Neurospora COP9 Signalosome Integrity Plays Major Roles for Hyphal Growth, Conidial Development, and Circadian Function

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 deneddylation 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

Mutations within the JAMM motif of CSN-5 abolish CSN–mediated deneddylation activity for Cul1, Cul3, and Cul4.

<p>(A) Amino acid alignment of conserved JAMM motifs of CSN-5 homologs from <i>Neurospora crassa</i> (Ncr), <i>Homo sapiens</i> (Hsa), <i>Arabidopsis thaliana</i> (Ath), <i>Drosophila melanogaster</i> (Dme), <i>Caenorhabditis elegans</i> (Cel), and <i>Schizosaccharomyces pombe</i> (Spb). (B) Predicted structure of the <i>N. crassa</i> 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, <i>csn-5^KO</i>, and CSN-5 complementation strains. (F) Western blot analysis showing the expression of Cul4 in the wild-type, <i>cul4^KO</i>, and <i>csn-5^KO</i> strains. (G) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 in the wild-type strain, <i>csn-5^KO</i>, and <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5 or <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5tri complementation strains. (H) Western blot analyses showing the expression of endogenous Cul4 in the wild-type strain, <i>csn-5^KO</i>, <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5 or <i>csn-5^KO</i>, 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).</p

Mutations in the JAMM motif of CSN-5 partially restore SCF-mediated FRQ degradation in the <i>csn-5^KO</i> strain.

<p>(A) Western blot analyses showing degradation of FRQ protein in <i>csn-5</i> mutant and the different CSN-5 complementation strains after addition of cycloheximide (10 mg/mL). Asterisks indicate nonspecific bands detected by FRQ antibody. (B) Densitometric analyses from four independent experiments showing the degradation of FRQ in different strains.</p

CSN-5–mutated CSN efficiently prevents degradation of components of the SCF^FWD-1 complex.

<p>(A–C) Western blot analyses with labeled antibodies showing degradation of Myc-Cul1 (A), Myc-SKP-1 (B), and FWD-1 (C) after addition of cycloheximide (10 mg/mL) in the wild-type, <i>csn-5^KO</i>, and different CSN-5 complementation strains. Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FWD-1 antibody. (D–F) Densitometric analyses from four independent experiments showing the degradation of Myc-Cul1 (D), Myc-SKP-1 (E), and FWD-1 (F).</p

CAND1 is not required for regulation of the circadian rhythm.

<p>(A–C) Normal conidiation rhythms of <i>cand1</i> mutants in dark–dark (A), light–dark (B), and temperature cycles (C) measured by race tube assays. At least four replicates were tested under each condition. Black lines indicate the growth fronts every 24 h.</p

CSN containing mutant CSN-5 efficiently prevents degradation of substrate receptors of CRLs.

<p>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, <i>csn-5^KO</i>, <i>csn-5^KO</i> 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).</p

CAND1 is not required for regulation of the SCF^FWD-1 ubiquitin ligase.

<p>Western blot analyses showing degradation of FRQ (A) and FWD-1 (C) in wild-type and <i>cand1^KO</i> strains after addition of cycloheximide (10 mg/mL). Densitometric analyses from four independent experiments showing the degradation of FRQ (B) and FWD-1 (D). Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FRQ antibody (A) or FWD-1 antibody (C).</p

Point mutations do not disrupt integrity of the CSN or interactions of the CSN with Cul1 and Cul4.

<p>(A) Immunoprecipitation assays confirming interactions between different versions of Flag-CSN-5 and Myc-CSN-6. Wild-type strain and wild-type strain expressing Myc-CSN-6 were used as negative controls. (B) The Myc-CSN-5, Myc-CSN-5H127A, Myc-CSN-5H129A, or Myc-CSN-5D140N in <i>csn-5^KO</i> properly incorporates into CSN complex. (C) Silver-stained SDS-PAGE showing the two-step purification of Myc-His-CSN-5, Myc-His-CSN-5H127A, Myc-His-CSN-5H129A, or Myc-His-CSN-5D140N in the <i>csn-5^KO</i> strains. A wild-type strain was used as the negative control. CSN subunits identified by mass spectrometry analysis in products of Myc-His-CSN-5 or Myc- His-CSN-5H127A are indicated. Asterisks indicate the two IgG bands. (D) Immunoprecipitation assays confirming the interaction between different versions of Flag-CSN-5 and Myc-Cul1. (E) Immunoprecipitation assays showing the interaction between different versions of Myc-CSN-5 and endogenous Cul4. The asterisk indicates a nonspecific cross-reacted protein band recognized by our Cul4 antiserum.</p

Rescue of growth and developmental defects in the <i>csn-5^KO</i> strain by the expression of JAMM-motif mutant CSN-5.

<p>(A) Wild-type, <i>csn-5^KO</i>, and the different CSN-5 complementation strains growing on slants containing QA. <i>csn-5^KO</i> strains produced significantly less conidia and aerial hyphae than wild-type or CSN-5 complementation strains. (B) Growth rates of the wild-type, <i>csn-5^KO</i>, and CSN-5 complementation strains, measured at 25°C 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.</p