COP9 Integrity Is Essential for Its Function Figure 6. CSN containing mutant CSN-5 efficiently prevents degradation of substrate receptors of CRLs. Western blot analyses with labeled antibody showing degradation of Myc-SCON-2 (A), Myc-FBP94 (C), Myc-BTB1 (E), and Myc-DCAF11 (G) after addition of cycloheximide (10 mg/ mL) in the wild-type, csn-5KO, csn-5KO complementation with CSN-5 and CSN-5H127A strains. Densitometric analyses from four independent experiments showing the degradation of Myc-SCON-2 (B), Myc-FBP94 (D), Myc-BTB1 (F), and Myc-DCAF11 (H). doi:10.1371/journal.pgen.1002712.g006 5KO strains expressing CSN-5 proteins with different point mu- tations in the JAMM metal-binding motif, the deneddylation activity was disrupted, while the assembly of the CSN complex and interactions between CSN and cullin proteins were not af- fected. Therefore, this system has great potential as a model for distinguishing between the two activities of the CSN. A recent study suggests that neddylated Cul1 and Cul3 are unstable in D. melanogaster csn mutant cells due to a defect in CSN deneddylation activity, whereas unneddylated cullins are stable in csn-5 mutant cells [33]. The results presented here show that un- stable forms of Cul1 in the csn-5 mutant were partially restored by expression of mutant CSN-5 protein without deneddylation ac- tivity. Like the stability of unneddylated Cul1 and Cul3 in D. melanogaster CSN-5–defective cells [33], unneddylated Cul1 re- mained stable in the csn-5H127A, csn-5H129A, and csn-5D140N strains, similar to that in the wild-type strain (Figure 5A), indicating that CSN integrity with catalytically dead CSN-5 effectively maintains the stability of cullins. Studies in D. melanogaster and A. nidulans CSN-5 mutants indicated that the CSN deneddylation activity is essential for cell differentiation and developmental initiation [11, 19,22,33]. However, in the A. thaliana fus6/C231 mutant (a CSN1 N-terminal deletion mutant), although the Cul1 neddylation still works in a wild-type pattern, it was lethal and exhibited severe gene expression defects [52]. This genetic evidence raises questions concerning whether the CSN has other important functions aside from its deneddylation activity. The accelerated degradation of PLoS Genetics | www.plosgenetics.org 10 May 2012 | Volume 8 | Issue 5 | e1002712