YAT transporters support growth on a range of amino acids.

Growth rates of Δ10AA expressing one of seven different wild-type YAT genes (AGP1, BAP2, CAN1, HIP1, LYP1, MMP1, PUT4) from pADHXC3GH and the empty vector control on 2 mM of each amino acid. Error bars represent the SEM (n ≥ 3). For the respective growth curves see Figure 1–figure supplement 1. For the respective growth rates see Supplementary Dataset 1.

Substrate range of transporters used in this study.

Positions of the substituted amino acids investigated in this study.

Side and top views of the AlphaFold models of AGP1 (AF-P25376) (A) and PUT4 (AF-P15380) (B) visualized in ChimeraX (1.3.0) [36]. The amino acids of interest are highlighted in different colors. The respective TMs are presented in circles. The predicted substrate binding site is represented as dashed circle.

Evolved AGP1-Cit variants and PUT4-Asp and -Glu variants.

The evolved variants support growth and uptake of the respective amino acids.

(A) Growth rate of the AGP1 variants and the vector control on 2 mM Cit. Error bars represent the SEM (n ≥ 3). (B) Uptake rate of 1 mM 14C-Cit by whole cells expressing different AGP1 variants or none (vector control). Error bars represent the SEM (n ≥ 3). Asterisks indicate the degree of significant difference (one-way ANOVA with a Dunett’s test; ** p < 0.01, * p < 0.05) between the AGP1 variants and wild-type. (C) Growth rate of the PUT4 variants and the vector on 2 mM Asp and Glu. Error bars represent the SEM (n ≥ 3). (D) Uptake rate of 1 mM 14C-Glu by whole cells expressing different PUT4 variants or none (vector control). Error bars represent the SEM (n ≥ 3). Asterisks indicate the degree of significant difference in pairwise comparisons between the transporter-expressing variants (Student’s t-test; ** p < 0.01, * p < 0.05).

Relative fitness of evolved AGP1 variants.

(A) Relative fitness of AGP1 variants and the control strain for the growth on each of 17 amino acids as the sole nitrogen source. The relative fitness was calculated separately for each amino acid by dividing the growth rate of the mutant by the mean growth rate of the wild-type AGP1. Error bars represent the SEM (n ≥ 3). For the respective growth curves see Figure 4–figure supplement 1. (B) Mean relative fitness of AGP1 variants per substrate as a function of the amino acid hydropathy index (x axis) and molecular volume (y axis). Color corresponds to relative fitness. The plot is based on the growth rate measurements of panel (A).

Effects of evolved AGP1 mutations on the growth and uptake of original substrates.

(A) Localization of the AGP1 variants in whole cells by fluorescence microscopy. (B) Growth rate of the AGP1 variants and the vector on 2 mM Glu. Error bars represent the SEM (n ≥ 3). (C-D) Uptake rate of 0.1 (C) and 2 mM (D) 14C-Glu in whole cells expressing different AGP1 variants or none (vector control). In the case of AGP1-G, the assay was performed with 2 mM 3H-Glu and is presented as independent experiment. Error bars represent the SEM (n ≥ 3). (E) Growth rate of the AGP1 variants and the vector control on 2 mM Phe. Error bars represent the SEM (n ≥ 3). (F-G) Uptake rate of 0.1 (F) and 2 mM (G) 14C-Phe in whole cells expressing different AGP1 variants or none (vector control). Error bars represent the SEM (n ≥ 3). Asterisks indicate the degree of significant difference (one-way ANOVA with a Dunett’s test; ** p < 0.01, * p < 0.05) between the AGP1 variants and wild-type. For the respective uptake curves see Figure 5–figure supplement 1.

Substrates of AGP1 strains with statistically significant relative fitness differences.

Effects of evolved PUT4-S mutation on the growth and uptake of original substrates.

(A) Localization of the PUT4 variants in whole cells by fluorescence microscopy. (B) Relative fitness of PUT4-S and the vector for the growth on 6 amino acids as the sole nitrogen source. The relative fitness was calculated separately for each amino acid by dividing the growth rate of the mutant by the mean growth rate of the wild-type PUT4. Error bars represent the SEM (n ≥ 3). For the respective growth curves see Figure 4–figure supplement 1. (C-E) Uptake rate of 10 μM 14C-Ala (C), GABA (D) or Gly (E) in whole cells expressing PUT4 variants or none (vector). Error bars represent the SEM (n ≥ 3). Asterisks indicate the degree of significant difference in pairwise comparisons between the transporter-expressing variants (Student’s t-test; ** p < 0.01, * p < 0.05). For the respective uptake curves see Figure 5–figure supplement 2.

The evolved PUT4-S mutation broadens the substrate range of the transporter.

(A) Growth rate measurements of PUT4 variants and the vector control on 2 mM of 18 different amino acids as the sole nitrogen source. Error bars represent the SEM (n ≥ 3). Asterisks indicate the degree of significant difference in pairwise comparisons (Student’s t-test; ** p < 0.01, * p < 0.05). For the respective growth curves see Figure 4–figure supplement 1. (B) Mean relative fitness of PUT4-S per substrate as a function of the amino acid hydropathy index (x axis) and molecular volume (y axis). Diamond shapes indicate novel substrates not found in the wild-type PUT4. Color corresponds to relative fitness. The plot is based on the same growth rate measurements as (A).

List of plasmids used in this study

S. cerevisiae strains used in this study.