The Saccharomyces cerevisiae amino acid transporter Lyp1 has a broad substrate spectrum

Abstract

The main mediators for the amino acid uptake in Saccharomyces cerevisiae are the permeases belonging to the yeast amino acid transporter family. Recently, we discovered that members of this family support growth on more amino acids than previously described. Here we study the substrate spectrum of Lyp1, the main transporter responsible for the uptake of lysine in yeast. We show that overexpressed Lyp1 supports growth on alanine, asparagine, leucine, methionine, phenylalanine, serine, and valine when these are provided as the sole source of nitrogen to a strain severely deficient for the uptake of amino acids. We show that alanine and serine compete with lysine for the common transport system, albeit with much lower affinity. Thus, Lyp1 has a much broader substrate spectrum than previously thought, which may be true for many amino acid transporters.

Keywords: Saccharomyces cerevisiae; competitive inhibition; growth assay; low‐affinity substrate; lysine permease; yeast amino acid transporter.

Fig. 1

Lyp1 supports growth on a range of amino acids. (A) Specific growth rates of S. cerevisiae Δ10AA expressing Lyp1 from pADHXC3GH or bearing the vector control on 2 mm of 20 different amino acids provided as the sole nitrogen source. Error bars represent the SEM (n = 6). Asterisks indicate the degree of significant differences in pairwise comparisons (Student's t‐test; **P < 0.01, *P < 0.05, ns = nonsignificant difference). For the respective growth curves, see Fig. S1. (B) Mean growth rates of Δ10AA expressing Lyp1 per substrate as a function of the amino acid hydropathy index (x axis) and molecular volume (y‐axis). Color corresponds to the corrected growth rate after subtraction of the vector control. The high‐affinity substrate Lys is shown as a blue diamond. The plot is based on (A).

Fig. 2

Growth rate as a function of amino acid concentration. Specific growth rates of S. cerevisiae Δ10AA expressing Lyp1 from pADHXC3GH or bearing the vector control on different concentrations of Ala (A), Ser (B), Phe (C), and Val (D) as the sole nitrogen source. The fit of the Monod model is plotted, and the dependence on amino acid concentration [x] for the cultures expressing Lyp1 is shown at the top of each panel. Error bars represent the SEM (n = 3). For the respective growth curves, see Fig. S3.

Fig. 3

Lyp1 supports the uptake of different amino acids. Uptake of 20 μm ¹⁴C‐Lys (A), 60 mm ¹⁴C‐Ala (B), and 40 mm ¹⁴C‐Ser (C) in S. cerevisiae Δ10AA expressing Lyp1 from pADHXC3GH or bearing the vector control (assay Buffer: KCP at pH 5). Error bars represent the SEM (n = 3). The degree of significant differences in (B, C) was calculated using a one‐sample t‐test with the uptake rate of the vector control as a fixed value. For the respective uptake curves, see Fig. S4.

Fig. 4

Kinetics of Lys transport in the presence and absence of Ala and Ser. (A) Uptake rate of ¹⁴C‐Lys by S. cerevisiae Δ10AA expressing Lyp1 from pADHXC3GH (assay Buffer: KCP at pH 5). The assay in KP at pH 6 is shown in the inset. The fit of the Michaelis–Menten model is plotted. (B) Lineweaver–Burk plot and linear regression for increasing concentrations of ¹⁴C‐Lys. (C, D) Dixon plot and linear regression for increasing concentrations of Ala in the presence of 1 and 10 μm ¹⁴C‐Lys (C), or Ser in the presence of 1 and 4 μm ¹⁴C‐Lys (D) (assay Buffer: KCP at pH 5). For the respective uptake rates, see Fig. S5. The plot equations and R ² values are shown on top of their respective graph. Error bars represent the SEM (n = 3).

Fig. 5

Docking of substrates on the predicted Lyp1 binding site. (A) Side view of Lyp1 (AF‐P32487). The volume box utilized to dock the substrates with AutoDock Vina (1.1.2) in Chimera (1.17.1) is shown in magenta. The box includes the residues expected to contribute to the substrate‐binding site, presented as sticks, according to the AdiC‐Arg structure. (B–D) Side view of the AlphaFold model of Lyp1 (AF‐P32487) and the docked Lys (PubChem CID: 5962) (B), Ala (PubChem CID: 5950) (C), or Ser (PubChem CID: 5951) (D), as predicted by AutoDock Vina (1.1.2) in Chimera (1.17.1). The hydrogen bonds are depicted as dashed lines, with the Lyp1 residues participating in them shown as brown sticks. Atoms are colored as gray: carbon; red: oxygen; blue: nitrogen; white: hydrogen. The respective TMs are mentioned in circles. Visualized in ChimeraX (1.8).