Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae

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.

An Alanine Aminotransferase Is Required for Biofilm-Specific Resistance of Aspergillus fumigatus to Echinocandin Treatment

“…While alaA may play a minor role in alanine biosynthesis, as evidenced by the 10% growth reduction, it is likely that an alternative pathway is utilized as the primary means of alanine biosynthesis. Specifically, the A. fumigatus genome encodes a putative alanine-glyoxylate aminotransferase (Afu1g09470), which generates alanine from pyruvate using glycine as an amino-group donor, and recent evidence in Saccharomyces cerevisiae suggests fungal GABA aminotransferase can generate alanine using GABA as the amino-group donor ( 42 ). Moreover, under low-oxygen conditions, an alternative pathway for alanine catabolism is clearly present that promotes growth in the absence of AlaA.…”

“…Recent studies in Saccharomyces cerevisiae have determined Alt1, the AlaA homolog, has moonlighting functions, or functions independent of an enzyme’s catalytic activity ( 42 , 43 ). To test if AlaA was functioning through its catalytic activity, we generated a catalytically inactive allele of alaA by changing the conserved catalytic lysine residue at position 322 to an alanine and adding a C-terminal green fluorescent protein (GFP) tag ( Fig.…”

“…In mammals it has been demonstrated that the localization of an alanine aminotransferase is predictive of whether it primarily serves a catabolic or anabolic function ( 45 ). In mammals as well as S. cerevisiae , mitochondrially localized alanine aminotransferases have been demonstrated to have a primarily catabolic role and impact mitochondrial function ( 42 , 45 , 46 ). The growth characteristics of alaA -deficient strains suggest a primarily catabolic role for the enzyme ( Fig.…”

“…AlaA is one of two enzymes encoded by the A. fumigatus genome capable of generating alanine, the other being a putative alanine-glyoxylate aminotransferase (Afu1g09470). It was also recently demonstrated that the GatA (Afu5g06680) homolog in S. cerevisiae , Uga1, is capable of generating alanine using GABA as the amino-group donor, making GatA a potential third enzyme capable of generating alanine ( 42 ). Despite alaA being critical for numerous physiological aspects of the A. fumigatus biofilm, these other two pathways appear to be sufficient for maintaining basal levels of alanine for protein production, as genetic disruption of alaA did not yield alanine auxotrophy.…”

H2O2 Induces Major Phosphorylation Changes in Critical Regulators of Signal Transduction, Gene Expression, Metabolism and Developmental Networks in Aspergillus nidulans

“…Linking nitrogen and carbon metabolism, the γ-amino butyric acid (GABA) shunt, a conserved metabolic route that bypasses two steps of the Krebs cycle and generates NAD(P)H, has been involved in stress response and ROS mitigation, as GABA accumulates under different stress conditions [212][213][214]. GABA is synthesized from glutamate by glutamate decarboxylase and converted by GABA aminotransferase and succinate semialdehyde dehydrogenase to succinate and NAD(P)H, which are linked to the Krebs cycle and the mitochondrial electron transport chain.…”