K63 polyubiquitination is a new modulator of the oxidative stress response.

Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.