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. 2014 Mar 5;4(3):130232. doi: 10.1098/rsob.130232

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© 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 6. — Low TPI activity increases PPP metabolite load and causes oxidant resistance and heat sensitivity. (a) Concentrations of glycolytic and PPP metabolites in the human TPI_Ile170Val and TPI_Ile170Thr mutants relative to yeast expressing human wild-type TPI. PPP and glycolytic metabolites were quantified by LC-MS/MS. PPP metabolites are increased in the TPI mutants. Absolute values are given in the electronic supplementary material, figure S4. (b) TPI_Ile170Val and TPI_Ile170Thr mediate increased tolerance to oxidizing agents. Overnight cultures of the indicated yeast strains were diluted to an OD₆₀₀ = 3 and spotted onto SC^−His agar plates containing the oxidants. Glucose 6-phosphate dehydrogenase (Zwf1) encodes the enzyme for the first step in the non-reversible oxidative PPP shunt and produces NADPH. Its deletion abolishes the oxidant resistance phenotype of cells expressing TPI_Ile170Val or TPI_Ile170Thr. Sol3 and Sol4 catalyse the second step of the PPP and their deletion reduced oxidant resistance on H₂O₂; a protective effect of TPI_Ile170Val was detected in Δsol3 yeast while causing H₂O₂ sensitivity in Δsol4 yeast. (c) TPI mutants are heat-sensitive. Overnight cultures were diluted to an OD₆₀₀ = 0.2 and exposed, or not exposed, to 50°C for 5 min and growth was monitored for 25 h after heat exposure. The duration until growth was re-established (lag phase) was used as an inverse indicator for heat resistance. The lag phase was prolonged in the 50°C exposed TPI mutants compared with isogenic yeast cells expressing wild-type TPI.