COP9 Integrity Is Essential for Its Function Figure 2. Rescue of growth and developmental defects in the csn-5KO strain by the expression of JAMM-motif mutant CSN-5. (A) Wild-type, csn-5KO, and the different CSN-5 complementation strains growing on slants containing QA. csn-5KO strains produced significantly less conidia and aerial hyphae than wild-type or CSN-5 complementation strains. (B) Growth rates of the wild-type, csn-5KO, and CSN-5 complementation strains, measured at 25uC by race tube assays in constant darkness after 1 d of light treatment. (C–E) Rescue of conidiation rhythms in the different CSN-5 complementation strains, measured by race tube assay in dark–dark (C), light–dark (D), and temperature cycles (E). At least four replicates were tested under each condition. Black lines indicate the growth fronts every 24 h. doi:10.1371/journal.pgen.1002712.g002 Taken together, these results confirm that the integrity of the CSN complex is not affected by mutations within the JAMM motif of CSN-5 in N. crassa. Next, we examined whether CSN complexes with mutant CSN- 5 subunits can still interact with Cul1 protein. As shown in Figure 3D, both wild-type CSN-5 and each of the mutant CSN-5 proteins co-immunoprecipitated with Cul1 protein. We further examined whether CSN complexes with mutant CSN-5 subunits can also interact with Cul4 protein in vivo by IP/western blotting experiments. As shown in Figure 3E, the Myc-tagged wild-type CSN-5 co-immunoprecipitated with the neddylated and unned- dylated Cul4, indicating that the N. crassa CSN complex can in- teract with all species of Cul4 in vivo. Similarly, the Myc-tagged mutant CSN-5s also co-precipitated with Cul4 (Figure 3E), further confirming that mutations within the JAMM metal-binding motif of CSN-5 do not interfere with interaction between CSN and cullins. These results strongly suggest that the point mutations within the JAMM metal-binding motif abolish NEDD8 isopepti- dase activity but have no effect on the integrity of the CSN or on its interactions with cullins. CSN-5 with mutations in the metal-binding motif of JAMM domain can partially restore SCF-mediated FRQ degradation in the csn-5KO strain In N. crassa, the clock protein FREQUENCY (FRQ) is a negative regulator in the negative feedback loop that controls the circadian clock under constant conditions [34,35]. Impaired FRQ degradation in csn-2 mutants results in the loss of circadian rhythm [24]. To investigate whether the mutant CSN-5s can rescue cir- cadian rhythm defects in the csn-5KO strain, we examined the de- gradation of FRQ protein in the wild-type and csn-5KO strains expressing wild-type CSN-5 or mutant CSN-5s after addition of the protein synthesis inhibitor cycloheximide (CHX). In the wild- type strain, the FRQ was gradually degraded after CHX treat- ment, with a half-life of about 2.5 h (Figure 4A and 4B). However, in the csn-5KO strain, the degradation of FRQ was mostly blocked (Figure 4A and 4B), similar to what was observed in the csn-2KO strain and the fwd1RIP mutant [24,36]. As shown in Figure 4A and 4B, the expression of Myc-tagged wild-type CSN-5 in the csn-5KO strain restored the degradation of FRQ to wild-type levels, so that the conidiation period on race tubes was similar to that of the wild- type strain (Figure 2C). We next checked FRQ degradation in the csn-5KO strain expressing CSN-5 proteins with mutations in the JAMM metal-binding site. As shown in Figure 4A and 4B, the expression of Myc-tagged CSN-5H127A, CSN-5H129A, or CSN- 5D140N in the csn-5KO strain partially rescued the degradation of FRQ in the csn-5KO strain. FRQ was degraded slightly slower in the mutants than the wild-type strain or the csn-5KO strain com- plemented by wild-type CSN-5, with a half-life of ,5 h, consistent with the prolonged period of the conidiation rhythms in the csn- 5KO strains expressing the mutant CSN-5, indicating that both deneddylation activity and integrity of CSN are needed in this process. Taken together, these results demonstrate that CSN-5 with point mutations in the JAMM metal-binding site partially restore the SCF-mediated FRQ degradation. Catalytically dead CSN-5 partially stabilizes the SCFFWD-1 complex Previous studies showed that FRQ ubiquitination and degra- dation is mediated by the SCFFWD-1 E3 ligase complex [24,36], and that the stability of E3 ligase components is controlled by CSN in vivo [3,7,16,24]. Because the ectopic expression of mutated CSN-5 partially rescued both the circadian conidiation rhythm and FRQ degradation in the csn-5KO strain, we decided to check whether CSN with mutant CSN-5 can prevent the degradation of components of the SCFFWD-1complex. As shown in Figure 5A, Myc-Cul1 was stable after induced expression of Myc-CSN-5 in the csn-5KO strain, with a half-life of .9 h in the presence of CHX, similar to that of the wild-type strain. In the csn-5 mutant, however, both the neddylated and unneddylated Myc-Cul1 be- came very unstable, with a half-life about 1.5 h (Figure 5A and 5D) [16]. Interestingly, the expression of JAMM mutant CSN-5 had a differential effect on the neddylated and unneddylated Cul1. In mutant CSN-5 transformants, the stability of neddylated Cul1 was only partially rescued, with a half-life of .3 h in the presence of CHX (Figure 5A and 5D), whereas the stability of unneddylated Cul1 was almost rescued, with a half-life of .12 h (Figure 5A and 5D). These data indicate that although CSN containing JAMM mutated CSN-5 fails to cleave NEDD8 from neddylated Cul1, it still functions to protect hyperneddylated and unneddylated Cul1 from degradation to a certain extent. In N. crassa, deletion of csn-5 or csn-3 has no effect on the stability of SKP-1 protein in the SCFFWD-1 complex [16]. As expected, Myc-SKP-1 were very stable in the wild-type strain and the csn- 5KO strain and in the complementation strains with mutant CSN-5, with a half-life of .12 h (Figure 5B and 5E). FWD-1, the substrate-recruiting subunit of the SCFFWD-1 com- plex, was quite stable in the wild-type strain, whereas it became undetectable after only 3 h of CHX treatment in csn-5KO strain (Figure 5C and 5F). In the csn-5H127A, csn-5H129A, and csn-5D140N strains, however, FWD-1 signals could still be detected after 6 h of CHX treatment (Figure 5C and 5F), indicating that CSN with mutated CSN-5 partially functions to protect F-box proteins from degradation. This finding further confirms that regulation of SCF- mediated FRQ degradation by the CSN is a key step in the N. crassa circadian clock. Therefore, both the deneddylation activity and the integrity of the CSN are important for preventing the degradation of components of the SCFFWD-1 complex. CSN complex containing mutant CSN-5 efficiently prevents degradation of substrate receptors of CRLs We next asked whether CSN with mutated CSN-5 still functions to protect other CRL substrate receptors from degradation. N. crassa SCON-2, an F-box protein involved in regulating sulfur me- tabolism, was previously shown to interact with SKP-1 and is very unstable in a csn-2KO strain [24,37]. We compared the stability of Myc-SCON-2 in wild-type, csn-5KO and csn-5KO expressing wild- type CSN-5 or mutant CSN-5H127A strains. The half-life of Myc- SCON-2 was approximately 12 h in the wild-type and csn-5KO expressing wild-type CSN-5 strains in the presence of CHX. Myc- SCON-2 was very unstable in the csn-5 mutant and became PLoS Genetics | www.plosgenetics.org 6 May 2012 | Volume 8 | Issue 5 | e1002712