Author Summary Cullin-RING E3 ubiquitin ligases (CRLs) play important roles in regulating a wide range of processes, such as signal transduction, transcription, cell cycle progression, circadian rhythm, and development, via the ubiquitin-proteasome pathway. The activity and stability of CRLs is precisely controlled by the COP9 signalosome (CSN), an evolution- arily conserved multisubunit protein complex. Under the control of the CSN, CRL activity can be either downreg- ulated via cleavage of NEDD8 (an ubiquitin-like protein) from cullin proteins (deneddylation) or preserved by maintaining the stability of CRL components. We gener- ated point mutations of key residues in the JAMM domain of the CSN-5 subunit to disrupt CSN deneddylation activity, thereby creating a series of mutants containing the intact CSN complex but lacking deneddylation activity. Surprisingly, hyphal growth, conidial development, circa- dian rhythm, and stability of the SCFFWD-1 complex in these CSN-5 point mutants were comparable to that observed in wild-type N. crassa. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, deletion of the N. crassa ortholog of CAND1 (cullin-associated NEDD8- dissociated protein 1) had little effect on conidial development and the circadian clock. Our results suggest that the integrity of the CSN is important for growth and development in N. crassa. mutation in JAMM domain results in a block in fruiting body formation at the primordial stage, with a few other observed phenotypic changes, such as light-dependent signaling [22,23]. Although deneddylation is a major activity of the CSN, it alone cannot explain all of the phenomena described above. These ob- servations raise the possibility that the CSN may have other func- tional activities in addition to its deneddylation activity. Recent genetic evidence suggest that the CSN has one addi- tional major function: it controls the stability of CRL ubiquitin ligases in vivo by mediating assembly/disassembly of CRL com- plexes and by protecting substrate receptors in CRLs from de- gradation [3,24,25,26,27,28]. A recent structural and biochemical study showed that the protective effect of the CSN on DDB2 and CSA autoubiquitination in CRL4 complexes does not require CSN-5–mediated deneddylation activity [29]. However, both of the CSN activities occur when CSN associates with cullins in CRL E3 complexes. Furthermore, there is also a tight correlation be- tween CSN deneddylation activity and the ability of the complex to modulate the stability of CRLs [3]. Thus, it is difficult to deter- mine which function is more important for growth and devel- opment through regulation of CRL activity, or how these two functions cooperate with each other in regulating CRL dynamicity in eukaryotes. In A. thaliana, the MPN (Mpr-Pad1-N-terminal domain) subunits CSN-5 and CSN-6 are essential for the struc- tural integrity of the CSN holocomplex [12]. Several studies have shown that point mutations in the JAMM metal-binding site of CSN-5 do not interfere with the proper assembly of CSN complexes in S. pombe, A. thaliana, and A. nidulans [11,21,23]. In N. crassa, CSN-5 is not an essential gene; the deletion mutant can survive, and displays obvious growth and developmental defects, making it an excellent model system for investigating the dis- tinctions between the deneddylation and CRL complex assembly/ disassembly functions of the CSN [16]. The CSN takes part in a wide range of cellular and devel- opmental processes in N. crassa, including hyphal growth, conidial formation, light and temperature responses, and circadian clock COP9 Integrity Is Essential for Its Function function [16,24]. To further investigate its biological function in vivo, we created a series of point mutations in the JAMM metal- binding motif of the CSN-5 subunit to disrupt the deneddylation activity of the CSN complex. In those mutant strains, the integrity of the CSN and its interactions with Cul1 and Cul4 were not affected. Surprisingly, mutated CSN-5 almost retained the ability to restore the phenotypic defects of a csn-5KO strain and partially maintained the stability of the SCFFWD-1 complex, which was able to carry out degradation of the clock protein FREQUENCY (FRQ) in vivo. Moreover, the stability of four other substrate receptors of CRLs can be efficiently restored by the CSN con- taining mutant CSN-5. However, deletion of the CAND1ortholog in N. crassa had little effect on conidiation circadian rhythm and the degradation of FRQ. Our results suggest that the integrity of CSN plays major roles in hyphal growth, conidial development, and circadian function in N. crassa. Results Mutations within the CSN-5 JAMM metal-binding site abolish CSN–mediated deneddylation of cullins The N. crassa genome encodes seven COP9 signalosome sub- units (CSN-1–CSN-7) [16,24]. Several studies have shown that the JAMM metal-binding sites in the MPN domain of CSN-5 are required for metalloprotease activity in the CSN [11,21,23]. When the CSN-5 protein sequence was used in a BLAST search against protein databases, a highly conserved MPN domain in the N. crassa CSN-5 subunit was identified. As shown in Figure 1A, three con- served residues corresponding to His127, His129, and Asp140 lie within the putative metal-binding motif (EXnHXHX10D) of the N. crassa CSN-5 JAMM domain. To determine whether these conserved residues form the metalloprotease-like active site of JAMM, we used the JAMM domain of Archaeoglobus fulgidus as a template to generate the tertiary structure of N. crassa CSN-5 [30]. Because of the low similarity between these two JAMM domains, the generated structure was poor. Thus, we instructed SWISS- MODEL to automatically select a template protein for generating the structure of N. crassa CSN-5 [31]. SWISS-MODEL selected the pre-mRNA splicing factor Prp8 as template (Protein Data Bank [PDB] accession number 2P8R) for N. crassa CSN-5. The functional sites were mapped into predicted structure according to the structural alignment with AfJAMM (PDB accession number 1R5X). As shown in Figure 1B, His127, His129, and Asp140 within EXnHXHX10D of the N. crassa CSN-5 JAMM correspond- ed to the putative metal-binding motif as metalloprotease-like active site in AfJAMM [30,32]. To confirm the contribution of CSN-5 to CSN-mediated deneddylation of cullins, we mutated these three highly conserved amino acids (H127A, H129A or D140N) using site-directed mu- tagenesis. We then introduced quinic acid (QA)–inducible Myc- tagged wild-type CSN-5 or one of the three mutant CSN-5 con- structs into a csn-5KO strain expressing Myc-Cul1 protein. As shown in Figure 1C, Myc-CSN-5, Myc-CSN-5H127A, Myc-CSN- 5H129A, and Myc-CSN-5D140N were expressed in the csn-5KO strains in the presence of QA. Expression of Myc-tagged wild-type CSN-5 in the csn-5KO strain resulted in a decrease in hyperneddy- lated Cul1 to the level of the wild-type strain (Figure 1C), in- dicating that the Myc-tagged CSN-5 protein was functional for CSN deneddylation activity. In contrast, expression of mutant CSN-5 (H127A, H129A, or D140N) failed to decrease the hyper- neddylated Cul1 in the csn-5KO strain (Figure 1C). Similarly, hyperneddylation of Cul3 (Figure 1D) and Cul4 (Figure 1E) in the csn-5KO strain was rescued by expressing the Myc-tagged wild- type CSN-5, but not by any of the mutated Myc-CSN-5s. This PLoS Genetics | www.plosgenetics.org 2 May 2012 | Volume 8 | Issue 5 | e1002712