The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans

Abstract

Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism. Previously, missense alleles of creA and creB were analysed for genetic interactions, and here we extended this to complete loss-of-function alleles of creA and creB, and compared morphological and biochemical phenotypes, which confirmed genetic interaction between the genes. We investigated whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitination enzyme, using co-purifications of CreA and CreB, first using strains that overexpress the proteins and then using strains that express the proteins from their native promoters. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. Thus, CreA is not a direct target of CreB, and nor are proteins that form part of a stable complex with CreA a target of CreB. These results open up new questions regarding the molecular mechanism of CreA repressing activity, and how the ubiquitination pathway involving CreB interacts with this regulatory network.

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

“…CreA is a candidate target of CreB and CreD. However, recent studies suggested that CreA was not a direct target of CreB (24,29). Therefore, analysis of CreA stability in creB and creD disruption mutant strains is necessary to understand the mechanism of CCR regulation in filamentous fungi.…”

Carbon Catabolite Repression in Filamentous Fungi

“…In A. nidulans ubiquitination is involved during CCR, while in S. cerevisiae there are no such regulatory genes for this mechanism. Activation of CreA involves addition and subsequent removal of ubiquitin, mediated by CreD-HulA ubiquitin ligase complex and the CreB-CreC DUB complex, respectively ( Figure 3 ) [ 3 , 73 ]. The co-repressor proteins, Tup1 and Ssn6, of yeast have RcoA and SsnF homologues in A. nidulans respectively [ 74 ].…”

Nuclear export‐dependent degradation of the carbon catabolite repressor CreA is regulated by a region located near the C‐terminus in Aspergillus oryzae

“…Several studies that have examined the phosphorylation of the CreA counterpart in Sclerotinia sclerotiorum and T. reesei support this idea (Vautard‐Mey and Fèvre, ; Cziferszky et al, ). Recently, six phosphorylation sites of A. nidulans CreA have been identified by mass spectrometry (Alam et al, ). Further investigations of SnfA function and CreA phosphorylation are required for a better understanding of the CCR regulation mechanism in filamentous fungi.…”

“…Ries et al () detected ubiquitinated proteins when they applied western blotting analysis to immunoprecipitated A. nidulans CreA‐GFP protein samples using anti‐ubiquitin antibody. However, it is possible that other ubiquitinated proteins were co‐immunoprecipitated with CreA‐GFP or nonspecific immunoprecipitated proteins, because no ubiquitinated form of A. nidulans CreA was detected by mass spectrometry or western blotting (Alam et al, ). Therefore, it is not clear whether CreA is actually modified by ubiquitin, and we are still investigating the ubiquitination of the CreA protein in A. oryzae .…”

“…The reduced CreA abundance in the creB disruption mutant was consistent with our recent study that showed the transcriptional relief of α‐amylase gene expression from CCR by A. oryzae creB disruption (Tanaka et al, ). However, a recent report suggests that CreA is not a direct target for CreB, although interaction between CreA and CreB was observed when they were overexpressed (Alam et al, ). We failed to measure the half‐life of FLAG‐CreA in Δ creB and Δ creC because FLAG‐CreA was hardly detectable in these strains.…”