Abstract
Redoxins are involved in maintenance of thiol redox homeostasis, but their exact sites of action are only partly known. We have applied a combined redox proteomics and transcriptomics experimental strategy to discover specific functions of two interacting redoxins: dually localized glutaredoxin 2 (Grx2p) and mitochondrial peroxiredoxin 1 (Prx1p). We have identified 139 proteins showing differential postranslational thiol redox modifications when the cells do not express Grx2p, Prx1p, or both and have mapped the precise cysteines involved in each case. Some of these modifications constitute functional switches that affect metabolic and signaling pathways as the primary effect, leading to gene transcription remodeling as the secondary adaptive effect as demonstrated by a parallel high throughput gene expression analysis. The results suggest that in the absence of Grx2p, the metabolic flow toward nucleotide and aromatic amino acid biosynthesis is slowed down by redox modification of the key enzymes Rpe1p (D-ribulose-5-phosphate 3-epimerase), Tkl1p (transketolase) and Aro4p (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase). The glycolytic mainstream is then diverted toward carbohydrate storage by induction of trehalose and glycogen biosynthesis genes. Porphyrin biosynthesis may also be compromised by inactivation of the redox-sensitive cytosolic enzymes Hem12p (uroporphyrinogen decarboxylase) and Sam1p (S-adenosyl methionine synthetase) and a battery of respiratory genes sensitive to low heme levels are induced. Genes of the Aft1p-dependent iron regulon were induced specifically in the absence of Prx1p despite optimal mitochondrial Fe-S biogenesis, suggesting dysfunction of the mitochondria to the cytosol signaling pathway. Strikingly, requirement of Grx2p for these events places dithiolic Grx2 in the framework of iron metabolism.
Highlights
Cysteine represents the most important redox-responsive amino acid within proteins, largely due to the wide range of oxidation states that sulfur can occupy
Because the eliminated redoxins are thought to be involved in antioxidant defense and a basal level of ROS is produced endogenously under normal growth conditions [1], we checked for possible oxidative alterations
Redox proteomics coupled to transcriptomics analyses has provided insights on unprecedented regulatory mechanisms of metabolic flow and gene expression triggered by posttranslational modification of specific proteins in cells growing under optimal conditions
Summary
Cysteine represents the most important redox-responsive amino acid within proteins, largely due to the wide range of oxidation states that sulfur can occupy. Due to the chemical reactivity and regulatory logic, redox modifications of Cys are not spontaneously reduced when redox homeostasis is restored but are regenerated with the help of the redoxins (e.g. glutaredoxins (Grx) and thioredoxins (Trx)) In this investigation, we have studied the yeast antioxidant defense enzymes Grx2p and Prx1p. We have carried out transcriptome expression profiling and have detected important and specific metabolic remodeling triggered by the absence of Grx2p, Prx1p, or both, including iron regulon, heme, amino acid, and cell wall biosynthesis These findings show how deep the functions of the studied redoxins reach into the metabolic network, opening unexpected new doors to the knowledge of cellular adaptive redox mechanisms. Their relevance is enhanced by the fact that they occur in cells without apparent phenotypical symptoms of stress
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