Genetic and environmental factors influencing glutathione homeostasis in Saccharomyces cerevisiae

Abstract

Glutathione is an essential metabolite protecting cells against oxidative stress and aging. Here, we show that endogenously synthesized glutathione undergoes intercellular cycling during growth to stationary phase. Genome-wide screening identified approximately 270 yeast deletion mutants that overexcrete glutathione, predominantly in the reduced form, and identified a surprising set of functions important for glutathione homeostasis. The highest excretors were affected in late endosome/vacuolar functions. Other functions identified included nitrogen/carbon source signaling, mitochondrial electron transport, ubiquitin/proteasomal processes, transcriptional regulation, ion transport and the cellular integrity pathway. For many mutants the availability of branched chain amino acids and extracellular pH influenced both glutathione homeostasis and cell viability. For all mutants tested, the onset of glutathione excretion occurred when intracellular concentration exceeded the maximal level found in the parental strain; however, in some mutants prolonged excretion led to substantial depletion of intracellular glutathione. These results significantly contribute to understanding mechanisms affecting glutathione homeostasis in eukaryotes and may provide insight into the underlying cause of glutathione depletion in degenerative processes such as Parkinson's disease. The important implications of these data for use of the yeast deletion collection for the study of other phenomena also are discussed.

Figure 1.

Disruption of HGT1, encoding a high-affinity glutathione uptake transporter, and a broad range of other genes leads to overaccumulation of extracellular glutathione in stationary phase cultures of S. cerevisiae. (A; inset) The parental strain (BY4743) and hgt1 mutant were grown to stationary phase (2 d) in SD medium, and final cell yield (open bars; A₆₀₀) and extracellular glutathione (closed bars; μmol/A₆₀₀) were quantified. (B) Frequency distribution of extracellular glutathione accumulated by each of the deletion mutants analyzed in this study. Deletion mutants pregrown in YPD medium (3 d) were diluted (1/10) and inoculated in SD medium by using a 96 pin replicator (initial A₆₀₀ of <0.01). Cells were incubated at 30°C for 3 d (without agitation), and extracellular glutathione was quantified. High-range glutathione overexcretion occurred predominantly after deletion of genes encoding functions associated with the late endosome and vacuole (under these conditions the parental strain excreted 1-2 μM glutathione).

Figure 2.

Influence of growth phase on glutathione levels in the parental strain (BY4743) and representative glutathione-overexcreting mutants. The parent (squares) and representative glutathione-overexcreting mutants vps27 (circles), ira2 (triangles), and doa1 (crosses) were inoculated (A₆₀₀ of 0.01) in SD medium and incubated at 30°C. (A) Growth (open symbols; A₆₀₀) and medium pH (closed symbols; initial pH of medium was 4.5) and (B) intracellular glutathione (closed symbols; μM/A₆₀₀) and extracellular glutathione (open symbols; μM/A₆₀₀) were quantified at intervals between 15 and 44 h postinoculation as indicated.

Figure 3.

Overview of the cellular processes influencing cellular glutathione homeostasis leading to glutathione overaccumulation. The figure depicts the glutathione biosynthetic pathway, its position in cellular metabolism, and known and putative transport routes, some of which may mediate glutathione efflux. Boxed annotations indicate some of the cellular processes that are essential to prevent abnormal glutathione homeostasis.

Figure 5.

Effect of the relative availability of branched chain amino acids on intra- and extracellular glutathione, and propidium iodide staining during growth of the parent (BY4743) and ure2 and vps27 mutants. The parent (BY4743; squares), ure2 (circles), and vps27 (triangles) strains were inoculated (A₆₀₀ of 0.01) in SD medium supplemented with 1 time (open symbols) or 4 times (closed symbols) branched chain amino acids (see Figure 4 for concentrations). (A) Intracellular glutathione. (B) Growth (solid lines; left axis) and extracellular glutathione (dashed lines; right axis). (C) Propidium iodide staining were quantified between 14 and 60 h.

Figure 4.

Influence of the relative availability of BCAAs on extracellular glutathione accumulation by selected deletion mutants. The parent (BY4743) and deletion mutants shown were inoculated (initial A₆₀₀ of 0.01) in SD medium supplemented with leucine, isoleucine, and valine: 1 time, 131, 66, and 59 mg l^-1 (open bars); 2 times, 262, 112, and 118 mg l^-1 (shaded bars); 4 times, 524, 264, and 236 mg l^-1 (closed bars), respectively, and grown to stationary phase (2 d; 30°C) at which time extracellular glutathione was quantified.

Figure 6.

Effect of external pH on glutathione homeostasis, cell viability, and cell permeability in stationary phase. The parent (BY4743), vps27, ure2, pep3, and ino1 strains were inoculated (A₆₀₀ of 0.01) in SD medium buffered (25 mM piperazine-N,N-bis(3-propanesulfonic acid)/[2-(N-morpholino)ethane sulfonic acid]) to pH 3.5 or 6.0 and grown to stationary phase (48 h). Intracellular and extracellular glutathione (A) and cell viability (colony-forming units; %) and propidium iodide staining (%) (B) were quantified.