Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress

Abstract

We have isolated several mutants of Saccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide. Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway. Allelism of the pos10 mutation (POS for peroxide sensitivity) to the zwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously. The second mutation, pos18, was complemented by transformation with a yeast genomic library. The open reading frame of the isolated gene encodes 238 amino acids. No detectable ribulose 5-phosphate epimerase activity was found in the pos18 mutant, suggesting that the corresponding structural gene is affected in this mutant. For that reason the gene was renamed RPE1 (for ribulose 5-phosphate epimerase). RPE1 was localized to chromosome X. The predicted protein has a molecular mass of 25966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82. Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases of Escherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus and Solanum tuberosum. We have characterized RPE1 by testing enzyme activities in rpe1 deletion mutants and in strains that overexpress RPE1, and compared the hydrogen peroxide sensitivity of rpe1 mutants to that of other mutants in the pentose phosphate pathway. Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.

Dissecting the roles of osmolyte accumulation during stress

“…This was proposed to be important in plant antioxidant defence mechanisms during stress (Babiychuk et al 1995). Consistent with the need for NADPH to maintain glutathione (GSH) and ascorbate in the reduced state, it is known that the oxidative pentose phosphate pathway is an important component of antioxidative defence mechanisms (Juhnke et al 1996). Several lines of evidence indicate an important role for proline synthesis in potentiating pentose phosphate pathway activity Fig.…”

Isoenzyme replacement of glucose-6-phosphate dehydrogenase in the cytosol improves stress tolerance in plants

“…Defective yeast mutants were found to be susceptible to oxidative stress (8), and a role for G6PDH in supplying NADPH for free-radical scavenging has also been observed in stressed mammalian cells (9). In higher plants, the OPPP is a major source of reduction power (NADPH) required for anabolic biosyntheses and assimilatory processes in the cytosol, as well as in plastids, providing key intermediates for the shikimate pathway and nucleic acid biosynthesis (10,11).…”

Stress-Induced GSK3 Regulates the Redox Stress Response by Phosphorylating Glucose-6-Phosphate Dehydrogenase in Arabidopsis

“…In animals and yeast, the pentose phosphate pathway is the main source of NADPH for ROS removal (Pandolfi et al, 1995;Juhnke et al, 1996); however, in plants, the role of the OPPP during salt stress is unclear. Under normal growth conditions, photosynthesis supplies reducing equivalents; however, under stress conditions, when photosynthesis may be impaired, the OPPP might deliver reducing power for maintenance of the cellular redox balance.…”

“…In addition, exogenous stresses such as pathogen infection, certain drugs, or ingestion of fava beans (Vicia faba) can trigger hemolytic anemia in humans with G6PD deficiency, which is the most common enzymopathy. Similarly, yeast mutants defective in G6PD show an increased susceptibility to oxidative stress and fail to induce adaptation (Juhnke et al, 1996;Izawa et al, 1998). In plants, G6PD activity is present in the plastids and cytosol (Debnam and Emes, 1999).…”