Thermosensitive phenotype of yeast mutant lacking thioredoxin peroxidase

A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25-kDa protein acts as a peroxidase but requires a NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was thus named thioredoxin peroxidase (TPx). The protective role of TPx in the cellular defense against heat shock (42 or 48 degreesC), which may increase oxidative stress in cells sufficiently to form reactive oxygen species harmful to cellular function, was investigated in a wild-type and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. Upon exposure under aerobic conditions to heat shock there was a distinct difference between these two strains in growth kinetics and viability. The wild-type strain was more resistant to killing by heat than the mutant strain. In addition, the expression of the tsa gene in Escherichia coli caused an increase in thermotolerance. The expression of the tsa gene increased under heat shock; however, modulation of activities of other antioxidant enzymes, such as catalase, superoxide dismutase, glucose 6-phosphate dehydrogenase, and glutathione reductase as well as the total glutathione level, remained unaltered in both strains under heat shock. The induction of heat shock protein HSP104 was not significantly different in the two strains under heat shock. The results indicate that the lack of TPx expression may be solely responsible for the thermosensitive phenotype of tsa mutant cells. When the oxidation of 2', 7'-dichlorofluorescin was used to examine hydroperoxide production in yeast cells, tsa mutant cells showed a 2.5- to 3.5-fold increase in fluorescence upon exposure to heat stress compared to wild-type cells. The antioxidant, N-acetylcysteine, prevented intracellular peroxide formation in response to heat shock. The carbonyl content of extract, the indicative marker of oxidative damage to protein, from tsa mutant cells was higher than that from wild-type cells. These results suggest that TPx may play a direct role in cellular defense against heat shock, presumably functioning as an antioxidant protein.