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.