Abstract
The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.
Highlights
The γ-aminobutyric acid (GABA) shunt is a closed-loop process, which produces and consumes GABA (Figure 1A)
We found that when grown on ammonium–glucose, the wild-type strain generated an nicotinamide adenine dinucleotide phosphate (NADPH) pool, which increased twofold in the presence of GABA, whereas in the presence of both, GABA and alanine, a similar pool to that found on ammonium was observed, suggesting GABA shunt repression on alanine (Figure 1D)
An uga1 mutant strain grown on ammonium or in the presence of GABA or GABA + alanine showed a basal NADPH level similar to that found in the wild-type strain grown on ammonium, indicating that alanine accumulation could result in repression of the GABA shunt function
Summary
The γ-aminobutyric acid (GABA) shunt is a closed-loop process, which produces and consumes GABA (Figure 1A). We found that when S. cerevisiae is grown on GABA as the sole nitrogen source, ALT1 expression was induced, suggesting GABA-dependent alanine biosynthesis, as observed in A. thaliana (Cao et al, 2013b). We found that (i) besides α-ketoglutarate, Uga alanine transaminase uses pyruvate, generating alanine; (ii) on GABA as the sole nitrogen source, an alt mutant shows impaired growth and accumulates alanine; (iii) in the presence of alanine, expression of the genes whose products form part of the GABA shunt (UGA1, UGA2, and GAD1) is repressed and NADPH production is decreased as compared to that found in the wild-type strain grown on ammonium; and (iv) an alt mutant grown on either ammonium or GABA is respiratory deficient, and null expression of the mitochondrially encoded COX2, COX3, and ATP6 genes is observed. It can be proposed that Alt moonlighting function could protect cells from stress by safeguarding mtDNA, provoking expression of mitochondrially encoded genes enhancing respiratory metabolism
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