Mutations affecting the enzymes involved in the utilization of 4‐aminobutyric acid as nitrogen source by the yeast Saccharomyces cerevisiae

Abstract

We present genetic evidence for the enzymes 4-aminobutyrate : 2-oxoglutarate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase [NAD(P)+] (EC 1.2.1.16) constituting the functional pathway for the utilization of 4-aminobutyric acid as a nitrogen source by Saccharomyces cerevisiae. We show that the pathway is induced by 4-aminobutyric acid and that the presence of the pathway enzymes probably requires the integrity of a positive control element.In Pseudomonas~uorescens [l ,2] GABA utilization occursStep (1) is catalyzed by a GABA transaminase and step (2) by a succinate semialdehyde dehydrogenase. These enzyme activities have also been detected in the yeasts Torulopsis utilis and Saccharomyces cerevisiae and the latter is able to use GABA as sole nitrogen source [3]. In order to establish this route genetically as the functional pathway in GABA utilization as a nitrogen source and to study the regulation of GABA catabolism, we have isolated and studied mutants unable to use GABA as sole source of nitrogen. via the following reactions: MATERIALS AND METHODSStrains used in our work were wild type C1278b and strain 2512c, derived from the former but which has lost the general amino-acid permease activity (Gap) as a result of a mutation in the gap gene [4].Media. The standard minimal medium M (without nitrogen source) is described in reference [5]. Nitrogen sources were added to a final concentration of 0.1 %.Genetic techniques. Mutagenesis by ethylmethane sulfonate was carried out according to [6], using strain 2512c. Standard genetic techniques are described in [7] and [8].Preparation of cell extracts. 500 ml cultures were inoculated and grown at 29 T. Exponentially growing cultures were harvested at a cell density of about 0.35 x lo7 cells/ ml, centrifuged, washed and resuspended in 2ml of 0.1 M potassium phosphate buffer at pH 8.2 containing 0.2 mM EDTA and 0.05 mM dithiothreitol. Extracts were made with a French press. Centrifuged extracts were passed through a Sephadex G-25 column pre-conditioned with the same buffer. Partial purification of succinate semialdehyde dehydrogenaseIn certain assays it was necessary to use GABA-transaminase-free succinate semialdehyde dehydrogenase. The purification was carried out in the following manner: cell extracts were treated with 1 YO neutralized protamine sulfate (0.15 g/l g protein) for 20 min at 0°C under gentle stirring. The homogenate was centrifuged and the supernatant solution was treated with increasing amounts of ammonium sulfate. Both GABA transaminase and SSA dehydrogenase were contained in the 60 -80% ammonium sulfate fraction. This fraction was dialyzed against 20 mM potassium phosphate buffer at pH 7.5 with protectors. The separation of the two enzymes was carried out on a 5 x 5 0 m m column (Pharmacia FPLC system) filled with Pharmacia polyanionic resin Polyanion SI-HR5/5 no. 17.0549.01 and eluted with a 20-200 mM potassium phosphate buffer, pH 7.5 with protectors. Under these conditions the GABA transaminase activity elutes with the exclus...

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

“…Previous studies have demonstrated that S. cerevisiae mutants lacking UGA1, UGA2 and UGA3 are incapable of growth on minimal medium with GABA as the sole nitrogen source, but grow normally on minimal medium with ammonium sulphate as the sole nitrogen source (Ramos et al, 1985;Coleman et al, 2001). To test the extent of interchangeable function of AtGABA-T and ScGABA-TKG in the cytosol and mitochondria, Δuga1 mutant was transformed with plasmid constructs containing AtGABA-T, with and without the mitochondrial targeting peptide or containing ScGABA-TKG or ScGABA-TKG with the mitochondrial targeting peptide.…”

“…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).…”

Changes in Transcription and Metabolism During the Early Stage of Replicative Cellular Senescence in Budding Yeast

“…7). This decrease can be explained by reduced mRNA levels of the GLT1 gene, which encodes glutamate synthase, the enzyme that catalyzes the synthesis of glutamate from glutamine and ␣-ketoglutarate (30), and by increased mRNA levels of the GAD1, UGA1, and UGA2 genes, which encode components of the glutamate degradation pathway (31,32). Alternatively, there could also be post-transcriptional changes in enzyme stability or activity that led to the change of amino acids as well as TCA cycle intermediates observed in metabolomics analysis.…”

A Novel γ-Hydroxybutyrate Dehydrogenase

“…mutant of uga2 is unable to use GABA as a nitrogen source and is defective in SSADH activity, suggesting that UGA2 is the structural gene for this enzyme (15). To demonstrate that SSADH activity is attributable to UGA2 and resides at the YBR006w locus, a ura3 uga2 mutant (strain 22641c) was transformed by a centromere-based plasmid library representing the genome of strain ⌺1278b (16).…”

“…Wild type yeast ⌺1278b and its mutant strain 22641c (15) were maintained on yeast minimal medium (18). Competent yeast cells were made and transformed with the Arabidopsis cDNA expression library (19).…”