A peroxisomal glutathione transferase of Saccharomyces cerevisiae is functionally related to sulfur amino acid metabolism

Abstract

Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine beta-lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

FIG. 1.

Cellular localization of the Gto proteins. (A and B) Wild-type BY4741 cells or the respective Δpex5 (strain Y03603) and Δpex7 (strain Y04076) mutants transformed with the empty vector pUG35 or with the plasmids carrying the GFP-tagged GTO constructions (pMM440, pMM399, and pMM446, respectively, for GTO1, GTO2, and GTO3) were grown in oleic acid (YPOle) medium for 16 h at 30°C or in glucose (YPD) medium in exponential conditions and then observed by fluorescence microscopy. (C) MM572 cells (carrying a chromosomally integrated version of GTO2-3HA) were grown in YPD medium until late exponential phase and then diluted 1:100 in YPGly medium and cultured at 30°C until a concentration of 2.5 × 10⁷ cells/ml before cellular fractionation was achieved. The resulting fractions were analyzed by Western blotting, using anti-HA antibodies to detect Gto2 and anti-lipoic acid antibodies to detect the mitochondrial markers pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KGDH). Six micrograms of protein were loaded in each lane for total cell extracts (TE) and postmitochondrial supernatant (PM) fractions, and 3 μg was loaded for the mitochondrial (MIT), intermembrane space (IMS), and matrix (MTX) fractions.

FIG. 2.

Northern blot analysis of the expression of the GTO1, GTO2, GTO3, GTT1, and GTT2 genes after addition of different agents. Cells from a wild-type (W303-1A) or the respective isogenic Δyap1 (Wyap1) or Δmsn2 msn4 (Wmsn2msn4) strains were grown exponentially at 30°C in YPD medium, except in the cadmium experiments, in which cells were grown in SC medium. When cultures reached a concentration of 1.5 × 10⁷ cells per ml, the agent was added at the indicated concentration (time zero), and samples were obtained at the indicated times for expression analysis. A total of 25 μg of total RNA was tested run per lane.

FIG. 3.

Effect of different chemical agents on growth rate of S. cerevisiae wild-type cells (W303-1A) and gtt and gto mutant derivatives. The following mutant strains were studied: MML535 (Δgto1), MML538 (Δgto2), MML542 (Δgto3), MML686 (Δgto1 Δgto2 Δgto3), MML628 (Δgtt1), MML629 (Δgtt2), MMO661 (Δgtt1 Δgtt2),and MML716 (Δgto1 Δgto2 Δgto3 Δgtt1 Δgtt2). Conditions for growth were as described previously (80), and the respective agents were used at the indicated concentrations. Growth was recorded by measuring the optical density (600 nm) at 20-min intervals. Bars represent the ratios between the exponential growth rates of treated and untreated mutant cultures, and the values were normalized by dividing them by the corresponding ratio of the wild-type strain. The values of two independent experiments were averaged.

FIG. 4.

(A) Expression of GTO1 in glucose- and oleic acid-grown cells. Wild-type W303-1A cells transformed with pMM736 (expressing GTO1 under its own promoter with a N-terminal TAP tag-expressing sequence) were pregrown in SC medium plus glucose at 30°C to about 4 × 10⁷ cells/ml and then were diluted 10 times, and half of the culture was maintained in SC plus glucose, whereas the other half was shifted to SC plus oleic acid medium. Samples were obtained after 16 h at 30°C for Northern (upper two panels, U1 mRNA as a loading control) or Western (lower panel, 20 μg of total cell protein per lane) blot analysis. (B) The growth of wild-type W303-1A cells (▪) and Δgto1 mutant cells (strain MML535 [▴]) in YPOle medium at 30°C was recorded. Cells were pregrown in YPD medium to about 3 × 10⁷ cells/ml and diluted 10 times in YPOle medium (time zero). The optical density (600 nm) was measured at the indicated times and made relative to the unit value at time zero for the respective strain. The inset box shows the doubling times (in minutes) of the wild-type and mutant strains during exponential growth in YPD medium at 30°C.

FIG. 5.

Northern blot analysis of the expression of the indicated genes in Δgto1 and wild-type cells. Samples were obtained under the conditions described in Table 2. The small nuclear U1 RNA served as a loading control (25 μg of total RNA per lane).

FIG. 6.

Biochemical steps involved in the sulfur assimilation and biosynthesis of cysteine and methionine in S. cerevisiae and genes coding for the respective proteins. Genes boxed in white and gray correspond to upregulated and downregulated genes, respectively, in the Δgto1 mutant.

FIG. 7.

The absence of GTO1 causes an imbalance in the synthesis of reduced glutathione and a number of amino acids. (A) Intracellular millimolar concentrations of reduced (GSH) and oxidized (GSSG) glutathione in wild-type (W303-1A) and Δgto1 (MML535) cells. Absolute levels of both glutathione forms were determined in exponentially growing cells in YPD medium at 30°C. The intracellular glutathione concentration was calculated after the cell volumes were measured. Values are the means of three independent experiments (the standard deviation is indicated). (B) Growth rate of exponentially growing cultures at 30°C of wild-type (W303-1A [□]) and Δgto1 (MML535 [▪]) cells in SC and SD medium and in SC medium without the individual amino acids indicated. Values were determined relative to wild-type cells in SC medium, which have a growth rate of 0.62 doublings per h.

FIG. 8.

Functional relationship between Gto1 and Str3 cystathione β-lyase. (A) CLUSTAL W multiple alignment of a region of cystathionine β-lyase molecules from diverse organisms that included three conserved cysteine residues (position marked for the residues in S. cerevisiae Str3). The abbreviations for the organisms were as follows: Sc, Saccharomyces cerevisiae; Cg, Candida glabrata; Ca, Candida albicans; Bc, Botrytis cinerea; Nc, Neurospora crassa; Gb, Gibberella zeae; An, Aspergillus nidulans; At, Arabidopsis thaliana; St, Solanum tuberosum; Os, Oryza sativa; Lm, Leishmania major; Ll, Lactococcus lactis; Lmo, Listeria monocytogenes; Cac, Clostridium acetobutylicum; Ba, Bacillus anthracis. (B) Growth of wild-type (W303-1A) and Δgto1 (MML535) cells on plates containing medium B, with glucose and ammonium sulfate, cysteine, or cystathionine as the sulfur source, after 3 days (for ammonium sulfate and cysteine) or 5 days (for cystathionine) at 30°C. (C) Mutant str3 cells (strain MML826) transformed with plasmid pMM756 (carrying wild-type STR3 gene), pCM188 (void vector), pMM758 (STR3 with the C353S mutation), pMM760 (STR3 with the C362S mutation), or pMM762 (STR3 with the C387S mutation) were grown exponentially in SC medium, centrifuged and resuspended in B medium with cysteine at a concentration of 3 × 10⁶ cells/ml. Cultures were incubated at 30°C and growth (optical density at 600 nm) was recorded 24 h later. Values are expressed relative to the stre3 mutant transformed with the plasmid with wild-type STR3.