An eight-subunit COP9 signalosome with an intact JAMM motif is required for fungal fruit body formation

Abstract

Fruit body formation in filamentous fungi is a complex and yet hardly understood process. We show here that protein turnover control is crucial for Aspergillus nidulans development. Deletion of genes encoding COP9 signalosome (CSN) subunits 1, 2, 4, or 5 resulted in identical blocks in fruit body formation. The CSN multiprotein complex controls ubiquitin-dependent protein degradation in eukaryotes. Six CSN subunits interacted in a yeast two-hybrid analysis, and the complete eight-subunit CSN was recruited by a functional tandem affinity purification tag fusion of subunit 5 (CsnE). The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4. Mutations in the JAMM metalloprotease core of CsnE resulted in mutant phenotypes identical to those of csn deletion strains. We propose that a correctly assembled CSN including a functional JAMM links protein turnover to fungal sexual development.development ͉ filamentous fungi F ungal fruit bodies are sexual reproduction structures that generate meiotic spores. The model mold Aspergillus nidulans develops a closed spherical fruit body (cleistothecium) including different tissue types: Hülle cells surround and nurse the growing cleistothecium, pericarp cells develop the protecting wall, and inner ascogenous cells mature into sexual spores (1, 2). Massive reconstruction of vegetative hyphae is required to build the complex three-dimensional fruit body. The regulation of this development is hardly understood in any fungus (3). A genetic screen recently identified csnD and csnE resembling genes for subunits of the COP9 signalosome (CSN) of animals and plants to be essential for fruit body formation of A. nidulans (4).CSN is a multiprotein complex composed of proteins containing PCI and MPN interaction domains (5, 6). Csn5/Jab1 is the only subunit conserved in all eukaryotes, and it carries an MPNϩ domain containing the JAMM motif conferring metalloprotease (deneddylation) activity (6, 7). CSN controls by its MPNϩ domain the activity of cullin-RING E3 ligases by cleaving the ubiquitin-like protein Nedd8/Rub1 from the cullin (8, 9). Neddylated E3 ubiquitin ligases are key mediators of posttranslational labeling of proteins for the proteasome (10). The CSN thus controls eukaryotic ubiquitin-dependent protein degradation.The complete eight-subunit CSN, composed of six PCI and two MPN domain proteins, was described for eukaryotes as humans (11), mice (12), plants (13), flies (14), and Dictyostelium (15). In fungi, definitive evidence for an eight-subunit CSN is lacking so far. CSN complex purification from Neurospora crassa revealed subunits 1-7, but subunit 8 was identified neither in the purification experiment nor in the genome sequence by bioinformatics means (16). In fission yeast subunits 6 and 8 have not been identified yet (17), and in the CSN-related complex of Saccharomyces cerevisiae only subunit Csn5 (yeast Rri1p) is well conserved (18).The fungal CSN complexes known to date are not essential for viability but are involved in cellu...

Lessons from Fungal F-Box Proteins

“…A. nidulans ⌬grrA mutants showed impaired ascosporogenesis, asexual conidiation, and sexual development, while displaying a normal vegetative growth. A similar phenotype was observed with CSN subunit mutants of A. nidulans (25,26). This suggests that CSN may be involved in the functioning of GrrA in A. nidulans.…”

“…Apparently, in fission yeast, the neddylation reaction is required for proper SCF function, but deneddylation is not, suggesting that in fission yeast an alternative deneddylation may be present. In Neurospora crassa, a deletion mutant for subunit 2 of CSN, ⌬csn-2, is viable but lacks a normal circadian rhythm and conidiation (64), while in Aspergillus nidulans, four CSN mutants, including one for subunit 5 (⌬csne), all lack fruiting body development (25,26). Together, these data suggest that, in filamentous fungi, proper recycling of the SCF is strictly required only for certain developmental processes, in accordance with the requirements of CSN in development in more-complex, multicellular organisms (reviewed in reference 179).…”

The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis

“…As it has been shown, CSN-mediated deneddylation prevents the assembly of a specific CRL [4]. In cooperation with the UPS the CSN participates in processes such as DNA repair [7], cell cycle [8], angiogenesis [9] and development [10][11][12]. However, its role in apoptosis is unknown.…”

Two Components of a velvet -Like Complex Control Hyphal Morphogenesis, Conidiophore Development, and Penicillin Biosynthesis in Penicillium chrysogenum

“…A multisubunit velvet complex could be hypothesized to function as a depot for regulatory proteins performing inducible and context-dependent cellular functions (58). Other examples of candidates for macromolecular complexes acting as depots for regulatory proteins are the proteasome and the COP9 signalosome, both of which are known to control major aspects of development (15,72).…”