Methionine sulphoxide reductases revisited: free methionine as a primary target of H₂O₂ stress in auxotrophic fission yeast

Abstract

SummaryAmino acid methionine can suffer reversible oxidation to sulphoxide and further irreversible over-oxidation to methionine sulphone. As part of the cellular antioxidant scavenging activities are the methionine sulphoxide reductases (Msrs), with a reported role in methionine sulphoxide reduction, both free and in proteins. Three families of Msrs have been described, but the fission yeast genome only includes one representative for two of these families: MsrA/ Mxr1 and MsrB/Mxr2. We have investigated their role in methionine reduction and H 2O2 sensitivity. We show here that MsrA/Mxr1 is able to reduce free oxidized methionine. Cells lacking each one of the genes are not significantly sensitive to different types of oxidative stresses, neither display altered life span. However, only when deletion of msrA/mxr1 is combined with deletion of met6, which confers methionine auxotrophy, the survival upon H2O2 stress decreases by 100-fold. In fact, cells lacking only Met6, and which therefore require addition of methionine to the growth media, are extremely sensitive to H2O2 stress. These and other evidences suggest that oxidation of free methionine is a primary target of peroxide toxicity in cells devoid of methionine biosynthetic capacity, and that an important role of Msrs is to recycle this oxidized free amino acid.

Global Fitness Profiling Identifies Arsenic and Cadmium Tolerance Mechanisms in Fission Yeast

“…This finding was unexpected because fission yeast was reported to lack the cystathionine β-synthase and cystathionine γ-lyase enzymes that convert methionine into cysteine ( Brzywczy and Paszewski 1994 ). However, we note that a previous study found that addition of cysteine or methionine to EMM2 was sufficient to rescue growth of met16 Δ cells, which lack a phosphoadenosine phosphosulfate reductase required for sulfate assimilation ( Garcia-Santamarina et al 2013 ). Furthermore we found that addition of methionine partially suppressed the growth defect of sua1 Δ cells ( Figure 4B ).…”

Increasing extracellular H2O2 produces a bi-phasic response in intracellular H2O2, with peroxiredoxin hyperoxidation only triggered once the cellular H2O2-buffering capacity is overwhelmed

“…Our previous studies have established that treatment with 0.2 mM H 2 O 2 causes the majority of Trx1 and Txl1 to become rapidly oxidized [11] , [12] . As broad specificity oxidoreductases, thioredoxin family proteins are important cofactors for many enzymes and for the reduction of other oxidized proteins that may also impact on intracellular H 2 O 2 levels [12] , [29] , [30] . Although, Tpx1 and Gpx1 were not important for buffering low levels of H 2 O 2 ( Fig.…”

Monitoring cytosolic H2O2 fluctuations arising from altered plasma membrane gradients or from mitochondrial activity

“…Strains SG61 and EP16 were obtained by insertion of an ura4 cassette in the trx1 and sty1 loci, respectively, of strain PN513 ( h − ura4-D18 leu1-32; our laboratory stock ) . Strain SG260 was obtained by mating MJ15 47 ( h + trx1::kanMX6 ) with SG257 48 ( h − trx3::natMX6 ). Strain EP160 was obtained by crossing CN513 49 ( h − ctt1::ura4 ade6-M216 leu1-32 ura4-D18 ) with JA368 ( h + ura4-D18 leu1-32; our laboratory stock) and selecting clones in MM supplemented with leucine but lacking uracile.…”