Biosynthetic and Iron Metabolism Is Regulated by Thiol Proteome Changes Dependent on Glutaredoxin-2 and Mitochondrial Peroxiredoxin-1 in Saccharomyces cerevisiae

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.

Thiol Redox Sensitivity of Two Key Enzymes of Heme Biosynthesis and Pentose Phosphate Pathways: Uroporphyrinogen Decarboxylase and Transketolase

“…Proteomics has developed into a powerful tool not only for the detection and identification of proteins, but recent advances have allowed the identification and quantification of posttranslational modifications on specific residues of redox proteins [45]. In a previous shotgun proteomic screen to detect potential Grx2p protein targets two proteins, Hem12p and Tkl1p, were identified as containing reversibly oxidized Cys residues in a ΔGrx2 strain but not in the corresponding WT [18]. Both proteins function at critical junctions in iron regulation and biosynthetic metabolism, respectively.…”

“…We confirmed that reversible redox modification of specific Cys residues of key glycolytic proteins allows a redirection of energy metabolites towards the PPP for NADPH production and antioxidant defense, as described earlier for glyceraldehyde-3-phosphate dehydrogenase [15–17]. In a subsequent redox shotgun proteomic screen using wild type (WT) yeast and a strain lacking the oxidoreductase glutaredoxin 2 (Grx2p), uroporphyrinogen decarboxylase (Hem12p) and transketolase (Tkl1p) were detected as containing reversibly oxidized Cys residues only in the strain lacking Grx2p, indicating they are involved in thiol disulfide exchange [18]. In the approach, proteins were tryptic digested and peptides containing reversibly oxidized Cys residues were affinity purified and analysed by MS/MS.…”

“…Shaking was set at 180 rpm and 1.5 L conical flasks containing 250 mL of media were used. Cells from three independent biological replicates were harvested and the reversibly oxidized Cys containing peptides were isolated and identified as described previously [14, 18]. Briefly, in cell extracts free thiols were initially blocked with N-ethylmaleimide (NEM), and reversibly oxidized thiols were then reduced and subsequently labelled with thiol specific, biotin-HPDP.…”

“…Initial mass spectrometry analysis was performed as before [18]. Samples containing the affinity purified peptides were subjected to complete evaporation in a Speedvac centrifuge and then suspended in 20  μ L of 5% acetonitrile/2% formic acid.…”

S-Bacillithiolation Protects Conserved and Essential Proteins Against Hypochlorite Stress inFirmicutesBacteria

“…Notably, the DAHP synthase Aro4p of yeast cells was found as a target for the Grx2 and showed increased thiol redox ratios at Cys76 and Cys244 in the grx2 mutant (35). The essential IMP dehydrogenase GuaB and the pyrophosphatase PpaC have been previously found to be Sglutathionylated in human T-lymphocytes and endothelial cells under diamide stress conditions (11,30).…”