Proteome-wide tagging with an H(2)O(2) biosensor reveals highly localized and dynamic redox microenvironments

Hydrogen peroxide (H(2)O(2)) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H(2)O(2) can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous “hotspots” of H(2)O(2) generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H(2)O(2) sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H(2)O(2)-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H(2)O(2) availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H(2)O(2) generation is much more localized and functionally differentiated than previously recognized.