Fig 1.
Glutathione biosynthesis pathway and two possible pathways for the synthesis of γ-glutamyl peptides.
A. Schematic depiction of glutathione biosynthesis. GCL, glutamate-cysteine ligase; GS, glutathione synthetase. B. Synthesis of γ-glutamyl peptides during glutathione degradation. γ-Glutamyltransferase (GGT) transfers γ-glutamyl residues from GSH to amino acids or peptides (X). C. Synthesis of γ-glutamyl peptides as a byproduct of GSH biosynthesis. GCL uses another amino acid (X) instead of cysteine as a substrate and produces a γ-glutamyl dipeptide (γ-Glu-X). Then, GS uses this peptide as a substrate and produces a tripeptide (γ-Glu-X-Gly).
Table 1.
Culture media used in this work.
Table 2.
Ions used for peptide identification by MS/MS analysis.
Fig 2.
Dependence of peptide concentration in yeast cell extracts on medium composition.
Cells of the WT strain were grown in SD or SDP (SD in which ammonium sulfate was replaced with proline as a nitrogen source) medium supplemented with γ-EV, VG, valine, or valine and glycine.
Fig 3.
Effect of deletion and overexpression of GSH2 on the γ-EVG concentrations in cell extracts.
Cells were cultivated in SD medium supplemented with γ-EV (SD+γEV). The data shown are the mean values of at least three independent determinations. As a control (WT), the strains S288C and S288C ura3Δ0 were used.
Fig 4.
Identification of the enzyme responsible for γ-EVG synthesis from VG.
A, Effect of overexpression of PTR2 and deletion of DUG1 on intracellular VG and γ-EVG concentrations. The strain S288C ura3Δ0 was used as a control (WT). B. Effect of GSH concentration on γ-EVG synthesis by the PADH1-PTR2 gsh1Δ strain. C. Effect of deletion of ECM38 or DUG2 on γ-EVG synthesis by the PADH1-PTR2 strain. In B and C, the level of γ-EVG in the PADH1-PTR2 strain cultivated in the corresponding medium was taken to be 100%.
Fig 5.
Identification of the enzyme responsible for the synthesis of γ-EV from valine.
The level of γ-EV in the WT strain cultivated in SD+V and SDV media was taken to be 100%. A, Effect of the GSH concentration in the medium and of deletions of ECM38 and DUG2 on γ-EV synthesis by the GSH1 deletion strain. B, Effect of GSH1 expression on γ-EVG synthesis in the strain with deletions of ECM38 and DUG2. The strains S288C ura3Δ227 and S288C ura3Δ0 were used as controls (WT).
Fig 6.
Synthesis of γ-EVG by a strain overexpressing GSH1 and GSH2.
All strains were cultivated in SD medium supplemented with valine (SD+V). Glycine was added to the medium where indicated.
Fig 7.
Effect of OPT1 deletion and overexpression on γ-EVG levels in cells grown in SD+γ-EVG medium.
The data shown are the mean values of at least three independent determinations. The strain S288C ura3Δ0 was used as a control (WT).
Fig 8.
Two pathways of γ-EVG synthesis in S. cerevisiae.
A. Synthesis from valine, glutamate and glycine. B. Synthesis from the dipeptide VG.