Neurospora COP9 Signalosome Integrity Plays Major Roles for Hyphal Growth, Conidial Development, and Circadian Function Zhipeng Zhou 1 , Ying Wang 1 , Gaihong Cai2, Qun He 1 * 1 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China, 2 National Institute of Biological Sciences, Beijing, China Abstract The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EXnHXHX10D) of the CSN-5 JAMM domain disrupted CSN dened- dylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCFFWD-1 complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa. Citation: Zhou Z, Wang Y, Cai G, He Q (2012) Neurospora COP9 Signalosome Integrity Plays Major Roles for Hyphal Growth, Conidial Development, and Circadian Function. PLoS Genet 8(5): e1002712. doi:10.1371/journal.pgen.1002712 Editor: Ning Wei, Yale University, United States of America Received October 31, 2011; Accepted March 29, 2012; Published May 10, 2012 Copyright: 2012 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project is supported by grant from the National Basic Research Program of China (973 Program) (Grant No. 2007CB814900) (www.most.gov.cn), grants from National Natural Science Foundation of China (30770029, 30970079) (www.nsfc.gov.cn) to QH. This project is also supported by Chinese Universities Scientific Fund (2011JS110) (www.moe.edu.cn) to YW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: qunhe@cau.edu.cn Introduction The COP9 signalosome (CSN) is an evolutionarily conserved multifunctional complex in eukaryotes; it is composed of eight sub- units (CSN1–CSN8) in plants and mammals [1]. The CSN was initially discovered to be an important regulator of photomorpho- genesis in Arabidopsis thaliana [2] and was later found to participate in a wide range of processes [1,3]. The CSN potentially influences these cellular pathways by regulating the activity of cullin-RING ubiquitin ligases (CRLs, e.g., CRL1, CRL3, and CRL4 complexes in most eukaryotes) [3,4,5]. CRLs are a big family of ubiquitin ligases that share a common cullin/RING-E2 module [6,7,8]. They are necessary for substrate ubiquitination in a cascade of enzymatic reactions involving E1, E2, and E3 [9]. Under the con- trol of the CSN-regulated ubiquitin-proteasome pathway, cells coordinate the expression of an array of genes involved in the regulation of growth and development in order to respond to en- vironmental signals, such as light, temperature, and changes in nutrient conditions [1,10]. Loss-of-function mutations in CSN sub- units result in dysfunction of hundreds of CRLs [3], which explains the pleiotropic phenotypes observed in CSN mutants [1,3,7]. In 2002, Deshaies and his colleagues first described that the CSN-5 metalloprotease (JAMM) motif is required for removing the ubiquitin-like protein NEDD8 from Cul1 [11]. Later studies confirmed that the isopeptidase activity of the CSN complex is responsible for cullin deneddylation in eukaryotes [1,3,4,5,12]. In this process, the CSN binds to CRL E3 ligase and cleaves NEDD8 from cullins via the catalytic activity of its CSN-5 subunit, and then inhibits CRL activity [5,13,14,15]. Thus, the deneddylation activity of CSN requires the metalloprotease motif located in the CSN-5 subunit and the functional core subunits of the CSN [11,16]. However, CSN-5–dependent metalloprotease activity is not essential in Schizosaccharomyces pombe, as no obvious phenotype was detected in csn-5 deletion strains [17,18]. The physiological importance of CSN deneddylation activity in development and cell differentiation was examined in Drosophila melanogaster, in which the lethality of csn-5D/D animals was rescued by expression of a CSN-5 transgene but no adult flies were re- covered upon equivalent expression of CSN-5 (D148N) (loss of deneddylation activity) [11,19]. In CSN-5-downregulated HeLa cells, however, the accelerated degradation of c-Jun was rescued equally by over-expression of either the JAMM domain mutant CSN-5D151N or wild-type CSN-5 [20]. These results suggest that the requirement for neddylation/deneddylation cycle of cullins is not absolutely necessary during normal growth and certain devel- opmental stages. In plants, genetic studies suggest that although neddylation/deneddylation cycle is not absolutely necessary during early embryonic development and germination, it is re- quired during seedling establishment and the later developmental stages [12,21]. In Aspergillus nidulans, deletion of csnE/csn-5 or PLoS Genetics | www.plosgenetics.org 1 May 2012 | Volume 8 | Issue 5 | e1002712