Expression of the H 2 O 2 Biosensor roGFP-Tpx1.C160S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H 2 O 2 Levels, Transmembrane Gradients, and Response to Metals

Intracellular hydrogen peroxide (H O ) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H O have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H O biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H O is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H O gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H O fluxes are very similar in different model organisms.