Abstract
ABSTRACTThe attachment of one or more ubiquitin molecules by SCF (Skp–Cullin–F-box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δfbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications.
Highlights
CreA::GFP A. nidulans wild type A. nidulans ⌬fbx23 A. nidulans ⌬fbx47 Glucose XylanGFP A. nidulans Fbx23::GFP A. nidulans casein kinase (CkiA)::GFP A. nidulans GskA::GFP aStrains were grown for 16 h at 22°C in minimal medium supplemented with either glucose or xylan before GFP cellular localization was assessed
A total of 74 Fbx (F-box) protein-encoding genes were previously identified in A. nidulans [35], and deletion strains were generated for each gene, with the exception of fbx25 (AN6359), which was shown to be essential, and fbx50 (AN10516), which was previously deleted and was identified as being required for the sexual development of ascospores [35]
The fbx deletion collection was subsequently screened for genes involved in carbon catabolite repression (CCR) by growing all 72 knockout strains in liquid minimal medium supplemented with xylose as a single carbon source and increasing concentrations of the glucose analogue 2-deoxy-glucose (2DG) in the presence of the cellular growth indicator alamarBlue
Summary
CreA::GFP A. nidulans wild type A. nidulans ⌬fbx A. nidulans ⌬fbx Glucose XylanGFP A. nidulans Fbx23::GFP A. nidulans CkiA::GFP A. nidulans GskA::GFP aStrains were grown for 16 h at 22°C in minimal medium supplemented with either glucose or xylan before GFP cellular localization was assessed. In the ⌬fbx strain, CreA::GFP was observed in the nuclei under both derepressing and repressing conditions These results suggest that Fbx is required for the degradation or the nuclear translocation of CreA or both in the absence of glucose. CreA::GFP nuclear localization was reduced in glucose and increased in xylan in the ⌬fbx strain compared to the wild-type strain, suggesting that this protein is required for correct CreA cellular localization (Table 1). Fluorescence of Fbx23::GFP was absent under CCRderepressing conditions, confirming the degradation under those conditions, whereas the intensity of Fbx23::GFP fluorescence increased upon the addition of glucose (Fig. S4) These results suggest that the predicted E3 ligase SCF complex-mediated ubiquitylation predominantly takes place in the cytoplasm under the conditions specified here and confirm that the Fbx protein complex is degraded in the presence of xylan (Fig. 4A; see Fig. S4)
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