indicates that the metal-binding motif of JAMM is essential for CSN-mediated deneddylation of cullins. Because all of the Cul3 and Cul4 was neddylated while not all of the Cul1 was neddylated in the csn-5KO strain and csn-5KO strains complemented by JAMM-domain mutant CSN-5, we rechecked Cul1 modification in the csn mutants. As shown in Figure S1, c- Myc antibody detected three specific protein bands in first generation of csn-5KO or csn-6KO transformants and two specific bands in the csn-1KO transformants. In most positive transformants, there was slightly less unneddylated Cul1 than neddylated Cul1, but the signal remained strong. This is different from the studies in yeast, plants, and fruit fly in which deletion of csn-5 results in hyperneddylation of Cul1 [11,21,33]. Possible explanations are that N. crassa genome codes for another deneddylase in addition to CSN complex or there is large amount of newly synthesized Cul1 proteins. We next examined the neddylation of Cul4 using a polyclonal antibody that recognizes the N terminus of N. crassa Cul4. As shown in Figure 1F, only the neddylated Cul4 was de- tected in csn-5KO strain, while in the wild-type strain, most of the detected Cul4 was unneddylated. Next, we transferred endoge- nous csn-5 promoter-driven constructs of either wild-type CSN-5 or CSN-5 with JAMM triple point mutations (H127A, H129A, and D140N) (hereafter referred to as CSN-5tri) into a csn-5KO strain expressing Myc-Cul1 protein. Myc-CSN-5 and Myc-CSN- 5tri were expressed in the csn-5KO strains (Figure 1G). Similar to what we observed in csn-5KO expressing CSN-5 with a single point mutation (Figure 1C), expression of the CSN-5tri failed to decrease the hyperneddylation of Cul1 in the csn-5KO strain (Figure 1G) as well. Interestingly, the amount of unneddylated Cul1 in csn-5KO strains expressing either single (Figure 1C) or triple (Figure 1G) point mutant CSN-5 was less than that in a csn-5KO strain. Furthermore, expression of CSN-5tri in the csn-5KO strain also failed to decrease hyperneddylated Cul4 to the levels observed in the wild-type or csn-5KO strain complemented with wild-type CSN- 5 (Figure 1H). Taken together, these data confirm that the JAMM domain metal-binding motif of N. crassa CSN-5 is essential for the deneddylation activity of the CSN. The rescue of growth and developmental defects in the csn-5KO strain by CSN-5 with JAMM domain mutations To examine whether the JAMM metal-binding site of CSN-5 functions in growth and development, we analyzed the phenotypes of the csn-5KO strain expressing either Myc-tagged wild-type or mutant CSN-5. On minimal slants with QA, the csn-5KO strain produced fewer aerial hyphae and conidia than the wild-type strain (Figure 2A). Expression of wild-type CSN-5 in the csn-5KO strain restored aerial hyphal growth and conidial formation to levels similar to those in the wild-type strain (Figure 2A). Sur- prisingly, when csn-5KO, Myc-CSN-5H127A; csn-5KO, Myc-CSN- 5H129A; and csn-5KO, Myc-CSN-5D140N strains (hereafter re- ferred to as csn-5H127A, csn-5H129A, and csn-5D140N, respectively) were grown in minimal slants containing QA, the transformants exhibited hyphal formation and conidiation that were the same as the wild-type strain and the csn-5KO, Myc-CSN-5 strain (Figure 2A). We next measured the growth rates of the wild-type strain, the csn- 5KO strain, and the transformants by race tube assay in constant darkness. Interestingly, the growth of csn-5H127A, csn-5H129A, and csn-5D140N strains was slightly faster than that of the wild-type strain (approximately 4.2 cm per day vs. 3.7 cm per day, res- pectively) and the csn-5KO, Myc-CSN-5 strain (Figure 2B). These results suggest that these CSN-5s with a point mutation within the JAMM metal-binding motif function similarly as the wild-type CSN-5 on N. crassa growth and conidiation. In QA-containing COP9 Integrity Is Essential for Its Function race tubes, the conidiation rhythms of the csn-5H127A, csn-5H129A, and csn-5D140N strains (a period of about 22.5 h) were pretty much (only slightly longer) to those of the wild-type and csn-5KO, Myc- CSN-5 strains (a period about 22.2 h) (Figure 2C) in constant darkness after light entrainment. To characterize the effect on light response of each CSN-5 point mutation, we further examined the light-entrained conidiation rhythm of each csn-5KO transformant during light–dark (LD) cycles (12 h light/12 h dark). As shown in Figure 2D, although the LD cycles entrained the conidiation rhythm of the csn-5KO strains expressing wild-type CSN-5 or mu- tant CSN-5, however, the conidiation bands of the csn-5H127A, csn- 5H129A, and csn-5D140N strains were broader than those of the wild- type and csn-5KO, Myc-CSN-5 strains. Similarly, 12 h 27uC/12 h 22uC temperature cycles entrained the conidiation rhythm of the csn-5H127A, csn-5H129A, and csn-5D140N strains, but not the patterns of conidiation bands (Figure 2E). Taken together, these results suggest that point mutations within CSN-5 are functional in growth and conidiation, and partially functional in circadian rhythm, light response, and temperature-entrained clock process. JAMM domain mutations do not disrupt CSN complex integrity or its interactions with Cul1 and Cul4 The loss of deneddylation activity of the JAMM domain mu- tations may be due to the disruption of the CSN complex. To examine this, we tested the interactions between the CSN-6 subunit and wild-type or mutant CSN-5s. Myc-tagged CSN-6 was co-expressed with Flag-tagged CSN-5 or mutant CSN-5 proteins in csn-5KO strains. As shown in Figure 3A, the Flag-tagged CSN-5 strongly interacted with Myc-tagged CSN-6 in an immunoprecip- itation reaction, suggesting that they were both in the intact CSN complexes. As expected, the Flag antibody pulled down the Myc- tagged CSN-6 protein in the csn-5KO strain co-expressing Myc- CSN-6 and each of the mutant Flag-CSN-5 proteins (Figure 3A), similar to what was observed in the csn-5KO strain co-expressing CSN-6 and wild-type CSN-5. This result indicates that the point mutations within the CSN-5 JAMM metal-binding motif did not affect the interactions between the CSN-5 and CSN-6 subunits and those two MPN proteins within PCI (Proteasome, COP9, eukaryotic Initiation factor 3) complexes may form dimers. To further examine whether Myc-His-tagged CSN-5 point mutants are incorporated into a larger molecular mass CSN complex, we performed gel filtration and followed by Western blot analysis. As shown in Figure 3B, like wild-type CSN-5, CSN-5H127A, CSN- 5H129A, and CSN-5D140N fusion proteins were eluted in larger molecular mass fractions, suggesting that each of the Myc-tagged CSN-5 point mutants can be incorporated into the intact CSN complex. Using protein affinity purification followed by Mass Spectrom- etry analysis, we further examined whether the CSN complex is properly assembled with CSN-5 point mutants. Myc-His-tagged CSN-5H127A, CSN-5H129A, CSN-5D140N, or wild-type CSN- 5 was purified on a nickel column followed by immunoprecipi- tation with a c-Myc monoclonal antibody. As shown in Figure 3C, similar immunoprecipitated protein profiles were detected in the Myc-His-CSN-5H127A, Myc-His-CSN-5H129A, Myc-His-CSN- 5D140N, and Myc-His-CSN-5 (wild-type CSN-5) samples, but not in the wild-type strain (negative control). Liquid chromatography– mass spectrometry/mass spectrometry (LC-MS/MS) analysis of excised gel bands led to the identification of seven subunits, from CSN-1 to CSN-7a, in the Myc-His-CSN-5 purified products and in the Myc-His-CSN-5H127A purified products (Figure 3C). PLoS Genetics | www.plosgenetics.org 4 May 2012 | Volume 8 | Issue 5 | e1002712