The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCFFWD-1 complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes.
Protein degradation is precisely controlled in cells. The ubiquitin-mediated protein degradation pathway is highly conserved in eukaryotes, and the activity of ubiquitin ligases is regulated by the Cop9 signalosome (CSN), a multisubunit complex that is evolutionarily conserved from yeast to humans. Determining how the CSN complex functions biologically is crucial for understanding regulation of the ubiquitin-mediated protein degradation pathway. The filamentous fungus N. crassa is commonly used to study protein degradation. Its CSN complex contains seven subunits (CSN-1 to CSN-7). In this study, we generated knockout mutants of individual CSN subunits and observed the phenotypes of each mutant. We demonstrated that six of the seven CSN subunits were essential for cleaving the ubiquitin-like protein Nedd8 from cullin proteins (which act as scaffolds for ubiquitin ligases). In contrast, loss of the CSN-3 subunit had no effect on cullin neddylation. We also found that each CSN subunit had distinct roles in maintaining the stability of key components of cullin-based ubiquitin ligases. In summary, we systematically investigated the unequal contributions of CSN subunits to deneddylation and the maintenance of cullin-based ubiquitin ligases in N. crassa. Our work establishes a framework for understanding the function of CSN subunits in other eukaryotes.