Redox homeostasis as a basic requirement for cullin NEDDylation

Cullin–RING ligases (CRLs) are inclusive class of modular ubiquitin E3 ligases that mediate ~20% of protein ubiquitination. CRLs control almost all cellular aspects, including the cell cycle, transcription, the stress response, self-incompatibility, pathogen defence, DNA damage repair and survival. Hence, it is surprising that all CRLs are activated through a single mechanism: covalent modification of their cullin backbone subunit by a small ubiquitin-like polypeptide, named NEDD8/Rub1 (a.k.a. NEDDylation).

s.cerevisiae is an important model organism for the study of dynamic of cullin NEDDylation cycles, since the mechanism is highly conserved in this organism, however, neither require for the turnover of substrates nor for viability. We found that the S. cerevisiae NEDD8/Rub1 pathway is sensitive to reactive oxygen species (ROS), generated in the mitochondria during respiration. Redox homeostasis breaks down naturally in yeast during the diauxic shift, a physiological transition from primarily anaerobic glycolysis to mitochondrial respiration accompanied by the production of ROS. We were able to recognize the high ROS at the diauxic shift as a checkpoint of CRLs NEDDylation, including a reversible loss of thioester formation between NEDD8 and the cognate E2 enzyme. We are currently deciphering whether inactivation of the NEDDylation cascade involves direct modification of enzymes residues by ROS, or through another mechanism, upstream to these enzymes. One way or another, our study adds the NEDDylation cascade to the list of cellular pathways regulated by redox homeostasis.