Functional Genomics of Gaba Metabolism in Yeast Thermotolerance
Type of DegreeDissertation
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?-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid which accumulates rapidly in response to diverse environmental stresses. The GABA shunt is a pathway involving three enzymes, glutamate decarboxylase (GAD, encoded by GAD1), GABA aminotransferase (GABA-T, encoded by UGA1), and succinate semialdehyde dehydrogenase (SSADH, encoded by UGA2). These three enzymes acting in concert convert glutamate to succinate. GABA specific permease (encoded by UGA4) mediates the transportation of GABA into cells. The GABA specific transcription factor (encoded by UGA3) regulates the expression of GABA genes (UGA1, UGA2 and UGA4). We have constructed deletion mutants of each of these genes in yeast and have found that mutants of GAD, GABA-T and SSADH are more susceptible to stress induced by lethal temperature (45°C) than wild type yeast cells. Additionally, set of the combinations of double and triple mutants were examined. With a pretreatment at 40°C (a non-lethal temperature) for 30 min, the mutants retained susceptibility to stress at 50°C compared to the wild type. The levels of accumulated ROS were correlated to the susceptibility of heat stress. In addition, in the uga1 and uga2 mutants, GABA and a-ketoglutarate accumulated markedly higher compared to the wild-type, while glutamate accumulated at higher levels in gad1 mutant. Deletion mutations of UGA3 and UGA4 grown showed heat tolerant to 45ºC with overexpression of antioxidant genes superoxide dismutase compared to the wild-type. However, ?uga3 mutant strain grown in minimal-GABA medium showed heat sensitive phenotype while ?uga4 maintained heat tolerance. RT-PCR analysis showed that the expression of all GABA shunt genes and UGA3 and UGA4 genes were GABA inducible and were also up-regulated by lethal heat at 45ºC. In addition, acidic pH in the growth medium induced the expression of UGA1, UGA2 and UGA4 but not that of GAD1 and UGA3. Under heat stress, deletion of UGA3 suppressed the expression of UGA1 and UGA4 but not on GAD1 and UGA2, while deletion of UGA4 did not affect the expression of all GABA shunt genes and UGA3. Additionally, the antioxidant genes superoxide dismutase (encoded by SOD1 and SOD2) were found to be gradually induced by heat in the wild-type strain but overexpressed in the ?uga3 and ?uga4 mutant strains. Bioinformatic programme ""TargetP and pSORT' predicts that GABA transaminase from Arabidopsis is localized in mitochondria with a 54 nucleotide mitochondrial transit peptide sequence, yeast GABA transaminase is localized in cytosol. We constructed vectors expressing ScGABA-TKG and AtGABA-TP in both yeast cytosol and mitochondria to complement yeast GABA transaminase mutant ?uga1 and succinate semialdehyde dehydrogenase mutant ?uga2 phenotypes: GABA growth defect, thermo sensitivity and heat induced production of reactive oxygen species (ROS). Our studies revealed that plant AtGABA-TP is functionally interchangeable with yeast ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS whether located in mitochondria or cytosol in yeast. However, yeast GABA-TKG, whether located in mitochondria or in the cytosol, displayed much stronger effect. The yeast succinic semialdehyde dehydrogenase gene (SSADH; EC 220.127.116.11) was cloned and overexpressed in E. coli., and kinetically characterized. It has a molecular mass of subunit around 54 kDa, the purified enzyme has a tetramer molecular mass of 200 kDa. The recombinant protein was highly specific for succinate semialdehyde, can use both NAD+ and NADP+ as a cofactor but with higher affinity to NAD+. The enzyme activity can be inhibited by substrate SSA, product NADH and adenine nucleotides AMP, ADP and ATP.