Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes

In this study, we set out to investigate the key role of Rad6 in regulating cellular response to stress in budding yeast as part of the RTU. Rad6 is small (20 kDa), highly conserved, and a multifunctional E2 involved in DNA repair and in the ubiquitination of substrates for the N-end rule pathway (Dohmen et al., 1991; Hoege et al., 2002). First, we found that Rad6 promotes K63 ubiquitination of ribosomes in vitro and in vivo. Using cryo-EM and molecular modeling, we identified key protein features important for enzyme-substrate association. We also elucidated a negative-feedback mechanism by which Rad6 is redox inhibited in a reversible fashion by forming a disulfide with the E1 ubiquitin-activating enzyme (Uba1), thus limiting the amount of K63-ubiquitin modification. We further showed that Rad6 function in the K63 ubiquitination of ribosomes can be complemented by its human orthologue UBE2A, which can also be redox regulated by disulfide formation in human cells. To cope with the harms of oxidative stress, cells must orchestrate a global inhibition of translation while increasing the synthesis of antioxidant, repair, and stress-related proteins. Here, we showed that Rad6 is necessary for reprogramming translation and for adequate synthesis of antioxidant proteins, suggesting a central role for this E2 enzyme in protein synthesis during the stress response.

 

Cell Reports, 2022.

Abstract

Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed that K63-linked polyubiquitinated conjugates heavily accumulate in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated conjugates accumulate mostly because of redox inhibition of the deubiquitinating enzyme Ubp2; however, the role and regulation of ubiquitin-conjugating enzymes (E2) in this pathway remained unclear. Here, we show that the E2 Rad6 associates and modifies ribosomes during stress. We further demonstrate that Rad6 and its human homolog UBE2A are redox regulated by forming a reversible disulfide with the E1 ubiquitin-activating enzyme (Uba1). This redox regulation is part of a negative feedback regulation, which controls the levels of K63 ubiquitination under stress. Finally, we show that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress, thus providing an additional physiological role for this multifunctional enzyme.