COP9 Integrity Is Essential for Its Function Figure 1. Mutations within the JAMM motif of CSN-5 abolish CSN–mediated deneddylation activity for Cul1, Cul3, and Cul4. (A) Amino acid alignment of conserved JAMM motifs of CSN-5 homologs from Neurospora crassa (Ncr), Homo sapiens (Hsa), Arabidopsis thaliana (Ath), Drosophila melanogaster (Dme), Caenorhabditis elegans (Cel), and Schizosaccharomyces pombe (Spb). (B) Predicted structure of the N. crassa CSN-5 JAMM domain. The structure was generated by the SWISS-MODEL using the structure of the pre-mRNA splicing factor Prp8 as template (PDB accession number 2P8R), and the functional sites (His127, His129, and Asp140) were mapped according to the structure alignment with the AfJAMM structure (PDB accession number 1R5X). (C–E) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 (C), Myc-Cul3 (D), and Myc-Cul4 (E) in the wild-type strain, csn-5KO, and CSN-5 complementation strains. (F) Western blot analysis showing the expression of Cul4 in the wild- type, cul4KO, and csn-5KO strains. (G) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 in the wild-type strain, csn-5KO, and csn-5KO, pcsn-5-Myc-CSN-5 or csn-5KO, pcsn-5-Myc-CSN-5tri complementation strains. (H) Western blot analyses showing the expression of endogenous Cul4 in the wild-type strain, csn-5KO, csn-5KO, pcsn-5-Myc-CSN-5 or csn-5KO, pcsn-5-Myc-CSN-5tri complementation strains. The asterisk indicates a nonspecific cross-reacted protein band recognized by our Cul4 antiserum (in F and H). doi:10.1371/journal.pgen.1002712.g001 PLoS Genetics | www.plosgenetics.org 3 May 2012 | Volume 8 | Issue 5 | e1002712