A second component of the SltA-dependent cation tolerance pathway in Aspergillus nidulans.

The transcriptional response to alkali metal cation stress is mediated by the zinc finger transcription factor SltA in Aspergillus nidulans and probably in other fungi of the pezizomycotina subphylum. A second component of this pathway has been identified and characterized. SltB is a 1272 amino acid protein with at least two putative functional domains, a pseudo-kinase and a serine-endoprotease, involved in signaling to the transcription factor SltA. Absence of SltB activity results in nearly identical phenotypes to those observed for a null sltA mutant. Hypersensitivity to a variety of monovalent and divalent cations, and to medium alkalinization are among the phenotypes exhibited by a null sltB mutant. Calcium homeostasis is an exception and this cation improves growth of sltΔ mutants. Moreover, loss of kinase HalA in conjunction with loss-of-function sltA or sltB mutations leads to pronounced calcium auxotrophy. sltA sltB double null mutants display a cation stress sensitive phenotype indistinguishable from that of single slt mutants showing the close functional relationship between these two proteins. This functional relationship is reinforced by the fact that numerous mutations in both slt loci can be isolated as suppressors of poor colonial growth resulting from certain null vps (vacuolar protein sorting) mutations. In addition to allowing identification of sltB, our sltB missense mutations enabled prediction of functional regions in the SltB protein. Although the relationship between the Slt and Vps pathways remains enigmatic, absence of SltB, like that of SltA, leads to vacuolar hypertrophy. Importantly, the phenotypes of selected sltA and sltB mutations demonstrate that suppression of null vps mutations is not dependent on the inability to tolerate cation stress. Thus a specific role for both SltA and SltB in the VPS pathway seems likely. Finally, it is noteworthy that SltA and SltB have a similar, limited phylogenetic distribution, being restricted to the pezizomycotina subphylum. The relevance of the Slt regulatory pathway to cell structure, intracellular trafficking and cation homeostasis and its restricted phylogenetic distribution makes this pathway of general interest for future investigation and as a source of targets for antifungal drugs.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in Aspergillus nidulans.

A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

SAGA complex components and acetate repression in Aspergillus nidulans.

Alongside the well-established carbon catabolite repression by glucose and other sugars, acetate causes repression in Aspergillus nidulans. Mutations in creA, encoding the transcriptional repressor involved in glucose repression, also affect acetate repression, but mutations in creB or creC, encoding components of a deubiquitination system, do not. To understand the effects of acetate, we used a mutational screen that was similar to screens that uncovered mutations in creA, creB, and creC, except that glucose was replaced by acetate to identify mutations that were affected for repression by acetate but not by glucose. We uncovered mutations in acdX, homologous to the yeast SAGA component gene SPT8, which in growth tests showed derepression for acetate repression but not for glucose repression. We also made mutations in sptC, homologous to the yeast SAGA component gene SPT3, which showed a similar phenotype. We found that acetate repression is complex, and analysis of facA mutations (lacking acetyl CoA synthetase) indicates that acetate metabolism is required for repression of some systems (proline metabolism) but not for others (acetamide metabolism). Although plate tests indicated that acdX- and sptC-null mutations led to derepressed alcohol dehydrogenase activity, reverse-transcription quantitative real-time polymerase chain reaction showed no derepression of alcA or aldA but rather elevated induced levels. Our results indicate that acetate repression is due to repression via CreA together with metabolic changes rather than due to an independent regulatory control mechanism.

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.

Molecular characterization and analysis of the acrB gene of Aspergillus nidulans: a gene identified by genetic interaction as a component of the regulatory network that includes the CreB deubiquitination enzyme.

Mutations in the acrB gene, which were originally selected through their resistance to acriflavine, also result in reduced growth on a range of sole carbon sources, including fructose, cellobiose, raffinose, and starch, and reduced utilization of omega-amino acids, including GABA and beta-alanine, as sole carbon and nitrogen sources. The acrB2 mutation suppresses the phenotypic effects of mutations in the creB gene that encodes a regulatory deubiquitinating enzyme, and in the creC gene that encodes a WD40-repeat-containing protein. Thus AcrB interacts with a regulatory network controlling carbon source utilization that involves ubiquitination and deubiquitination. The acrB gene was cloned and physically analyzed, and it encodes a novel protein that contains three putative transmembrane domains and a coiled-coil region. AcrB may play a role in the ubiquitination aspect of this regulatory network.

Regulation by carbon and nitrogen sources of a family of cellulases in Aspergillus nidulans.

The total amount of Aspergillus nidulans secreted cellulases is affected by both the carbon and nitrogen source present in the medium, and is regulated directly and/or indirectly by the carbon metabolism regulators, CreA, CreB, and CreC, and the global nitrogen metabolism regulator, AreA. We have characterized two A. nidulans genes that encode exo-cellulases, and one gene that encodes an endo-cellulase which is additional to the previously described endo-cellulase encoding gene, eglA. The putative regulatory regions 5(') of all the genes contain potential binding sites for the global carbon and nitrogen regulatory proteins, CreA and AreA. The sequences 5(') of eglA and eglB also contain potential consensus binding sites for XlnR which is involved in induction in Aspergillus niger, but none of the 5(') sequences contains an exact copy of the AceII DNA binding consensus sequence involved in induction in Trichoderma reesei, and thus it is likely that they may be induced by different pathway specific regulatory proteins.

The WD40-repeat protein CreC interacts with and stabilizes the deubiquitinating enzyme CreB in vivo in Aspergillus nidulans.

Genetic dissection of carbon catabolite repression in Aspergillus nidulans has identified two genes, creB and creC, which, when mutated, affect expression of many genes in both carbon catabolite repressing and derepressing conditions. The creB gene encodes a functional deubiquitinating enzyme and the creC gene encodes a protein that contains five WD40 repeat motifs, and a proline-rich region. These findings have allowed the in vivo molecular analysis of a cellular switch involving deubiquitination. We demonstrate that overexpression of the CreB deubiquitinating enzyme can partially compensate for a lack of the CreC WD40-repeat protein in the cell, but not vice versa and, thus, the CreB deubiquitinating enzyme acts downstream of the CreC WD40-repeat protein. We demonstrate using co-immunoprecipitation experiments that the CreB deubiquitinating enzyme and the CreC WD40-repeat protein interact in vivo in both carbon catabolite repressing and carbon catabolite derepressing conditions. Further, we show that the CreC WD40-repeat protein is required to prevent the proteolysis of the CreB deubiquitinating enzyme in the absence of carbon catabolite repression. This is the first case in which a regulatory deubiquitinating enzyme has been shown to interact with another protein that is required for the stability of the deubiquitinating enzyme.