COP9 Integrity Is Essential for Its Function Figure 5. CSN-5–mutated CSN efficiently prevents degradation of components of the SCFFWD-1 complex. (A–C) Western blot analyses with labeled antibodies showing degradation of Myc-Cul1 (A), Myc-SKP-1 (B), and FWD-1 (C) after addition of cycloheximide (10 mg/mL) in the wild- type, csn-5KO, and different CSN-5 complementation strains. Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FWD-1 antibody. (D–F) Densitometric analyses from four independent experiments showing the degradation of Myc-Cul1 (D), Myc-SKP-1 (E), and FWD-1 (F). doi:10.1371/journal.pgen.1002712.g005 key residues in the metal-binding motif (EXnHXHX10D) of the CSN-5 disrupted CSN deneddylation activity without interfering with the CSN assembly. We demonstrated that those mutant CSN-5s could almost restore the growth and conidiation defects of the csn-5KO strain. Furthermore, even without the deneddylation activity, the CSN partially maintained the stability of the SCFFWD-1 complex and partially restored the degradation of clock protein FRQ in vivo. Finally, we also showed that CSN containing mutant CSN-5 could efficiently prevent the degradation of the substrate receptors of CRLs. In addition, deletion of the CAND1 ortholog in N. crassa had little effect on the circadian rhythm of conidiation. Thus, our results suggest that maintenance of CRL stability by the CSN integrity is even more crucial in hyphal growth, conidial de- velopment, and circadian function in N. crassa. Both deneddylation activity and integrity of CSN are required for maintenance of CRL stability As the key regulator of CRLs, both deneddylation and main- tenance of CRL stability by the CSN occurs when the CSN binds to CRLs. Thus, it is difficult to distinguish which function is more important for maintaining the proper function of CRLs in eu- karyotes. To precisely determine the function of the CSN in maintaining the stability of CRLs, we sought to separate the two functional aspects of CSN from one other in N. crassa. In those csn- PLoS Genetics | www.plosgenetics.org 9 May 2012 | Volume 8 | Issue 5 | e1002712