Protection of substrate receptors by the CSN has been described for the CRLs in vivo [24,25,26,27,28]. We found that CSN with mutated CSN-5 had a contribution to the stabilities of five re- ceptor proteins of CRLs in vivo. These results provide evidence for the idea that the abundance of adaptor modules (rather than cycles of neddylation/deneddylation and CAND1 binding) drives CRL network organization [51]. This possibility is supported by our genetic observations that the csn-5H127A, csn-5H129A, and csn-5D140N strains exhibited normal growth and conidiation phenotypes. The integrity of CSN was maintained in these JAMM mutation com- plementation strains, thus it can serve as a platform to recruit other proteins for regulating the activities of CRLs, such as the recruit- ment of UBP12 in yeast, as well as USP15 in human [14,53]. In addition, a non-catalytic CSN itself may stabilize the substrate receptors of CRLs. A very recent study has shown that the protective effect of the CSN on DDB2 and CSA autoubiquitina- tion is independent of CSN-5 mediated deneddylation in vitro [29]. These results suggest that the partial rescue of stability of substrate receptors by the catalytically dead CSN is mainly dependent on its protective effect. Therefore, the stability of cullins and some sub- strate receptors of CRLs are dependent on both deneddylation activity and integrity of the CSN in N. crassa. Maintaining stability of the SCFFWD-1 complex is the key process in circadian rhythm regulation The csn-5KO strain exhibits abnormal conidiation rhythms in DD, which cannot be entrained by either LD or temperature cycles, indicating that light and temperature regulation of the conidiation process is impaired in this mutant [16]. We found that degradation of the clock protein FRQ is impaired in the csn-5KO strain, especially when protein synthesis is completely blocked. To further characterize the molecular mechanism of how the CSN regulates the conidiation rhythm, we focused on the SCFFWD-1 ubiquitin ligase, which controls the N. crassa circadian rhythm by ubiquitinating FRQ [36]. Our results demonstrated that defective FRQ degradation in the csn-5KO strain is due to the drastically reduced stability and levels of FWD-1 and Cul1 proteins in the SCFFWD-1 complex. Ectopic expression of mutant CSN-5 without deneddylation activity restored the defects of growth and coni- diation in the csn-5KO strain, and almost restored the defects of the circadian conidiation rhythm in DD and FRQ degradation in the csn-5KO strain. Our data further showed that the low levels of FWD-1 in the csn-5KO strain were dramatically increased after expression of each of the CSN-5 proteins with point mutations in the JAMM metal-binding site, however, the increased stability and levels of the components in the SCFFWD-1 ubiquitin ligase are not enough to fully restore the degradation of FRQ to wild-type level, indicating that regulation of FRQ degradation plays a key role in maintaining the precise period length of conidiation rhythm in N. crassa. This is further supported by the finding that accelerated degradation of c-Jun in HeLa cells in which CSN-5 is down- regulated can be rescued equally by over-expression of the deneddylation mutant CSN-5D151N or wild-type CSN-5; how- ever, accelerated c-Jun degradation is not rescued in CSN-1– or CSN-3–downregulated cells by over-expression of wild-type CSN- 5 [20]. Furthermore, the degradation of EB1 (microtubule-end- binding protein 1) is accelerated by over-expression of wild-type CSN-5 or CSN-5D151N in HeLa cells [20]. These results suggest that the integrity of CSN might contribute more to regulating the stability of some substrates of CRLs. Current models suggest that the CRL complex is controlled by cycles of CRL deneddylation and CAND1 binding [7]. Our experiments further suggested that CAND1, a putative regulator of CRLs, is not required for maintenance of SCFFWD-1 ubiquitin ligase activity and circadian COP9 Integrity Is Essential for Its Function rhythm in N. crassa. These data provide additional evidence that the CSN is an important regulator of the circadian clock in N. crassa through maintenance of SCFFWD-1 ubiquitin ligase stability. In conclusion, the results of our experiments indicate that even without deneddylation activity, the N. crassa CSN can still regulate hyphal growth, conidial development, and circadian function by regulating the activities of E3 ubiquitin ligases. Because the func- tion of the CSN in the regulation of CRL activities is conserved in higher eukaryotes, we propose that the CSN may have a similar role in plants and animals. Materials and Methods Strains and culture conditions The N. crassa strain 87-3 (bd, a) was used as the wild-type strain in this study. The bd ku70RIP strain, which was generated pre- viously [54], was used as the host strain for creating the cand1 knockout mutants. We also used csn-5KO, csn-2KO and csn-5KO, his-3 strains that were generated previously [16]. The 301-6 (bd, his-3, A) strain and the csn-5KO, his-3 strain were used as the host strains for the his-3 targeting construct transformation [24]. Liquid culture conditions were the same as described previously [34]. For QA- induced protein expression, 0.01 M QA (pH 5.8) was added to liquid medium containing 16 Vogel’s medium, 0.1% glucose, and 0.17% arginine. The medium for the race tube assay contained 16 Vogel’s, 0.1% glucose, 0.17% arginine, 50 ng/mL biotin, and 1.5% agar [55]. For race tubes containing QA (1023 M), glucose was excluded from the medium. Plasmids All three JAMM point mutations of CSN-5 were generated using the Quikchange Site-Directed Mutagenesis Kit (Stratagene). pUC19-CSN-5 was used as the template for mutagenesis. After- wards, the mutated CSN-5 DNA fragments were subcloned into either the pqa-5Myc-6His or pqa-3Flag vectors. The triple point mutant CSN-5 (H127A, H129A and D140N) generated from pUC19-CSN-5 was subcloned into the endogenous csn-5 promot- er-driven vector pcsn-5-Myc-His-CSN-5, resulting in pcsn-5-Myc- His-CSN-5tri. The previously constructed plasmids pqa-Myc- Cul1, pqa-Myc-His-Cul3, pqa-Myc-His-Cul4, pqa-Myc-His-CSN- 6, pqa-Myc-His-SCON-2, pqa-Myc-His-FBP94, pqa-Myc-His- BTB1, and pqa-Myc-His-DCAF11 were also used for his-3 tar- geting transformation in the csn-5KO, his-3 and 301-6 (bd, his-3, A) strains [16] and cotransformation in the csn-5H127A, csn-5H129A, and csn-5D140N strains. Generation of antiserum against Cul4 GST-Cul4 (containing Cul4 amino acids 1–113) fusion protein was expressed in RIL cells and the recombinant protein was purified and used as the antigen to generate rabbit polyclonal antiserum, as described previously [56]. Purification of Myc-His-CSN-5 and mutant Myc-His-CSN-5 proteins from N. crassa The csn-5KO, Myc-His-CSN-5H127A, csn-5KO, Myc-His-CSN- 5H129A, or csn-5KO, Myc-His-CSN-5D140N strain, wild-type strain (negative control), and csn-5KO, Myc-His-CSN-5 strain (po- sitive control) were cultured for approximately 24 h in constant light (LL) in liquid medium containing QA (0.01 M QA, 16 Vogel’s medium, 0.1% glucose, and 0.17% arginine). Approxi- mately 10 g of tissue from each strain grown in LL was harvested. The purification procedure was the same as described previously [16]. Fractions containing purified Myc-tagged CSN proteins were immunoprecipitated by adding 25 mL of c-Myc monoclonal PLoS Genetics | www.plosgenetics.org 12 May 2012 | Volume 8 | Issue 5 | e1002712