Involvement of GDH3-encoded NADP+-dependent glutamate dehydrogenase in yeast cell resistance to stress-induced apoptosis in stationary phase cells

Abstract

Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP(+)-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.

FIGURE 1.

Deletion of GDH3 results in increased sensitivity of yeast cells to thermal, oxidative, and aging stress. A, survival rates of yeast strains exposed to thermal or oxidative stress. Yeast cells (WT, Δgdh1, Δgdh3, Δgdh1Δgdh3/YCp111, Δgdh1Δgdh3/YCpGdh1, and Δgdh1Δgdh3/YCpGdh3) grown in SCD for 2 days were exposed to thermal (50 °C, 30 min) or oxidative (10 mm H₂O₂, 1 h) stress. Surviving cells were evaluated by CFU assays. Values are the means ± S.E. of three independent experiments. *, p < 0.001 (two-tailed Student's t test versus WT). B, survival rates of yeast strains during chronological aging. The yeast strains listed in A were grown in SCD for 13 days. Surviving cells were evaluated as described in A. ■, WT; □, Δgdh1; ●, Δgdh3; ○, Δgdh1Δgdh3/YCp111; ♦, Δgdh1Δgdh3/YCpGdh1; ♢, Δgdh1Δgdh3/YCpGdh3.

FIGURE 2.

Yeast Gdh3-null cells display apoptotic markers under stress conditions. A, ROS accumulation in yeast cells exposed to oxidative stress. Yeast cells (WT, Δgdh1, Δgdh3, and Δgdh1Δgdh3) grown in SCD for 2 days were exposed to 10 mm H₂O₂ for 1 h. Cells were stained with DCFH-DA and examined by fluorescence microscopy. Bar, 10 μm. DIC, differential interference contrast. B, nuclear or DNA fragmentation in yeast cells exposed to oxidative stress. Yeast cells prepared and treated as described in A were stained with DAPI or TUNEL reagent and examined by fluorescence microscopy. Bar, 10 μm. C, phosphatidylserine externalization in yeast cells exposed to oxidative stress. Yeast strains prepared and treated as described in A were stained with FITC-annexin V and PI and examined by fluorescence microscopy. Bar, 10 μm. D, percentage of yeast cells exhibiting the typical hallmarks of apoptosis after exposure to oxidative stress (10 mm H₂O₂, 1 h). The number of cells with accumulated ROS (DCFH-DA(+)), fragmented nuclei (DAPI(+)), and fragmented DNA (TUNEL(+)) were determined from ∼500–700 cells in three independent experiments. Values are the mean ± S.E. *, p < 0.001; **, p < 0.005 (two-tailed Student's t test versus WT).

FIGURE 3.

Exogenous supply of GSH or glutamate suppresses the hypersusceptibility of yeast Gdh3-null cells to stress-induced apoptosis mediated by ROS accumulation. A, effect of exogenous GSH and glutamate on survival of yeast cells exposed to oxidative stress. Yeast strains (WT, Δgdh1, Δgdh3, and Δgdh1Δgdh3) grown in SCD with or without 10 mm GSH or glutamate for 2 days were exposed to 10 mm H₂O₂ for 1 h. Surviving cells were evaluated by CFU assays. Values are the mean ± S.E. of three independent experiments. B, effect of exogenous GSH and glutamate on ROS accumulation in yeast cells exposed to oxidative stress. Yeast cells prepared and treated as described in A were stained with DCFH-DA and examined by fluorescence microscopy. The number of DCFH-DA-positive cells was estimated in fluorescence images and total cells in the corresponding differential interference contrast images. Approximately 500–700 cells were observed in three independent experiments. Values are the mean ± S.E. *, p < 0.005 (two-tailed Student's t test versus untreated).

FIGURE 4.

Deletion of GDH2 suppresses the hypersusceptibility of yeast Gdh3-null cells to stress-induced apoptosis mediated by ROS accumulation. A, effect of Δgdh2 mutation on survival of yeast cells exposed to thermal or oxidative stress. Yeast strains (WT, Δgdh1, Δgdh2, Δgdh3, Δgdh2Δgdh3, WT/YEpGDH2, Δgdh1/YEpGDH2, Δgdh2/YEpGDH2, and Δgdh3/YEpGDH2) grown in SCD for 2 days were exposed to either thermal (50 °C, 30 min) or oxidative stress (10 mm H₂O₂, 1 h). Surviving cells were evaluated by CFU assays. Values are the means ± S.E. of three independent experiments. *, p < 0.001 (two-tailed Student's t test versus Δgdh3). B, effect of the Δgdh2 mutation on ROS accumulation in yeast cells exposed to thermal or oxidative stress. Yeast cells prepared and treated as described in A were stained with DCFH-DA and examined by fluorescence microscopy. The number of DCFH-DA-positive cells was estimated in fluorescence images and total cells in corresponding differential interference contrast images. Approximately 500–700 cells were observed in three independent experiments. Values are the mean ± S.E. *, p < 0.001; **, p < 0.05 (two-tailed Student's t test versus Δgdh3).

FIGURE 5.

GDH3 transcription occurs exclusively during stationary phase, whereas GDH1 transcription is consistent throughout all growth periods. Yeast strains (WT/YEpP_GDH1-LacZ and WT/YEpP_GDH3-LacZ) were grown in SCD, and samples were taken after 24, 48, and 72 h of culture. GDH1 and GDH3 transcription levels were estimated by measuring β-gal activity. β-Gal activity was calculated using the following equation: β-gal activity = (1000 × A₄₂₀)/(t × V × OD₆₆₀), where t = time (in minutes) of incubation and V = volume of cells (ml) used in the assay. Three independent experiments were performed in triplicate. Values are the mean ± S.E. *, p < 0.001 (two-tailed Student's t test versus YEpP_GDH3-LacZ at 24 h).

FIGURE 6.

Gdh3 protein is stable throughout all growth stages, whereas Gdh1 is subjected to stationary phase-specific degradation. A, immunoblot analysis of the Gdh1 and Gdh3 proteins in yeast cells at different growth stages. Yeast strains (WT, Δgdh1, Δgdh3, Δgdh1Δgdh3/YCp111, Δgdh1Δgdh3/Gdh1, Δgdh1Δgdh3/YCpGdh3) were grown in SCD, and samples were taken after 24 and 48 h of culture. Cell extracts were immunoblotted and probed with anti-Gdh1, anti-Gdh3 and anti-tubulin antibodies. B, immunoblot analysis of the FLAG-tagged Gdh1 and Gdh3 proteins in yeast cells carrying the promoter-swapped derivatives of GDH1 and GDH3 at different growth stages. Yeast strains (Δgdh1Δgdh3/YCpGdh1-FLAG, Δgdh1Δgdh3/YCpGdh3-FLAG, Δgdh1Δgdh3/YCpP_GDH1-Gdh3-FLAG, and Δgdh1Δgdh3/YCpP_GDH3-Gdh1-FLAG) were grown in SCD and samples were taken after 12, 24, 48, and 72 h of culture. Cell extracts were immunoblotted and probed with anti-FLAG and anti-tubulin antibodies.

FIGURE 7.

Ectopic expression of gdh3_P::gdh1_ORF cannot prevent stress-induced apoptosis in Δgdh1Δgdh3 cells, whereas ectopic expression of gdh1_P::gdh3_ORF confers stress resistance throughout all growth stages. Yeast strains (WT, Δgdh1, Δgdh3, Δgdh1Δgdh3, Δgdh1Δgdh3/YCpGDH1, Δgdh1Δgdh3/YCpGDH3, Δgdh1Δgdh3/YCpP_GDH1-GDH3, and Δgdh1Δgdh3/YCpP_GDH3-GDH1) grown in SCD for 24 or 48 h were exposed to oxidative stress (10 mm H₂O₂, 1 h). Surviving cells were evaluated by CFU assays. Values are the mean ± S.E. of three independent experiments. *, p < 0.001; **, p < 0.005 (two-tailed Student's t test versus WT).

FIGURE 8.

Gdh1, but not Gdh3, is subjected to stationary phase-specific degradation in which the Lys-426 residue in the Box420_Gdh1 region plays an essential role. A, amino acid sequences of the C-terminal regions of Gdh1 and Gdh3. The two isoenzymes share an extremely high degree of homology throughout their amino acid sequences except for in the Box420_Gdh1 and Box420_Gdh3 regions. Point mutations causing single amino acid substitutions in Gdh1 (K419A, K420A, K423A, and K426A) were introduced directly into YCpGdh1-FLAG. B, immunoblot analysis of the FLAG-tagged Gdh1 and its mutant derivatives. Yeast strains (BY4741) carrying YCpGdh1_K419A-FLAG, YCpGdh1_K420A-FLAG, YCpGdh1_K423A-FLAG, or YCpGdh1_K426A-FLAG were grown in SCD, and samples were taken after 12, 24, and 48 h of culture. Cell extracts were immunoblotted and probed with anti-FLAG and anti-tubulin antibodies.