Fig. 3.

vAmt is a functional ${\text{NH}}_4^{+}$ transporter that takes up alternative substrates, is localized to the cell membrane, and mediates an uptake at higher rate for low substrate concentrations than Ostreococcus Amt1.1 homolog when expressed in yeast. (A) Culture optical density (OD_600nm) of an ${\text{NH}}_4^{+}$ uptake defective yeast mutant (strain 31019b) transformed either with an empty vector or with a vAmt-containing vector in two ${\text{NH}}_4^{+}$ concentrations (100 $\mu$M and 500 $\mu$M). Error bars represent SEs based on culture triplicates. The lines represent local polynomial regression fits (see SI Appendix, Fig. S8 for the equivalent results of an additional experiment with O. tauri Amt1.1). Growth of the empty vector transformed mutant is facilitated by passive diffusion of ${\text{NH}}_4^{+}$ across the yeast membrane and/or scavenging of intracellular N stocks. (B) vAmt GFP fusion proteins expressed in yeast, cloned in frame in N terminus (Left) or in C terminus (Right). (Scale bars, 3-$\mu$m distances.) (C) [¹⁴C]-Methylammonium uptake rates in mutant strain 31019b complemented with vAmt or the O. tauri Amt1.1 homolog. Lines show Michaelis–Menten curves; error bars represent SEs based on culture triplicates. (D) Omnilog comparison of mean point estimates and their 95% confidence intervals for N sources showing significant increase in respiration (area under the curve) of vAmt-transformed 31019b yeast cultures compared with non–vAmt-transformed 31019b cultures; data were normalized by subtracting the negative control. See SI Appendix, Fig. S8 for complementation experiments confirming culture growth phenotypes in all of these results apart from D,L-a-amino butyric acid where complementation is not confirmed; see SI Appendix, Fig. S9 for OmniLog Phenotype Microarray respiration curves.