In this project, we tagged all proteins with genetically encoded redox biosensors to monitor their local redox environment inside living yeast cells. Specifically, we generated yeast libraries in which redox probes for H2O2 and thiol oxidation, respectively, were attached to all protein-coding open reading frames. The libraries were screened to identify proteins located within oxidizing microenvironments. Measuring the libraries under different conditions of nutrient availability allowed us to identify individual proteins and protein complexes undergoing metabolism-induced oxidation or reduction. In general, the redox state of protein-tethered probes differs substantially between individual proteins located within the same subcellular compartment. These protein context-dependent redox differences could not be observed with conventional probes and procedures. We identified both known and unknown intracellular hotspots of H2O2 generation. Changes in nutrient availability caused changes in the redox environment for highly distinct sets of proteins, which are often metabolic enzymes. We also observed that opposing redox changes occur in parallel within the same subcellular compartment. By crossing the fusion protein library with a deletion library, we identified oxidant-generating and consuming enzymes controlling context-specific protein oxidation and reduction. Taken together, these findings strongly support the notion that nanoscale redox domains exist on the level of individual protein complexes, which potentially are part of biomolecular condensates. These findings show that intracellular thiol oxidation generation is much more regulated, localized and functionally differentiated, i.e. protein-specific, than previously recognized. The project also involved the development of new genetically encoded redox probes providing specific advantages over previous probes. The tools developed in this project, i.e. libraries, strains, fusion constructs and probes, are available to the community upon request. The project also undertook step towards transferring these strategies to mammalian cells. The role of peroxiredoxins as transmitters of oxidation was further investigated and consolidated. Based on screening results, we studied individual redox-regulated proteins in more detail. One example is the sulfur transferase MPST which was identified as an enzyme mediating redox changes in other proteins. This involves a distinct kind of thiol modification, called persulfidation. Another key example is asparagine synthetase which was revealed to undergo thiol oxidation while undergoing condensation, suggesting a causal connection between localized redox regulation and phase separation. This is an example of how the project has opened new and unexpected research directions. Finally, the project contributed to the training of a new generation of redox biologists.
