Regulation of Aspergillus nidulans CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47

Abstract

The 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.

Broad substrate-specific phosphorylation events are associated with the initial stage of plant cell wall recognition inNeurospora crassa

“…For 16 of these, we were able to detect phospho-peptides under at least one condition. Since homologs for some of these phosphorylated proteins had been implicated in regulatory functions of D-Glc utilization and CCR in A. nidulans (Assis et al, 2018), the respective N. crassa deletion strains were tested in more detail. First, we tested the capability of the strains to grow on different carbon sources (Figure 4A).…”

“…To test for a potential role in CCR, we next evaluated the resistance of WT and the mutants to 2-deoxy-glucose (2-DG) and allyl alcohol (AA) (Figure 4B). 2-DG is a non-metabolizable analogue of D-Glc that is often used to indicate impaired glucose repression in filamentous fungi (Allen et al, 1989; Assis et al, 2018; Xiong, Sun, et al, 2014). When functional, 2-DG is phosphorylated and activates CCR, resulting in the inability of the strain to grow on alternative carbon sources.…”

“…The WT and deletion strains were obtained from the FGSC (http://www.fgsc.net) (McCluskey et al, 2010). The F-box genes were named following A. nidulans (Assis et al, 2018; Colabardini et al, 2012): fbx-9 ( spp-1 , ΔNCU03658, FGSC#21255, Fbx9, AN10061), fbx-19 (ΔNCU08642, FGSC#15478, Fbx19, AN4510), fbx-20 (ΔNCU06250, FGSC#15629, Fbx20, AN4535) and fbx-22 ( cdc4 , ΔNCU05939, FGSC#13190, Fbx22, AN5517). The corresponding deletion strain for cr-1 (NCU08377) is FGSC#11514.…”

“…Another protein class known to be involved in metabolic adjustments and CCR are F-Box proteins as part of the SCF complex (Skp, Cullin, F-box containing complex) (Assis et al, 2018; Colabardini et al, 2012; Jonkers and Rep, 2009; Skowyra et al, 1997). This multi-protein E3 ubiquitin ligase complex catalyzes the ubiquitination of proteins destined for the proteasomal degradation process (Assis et al, 2018; Welchman et al, 2005) and is, therefore, paving the way for new cellular or metabolic states. F-box proteins are found in all eukaryotes and display a large variety of functions.…”

“…Phosphorylation reactions are a central element of F-box protein-mediated degradation, since F-box target proteins are commonly first phosphorylated before being recognized and ubiquitinated (Jonkers and Rep, 2009). For A. nidulans, Fbx23 and Fbx47 were identified as being important for CCR, with the former working in a ubiquitin ligase complex that appears responsible for CreA transcription factor degradation under carbon catabolite-derepressing conditions (Assis et al, 2018). In T. reesei , the ubiquitin C-terminal hydrolase CRE2 and the E3 ubiquitin ligase LIM1 play a role in the regulation of cellulase gene expression (Denton and Kelly, 2011; Glass et al, 2013; Gremel et al, 2008).…”