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

Extent: 6p.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.Paraskevi Georgakopoulos, Robin A. Lockington, Joan M. Kell

SAGA complex purification.

<p>a) Tandem affinity purification of a strain containing SptC tagged with the TAP tag (Lane 1) and a strain with wildtype SptC (Lane 2). The gel regions that were purified are numbered, and the <i>S. cerevisiae</i> homologues of the SAGA complex components identified in <i>A. nidulans</i> by LC MS are shown on the right. b) Tandem affinity purification of the N-TAP<i>sptC</i>;MYC<i>acdX</i>;<i>nkuAΔ</i> strain grown in media containing either 1% glucose (Lane 1), 50 mM arabinose (Lane 2) or 50 mM sodium acetate pH 6.0 (Lane 3). LC-MS was performed for all three conditions in this experiment. c) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065221#pone-0065221-g002" target="_blank">Figure 2c</a> shows one of a further two repeat experiments, designed specifically to determine whether the differences in staining intensity around 50KDa in lane 3 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065221#pone-0065221-g002" target="_blank">Figure 2b</a> were robustly repeatable, showing that the apparent differences in part b are an artifact.</p

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p

The SAGA complex components present in the <i>A. nidulans</i> genome.

a<p>Functions of the <i>S. cerevisiae</i> SAGA complex subunits.</p>b<p><i>S. cerevisiae</i> homologues identified in <i>A. nidulans.</i></p>c<p><i>A. nidulans</i> accession number.</p><p>References: RfeE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065221#pone.0065221-Malavazi1" target="_blank">[38]</a>, AdaB and GcnE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065221#pone.0065221-ReyesDominguez1" target="_blank">[16]</a>, AcdX and SptC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065221#pone.0065221-Georgakopoulos1" target="_blank">[21]</a>.</p

Complementation of the <i>sptCΔ</i>^MYC<i>acdXnkuAΔ</i> by p^N−TAPSPTC.

<p><i>sptCΔ</i>^MYC<i>acdXnkuAΔ</i> protoplasts plated on osmotically stabilised minimum medium, after 3 days growth at 37°C. <b>A)</b> No DNA control. <b>B)</b> Transformed with p^{N<b>−</b>TAP}SPTC; arrow indicates complemented transformant.</p

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

46 p.-8 fig.-2 tab.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 drugsThis work was supported by the Spanish Ministerio de Economía y Competitividad (BFU2012-33142 to E.A.E), the Biotechnology and Biological Sciences Research Council (BB/F01189/X1 to H.N.A. and Elaine Bignell), the Wellcome Trust (084660/Z/08/Z to H.N.A. and Joan Tilburn), and by the Australian Research Council (to J.M.K).Peer reviewe

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

The definitive version is available at www.blackwell-synergy.comGenetic 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.Robin A. Lockington and Joan M. Kell