (D) Sequence alignment of AMT1;3 from Arabidopsis (At-Amt1;3) and Af-Amt1 from A. fulgidus. The residues belonging to TMH domains of the two pseudo-symmetric halves of AfAMT1 are shown in red and blue.C-terminal TMH-XI of AfAMT1 is shown in grey, with white font. Predicted TMHs of AMT1;3 are highlighted in grey, with black font. The corresponding residues identified in the suppressor screen of AmTrac-T464D are indicated in both sequences (yellow). (E) Correlation between transport efficiency (growth in 2 mM NH4Cl) and fluorescence change after addition of 1 mM NH4Cl of the suppressor mutants. Data are normalized to values obtained for AmTrac (=100) (mean ± SD; n = 3). (F) Fluorescence response of suppressors to addition of the indicated concentrations of NH4Cl. Data were normalized to water-treated controls (0) (mean ± SE; n = 3).