Involvement of GDH3-encoded NADP+-dependent Glutamate Dehydrogenase in Yeast Cell Resistance to Stress-induced Apoptosis in Stationary Phase Cells

Yong Joo Lee,Kyung Jin Kim,Hong Yong Kang,Hye-Rim Kim,Pil Jae Maeng

doi:10.1074/jbc.m112.375360

Yong Joo Lee, Kyung Jin Kim + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m112.375360

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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.

Highlights

Gdh1 and Gdh3 are glutamate-synthesizing isofunctional NADP-Glutamate dehydrogenase (GDH) in S. cerevisiae
Deletion of GDH3 Causes Increased Sensitivity to Thermal and Oxidative Stress in Stationary Phase Cells and Accelerates Chronological Aging—To address the differential roles of the two NADP-GDH, Gdh1 and Gdh3, we examined the resistance against heat and oxidative stress of NADP-GDH yeast mutants and their derivatives that harbor either ectopically expressed GDH1 or GDH3 genes
These results indicate that deletion of GDH3 causes increased sensitivity to both heat and oxidative stress in stationary phase cells, whereas impairment of GDH1 leads to no significant stress-sensitive phenotype

Summary

Background

Results: Stationary phase-specific GDH3 expression and degradation of Gdh were responsible for the Gdh3-dependent glutamate supply and resistance to stress-induced apoptosis in stationary phase. The NADϩ-dependent GDH (NAD-GDH; Gdh2) encoded by GDH2 catalyzes reversible oxidative deamination of glutamate to ␣-ketoglutarate and ammonia [15]. An alternative pathway for glutamate biosynthesis is accomplished by the combined activities of GLN1encoded glutamine synthetase and the GLT1-encoded glutamate synthase [17, 18] Both GDH3 and GLT1 are dispensable for yeast growth in minimal glucose medium containing ammonia as a sole nitrogen source, indicating that Gdh is the primary enzyme for glutamate biosynthesis [16]. Our results indicate that Gdh, but not Gdh, is responsible for tolerance to stress-induced apoptosis in stationary phase cells, as there is stationary phase-specific expression of GDH3 and degradation of Gdh

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION