COP9 Integrity Is Essential for Its Function Figure 4. Mutations in the JAMM motif of CSN-5 partially restore SCF-mediated FRQ degradation in the csn-5KO strain. (A) Western blot analyses showing degradation of FRQ protein in csn-5 mutant and the different CSN-5 complementation strains after addition of cycloheximide (10 mg/mL). Asterisks indicate nonspecific bands detected by FRQ antibody. (B) Densitometric analyses from four independent experiments showing the degradation of FRQ in different strains. doi:10.1371/journal.pgen.1002712.g004 strain complemented with Myc-CSN-5, whereas in the csn-5KO strain it became undetectable after only 6 h of CHX treatment. In the csn-5H127A strain, detection of FBP94 signal extended to 12 h after CHX treatment (Figure 6C and 6D). Therefore, CSN com- plex with mutated JAMM domain can partially function in main- taining the stability of other F-box–containing adaptor proteins. In a previous study, we determined that the N. crassa Cul3 pro- tein interacts with BTB1 protein, and both proteins become unstable in the csn-5KO strain [16]. The half-life of Myc-BTB1 was .12 h in the wild-type and csn-5KO expressing wild-type CSN-5 strains in the presence of CHX, whereas in the csn-5KO strain it became undetectable after 6 h of CHX treatment (Figure 6E and 6F). As expected, in the csn-5H127A strain, BTB1 signals were de- tectable at 12 h after CHX treatment (Figure 6E and 6F), indi- cating that CSN with the JAMM mutated CSN-5 still partially functions to protect the substrate adaptor proteins of CRL3 from degradation. We also investigated whether CSN with the JAMM mutated CSN-5 regulates the substrate receptor protein of CRL4 in a similar manner. N. crassa Cul4 was previously shown to inter- act with DCAF11, a putative substrate receptor of CRL4DCAF11 [16,38]. As shown in Figure 6G and 6H, the half-life of Myc- DCAF11 was .12 h in the wild-type and csn-5KO expressing wild- type CSN-5 strains in the presence of CHX, whereas in the csn-5KO strain it became undetectable after 6 h of CHX treatment [16]. As expected, in the csn-5H127A strain, the detection of DCAF11 signal was extended to 9 h after CHX treatment (Figure 6G and 6H). Taken together, these in vivo results indicate that the CSN complex containing mutant CSN-5 efficiently prevents degradation of sub- strate receptors of CRLs. CAND1 is not required for regulation of the circadian rhythm and SCFFWD-1 ubiquitin ligase Current models suggest that the activity and assembly of CRLs are controlled by cycles of CRL deneddylation and CAND1 bind- ing of deneddylated cullins [39,40,41,42,43,44,45,46]. In plants and worms, CAND1 mutants exhibit defects consistent with a positive role in regulating the function of a subset of CRLs [40,47,48,49]. However, in yeast and human cells, loss of CAND1 has little effect on the abundance of neddylated cullins, suggesting that the neddylation/ deneddylation cycle may function independently of CAND1 [50,51]. To test whether CAND1 is involved in maintaining the function of CRLs in N. crassa, we examined the role of CAND1 in the regulation of circadian conidiation rhythm and proper functioning of the SCFFWD-1 complex. We first measured the growth rates of the wild- type and cand1KO strains by race tube assay under constant darkness. The growth of the cand1KO strain (about 3.0 cm per day) was slightly slower than that of the wild-type strain (about 3.7 cm per day), suggesting that CAND1 is involved in regulating hyphal growth. After entrainment by light, like the wild-type strain, the cand1KO strain exhibited a robust circadian conidiation rhythm with a period of about 22 h at 25uC in constant darkness (Figure 7A), suggesting that CAND1 is not required for circadian rhythms in N. crassa. To test whether CAND1 functions in a manner similar to the CSN, we examined the conidiation rhythms of the cand1 mutant in LD cycles (12 h light/12 h dark). As shown in Figure 7B, the conidiation rhythms of the cand1KO strain were entrained by LD cycles, indicating that unlike CSN, CAND1 is not required in light regulation of the circadian clock. We also examined the responses of the cand1 mutant to temperature entrainment using race tube assays. As expected, in 12 h 27uC/12 h 22uC temperature cycles, as shown in Figure 7C, like the wild-type strain, the conidiation rhythm of the cand1KO strain was synchronized by the temperature cycles, indicating that CAND1 is not required for the temperature-entrained conidiation process. These results suggest that CAND1 does not play a significant role in the regulation of circadian rhythm in N. crassa. Deletion of cand1 also had no effect on degradation of the clock protein FRQ, which is the substrate of the SCFFWD-1 ubiquitin ligase complex in N. crassa (Figure 8A and 8B). We also examined the stability of FWD-1 of the SCFFWD-1 complex in the cand1KO strain. As shown in Figure 8C and 8D, FWD-1 was very stable in the cand1 mutant, as in the wild-type strain, with a half-life of .12 h. Together, these results suggest that CAND1 is not required for regulation of the circadian rhythm and for main- taining the proper function of the SCFFWD-1 complex in N. crassa. Discussion In eukaryotes, the COP9 signalosome (CSN) is a highly con- served multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins [1,3,7,11] and main- taining the stability of the CRL components [7,24]. In this study, we used mutation analysis to confirm that the JAMM metal- binding motif of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in N. crassa. Point mutations of the PLoS Genetics | www.plosgenetics.org 8 May 2012 | Volume 8 | Issue 5 | e1002712