Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis.

The COP9 signalosome (CSN) is a highly conserved eukaryotic protein complex which regulates the cullin RING family of ubiquitin ligases and carries out a deneddylase activity that resides in subunit 5 (CSN5). Whereas CSN activity is essential for the development of higher eukaryotes, several unicellular fungi including the budding yeast Saccharomyces cerevisiae can survive without a functional CSN. Nevertheless, the budding yeast CSN is biochemically active and deletion mutants of each of its subunits exhibit deficiency in cullins deneddylation, although the biological context of this activity is still unknown in this organism. To further characterize CSN function in budding yeast, we present here a transcriptomic and proteomic analysis of a S. cerevisiae strain deleted in the CSN5/RRI1 gene (hereafter referred to as CSN5), coding for the only canonical subunit of the complex. We show that Csn5 is involved in modulation of the genes controlling amino acid and lipid metabolism and especially ergosterol biosynthesis. These alterations in gene expression correlate with the lower ergosterol levels and increased intracellular zinc content which we observed in csn5 null mutant cells. We show that some of these regulatory effects of Csn5, in particular the control of isoprenoid biosynthesis, are conserved through evolution, since similar transcriptomic and/or proteomic effects of csn5 mutation were previously observed in other eukaryotic organisms such as Aspergillus nidulans, Arabidopsis thaliana and Drosophila melanogaster. Our results suggest that the diverged budding yeast CSN is more conserved than was previously thought.

The distinction between pleiotrotic and moonlighting roles of proteins is challenging; however, this distinction may be clearer when it comes to multiprotein complexes. Two examples are the proteasome lid and the COP9 signalosome (CSN), which are twin enzymes with 1:1 paralogy between subunits. In each complex, one out of eight subunits harbours a JAMM/MPN⁺ metalloprotease motif. This motif contributes the canonical activity of each complex: hydrolysis of covalently attached ubiquitin by Rpn11 in the proteasome lid and hydrolysis of ubiquitin-related 1 (Rub1/Nedd8) from Cullins by Csn5 in the CSN. In both complexes, executing this activity suggests pleiotropic effects and requires an assembled full complex. However, beyond canonical functions, both Rpn11 and Csn5 are involved in additional unique, complex-independent functions, herein referred to as moonlighting activities.

The class of Cullin–RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8–Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1–CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53–Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes.