Identification of SkpA-CulA-F-box E3 ligase complexes in pathogenic Aspergilli.

The ubiquitin proteasome system is critical for the regulation of protein turnover, which is implicated in the modulation of a wide array of biological processes in eukaryotes, ranging from cell senescence to virulence in plant and human hosts. Proteins to be marked for ubiquitination and subsequent degradation are bound by F-box proteins, which are interchangeable substrate-recognising receptors. These F-box proteins bind a wide range of substrates and associate with the adaptor protein Skp1 and the scaffold Cul1 to form Skp1-Cul1-F-box (SCF) complexes. SCF complex components are highly conserved in eukaryotes, ranging from yeast to humans. However, information regarding the composition of these complexes and the biological roles of F-box proteins is limited, specifically in filamentous fungal species like the genus Aspergillus. In this study, we have identified 51 and 55 fbx-encoding genes in the genomes of two pathogenic fungi, A. fumigatus and A. flavus, respectively. Immunoprecipitations of the HA-tagged SkpA adaptor protein revealed that 26 F-box proteins in A. fumigatus and 30 F-box proteins in A. flavus are involved in SCF complex formation during vegetative growth. These interactome data also revealed that a diverse array of SCF complex conformations exist in response to various exogenous stressors. Lastly, we have provided evidence that the F-box protein Fbx45 interacts with SkpA in both species in response to Amphotericin B. Orthologs of the fbx45 gene are highly conserved in Aspergillus species, but are not present within the genomes of organisms such as yeast, plants or humans. This suggests that Fbx45 could potentially be a novel F-box protein that is unique to specific filamentous fungi such as Aspergillus species.