GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

Abstract

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (a-ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45 C. Double and triple mutation constructs were compared for thermotolerance with the wild-type and single mutant strains. Although wild-type and all mutant strains were highly susceptible to brief heat stress at 50 C, a non-lethal 30 min at 40 C temperature pretreatment induced tolerance of the wild-type and all of the mutants to 50 C. The mutant strains collectively exhibited similar susceptibility at 45 C to the induced 50 C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild-type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N-tert-butyl-a-phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild-type. Measurement of intracellular GABA, glutamate and a-ketoglutarate during lethal heat exposure at 45 C showed higher levels of accumulation of GABA and a-ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from a-ketoglutarate to succinate during heat stress.

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria

“…All S. cerevisiae strains used in this study are listed in Table 1. The yeast strain W303-1A was obtained from American Type Culture Collection; Δuga1 was derived from W303-1A, as previously described (Cao et al, 2013). Yeast cells were grown at 30°C on either YPD medium or YNB minimal medium supplemented with required amino acids and various nitrogen sources.…”

“…Survival assays under lethal heat stress were performed as described previously (Cao et al, 2013), using strains listed in Table 2.…”

“…Intracellular ROS generated during heat stress were measured as described previously (Cao et al, 2013), using strains listed in Table 2 and the complementing strains outlined above.…”

“…Yeast mutants deficient in UGA1 or UGA2 are sensitive to oxidative stress (Coleman et al, 2001) and heat stress (Cao et al, 2013). Mutant strains lacking UGA1 or UGA2 are incapable of growth on minimal medium with GABA as the sole nitrogen source (Coleman et al, 2001;Cao et al, 2013). This growth defect was not observed during growth on ammonium sulphate as the sole nitrogen source (Ramos et al, 1985;Cao et al, 2013).…”

“…Mutant strains lacking UGA1 or UGA2 are incapable of growth on minimal medium with GABA as the sole nitrogen source (Coleman et al, 2001;Cao et al, 2013). This growth defect was not observed during growth on ammonium sulphate as the sole nitrogen source (Ramos et al, 1985;Cao et al, 2013). We have shown that yeast Δuga1 and Δuga2 deletion mutants are more sensitive to heat stress than wild-type growing either on GABA, ammonia or an amino-nitrogen rich medium, and the Δuga1 mutant is more sensitive to heat stress than the Δuga2 mutant (Cao et al, 2013).…”