During oxidative stress, K63-linked polyubiquitin chains accumulate in the cell and modify a variety of proteins including ribosomes. Knowledge of the precise sites of K63 ubiquitin is key to understanding its function during the response to stress. To identify the sites of K63 ubiquitin, we developed a new mass-spectrometry based method that quantified >1,100 K63 ubiquitination sites in yeast responding to oxidative stress induced by H2O2. We determined that under stress, K63 ubiquitin modified proteins involved in several cellular functions including ion transport, protein trafficking, and translation. The most abundant ubiquitination sites localized to the head of the 40S subunit of the ribosome, modified assembled polysomes, and affected the binding of translation factors. The results suggested a new pathway of post-initiation control of translation during oxidative stress and illustrated the importance of high-resolution mapping of noncanonical ubiquitination events.

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Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Among its non-proteolytic functions, K63 ubiquitin accumulates in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63 ubiquitin conjugates accumulate because of redox inhibition of the deubiquitinating enzyme Ubp2, however, the role and regulation of ubiquitin conjugating enzymes in this pathway remained unclear. Here we found that the E2 Rad6 binds and modifies elongating ribosomes during oxidative stress. We elucidated a mechanism by which Rad6 and its human homolog UBE2A are redox-regulated by forming reversible disulfides with the E1 activating enzyme, Uba1. We further showed that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress. Finally, we showed that Rad6 is required to induce phosphorylation of the translation initiation factor eIF2α, providing a novel link for K63 ubiquitin, elongation stalling, and the integrated stress response.