Integration of Fungus-Specific CandA-C1 into a Trimeric CandA Complex Allowed Splitting of the Gene for the Conserved Receptor Exchange Factor of CullinA E3 Ubiquitin Ligases in Aspergilli

Abstract

E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-amino-acid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution. IMPORTANCE Aspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.

COP9 Signalosome Interaction with UspA/Usp15 Deubiquitinase Controls VeA-Mediated Fungal Multicellular Development

“…Plasmid pME4654 carries the deletion cassette of csnE [28] and was used for the construction of A. nidulans strain AGB1169. Restriction digest of pME4654 with PmeI and the transformation of the linear fragment into AGB551 resulted in strain AGB1169.…”

“…Therefore, the 5′ flanking region of uspA together with the uspA ORF was amplified from genomic DNA of the wild type AGB551 with primers CM37/CM48. The PreScission cutting site, the linker, as well as the gfp, were amplified together with primers AMK82/AMK85 from pME4722 [28]. A SwaI cutting site is included in the overhang of primer AMK85, which allows the cloning in two steps.…”

“…The 4628 bp long fusion product was ligated into the SwaI restriction site of pME4313. Furthermore, the ORF of csnB was amplified from the genomic DNA of AGB551 with the primer pair CM163/CM164 and fused through PCR to the N-terminal half of YFP ( nYFP ), which was amplified with primers AMK163/CM94 from pME4685 [28]. This fusion product was ligated into the PmeI restriction site and resulted in the plasmid pME4708, which was transformed into AGB1014 and resulted in the strain AGB1170.…”

“…The plasmid pME4711 contained the cYFP:uspA fusion in its SwaI restriction site. Furthermore, csnF was amplified from the genomic DNA of AGB551 with the primer pair CM166/CM167 and fused N-terminally to nYFP that was amplified with AMK163/CM94 from pME4685 [28]. Both fragments were fused through a PCR and ligated into the PmeI restriction site.…”

“…Deneddylation induces a conformational change of the scaffolding protein and leads to CRL complex disassembly and short-term inability to modify proteins with ubiquitin [27]. Thereby, the adaptor/receptor exchange factor CandA/1 binds to unneddylated cullins, which consists of three proteins in A. nidulans [28]. The deletion of single CSN subunit encoding genes leads to embryonal lethality in higher eukaryotes [29,30,31].…”