CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA
Introduction
The major repressor protein regulating the carbon catabolite repression of genes involved in carbon metabolism in Aspergillus nidulans is CreA. The gene encoding CreA was identified by mutations that relieve repression of genes encoding enzymes for the use of alternative carbon sources when glucose is present (Arst and Cove, 1973, Bailey and Arst, 1975, Hynes and Kelly, 1977). CreA is a Cys2His2 zinc finger DNA binding protein (Dowzer and Kelly, 1989, Dowzer and Kelly, 1991), and the consensus binding motif was shown to be SYGGRG (Cubero and Scazzocchio, 1994, Cubero et al., 2000, Kulmberg et al., 1993). Both the zinc finger region of the protein and the DNA binding consensus sequence are very similar to those of Mig1p, which is involved in carbon catabolite repression in Saccharomyces cerevisiae (Nehlin and Ronne, 1990, Nehlin et al., 1991). It was thought that Mig1p acts by recruiting the co-repressor complex, Ssn6p/Tup1p, to promoters that are under carbon catabolite repression control (Nehlin et al., 1991, Treitel and Carlson, 1995, Tzamarias et al., 1995) and that cytoplasmic translocation of Mig1p, triggered by its phosphorylation by Snf1 kinase in the nucleus, was a key regulatory step for releasing glucose repression (De Vit and Johnston, 1999, De Vit et al., 1997). However, studies on the nuclear export of Mig1p showed that glucose still regulates Mig1p-dependent repression in a mutant that no longer has export activity, indicating that Mig1p is regulated both by nuclear localisation, and by modification of its ability to repress transcription (De Vit and Johnston, 1999). It has subsequently been shown that the Ssn6p–Tup1p complex is tethered to the GAL1 promoter DNA in both repressing and activating conditions (Papamichos-Chronakis et al., 2002), and that Mig1p is not required for the Ssn6p–Tup1p complex to be tethered to the promoter (Papamichos-Chronakis et al., 2002). It has also been shown that Mig1p is tethered to the GAL1 promoter under both repressing and derepressing conditions, and although Mig1p is not essential for the binding of the Ssn6p–Tup1p complex to the promoter, it is important for Ssn6p–Tup1p mediated glucose repression (Papamichos-Chronakis et al., 2002). The Hxk2p-dependent signalling pathway is also important in glucose sensing (Moreno et al., 2005), and Hxk2p is nuclear localised in response to glucose, and Mig1p is required to keep Hxk2p in the nucleus (Ahuatzi et al., 2004), and the two proteins directly interact (Ahuatzi et al., 2007). In glucose grown cells, Hxk2p interacts with both Mig1p and Snf1p to inhibit the phosphorylation of Mig1p (Ahuatzi et al., 2007). In this system, Snf1p-dependent phosphorylation of Mig1p abolishes the interaction with Ssn6p–Tup1p, and controls transcriptional repression/derepression, and it is the regulation of this interaction, not the Mig1p cytoplasmic localisation, that is the molecular switch that controls transcriptional repression/derepression of GAL1 (Papamichos-Chronakis et al., 2002). Hexose phosphorylating enzymes have also been shown to be important in signalling in A. nidulans, with strains lacking both HxkA and GlkA showing impairment of carbon catabolite repression (Flipphi et al., 2003).
In A. nidulans, mutations in creB and creC were also identified that shared some of the mutant phenotypes of creA (Hynes and Kelly, 1977) The CreB protein is a deubiquitinating enzyme, and CreC is a WD40 protein present in a complex with the CreB deubiquitinating enzyme (Kelly and Hynes, 1977, Lockington and Kelly, 2001, Lockington and Kelly, 2002, Todd et al., 2000). Thus it is likely that deubiquitination is involved in the carbon catabolite repression mechanism. There are no protein sequences in the S. cerevisiae genome that are apparent homologues of CreB and CreC, and this aspect of the regulation differs between A. nidulans and S. cerevisiae. Mutations in creD suppress some of the phenotypes of creB and creC mutations (Boase and Kelly, 2004, Kelly and Hynes, 1977) and CreD contains arrestin domains and PY motifs, and has been shown to interact with the HECT ubiquitin ligase, HulA in bacterial two hybrid experiments (Boase and Kelly, 2004). Like mutations in creD, mutations in acrB also suppress some of the phenotypes due to mutations in creB and creC (Boase et al., 2003), and thus AcrB may also be involved in this ubiquitination pathway. Although the components of both the deubiquitination and ubiquitination network have been identified, the direct targets of these have not been identified. It is probable that either the amount or the activity of CreA is regulated by ubiquitination, but whether this control is direct or indirect has not been demonstrated.
There has been some evidence that ubiquitination is a component of the S. cerevisiae carbon catabolite repression mechanism. In yeast, glucose activates the genes important for its uptake and metabolism, and this is regulated via Rgt1p (Ozcan et al., 1996). In the absence of glucose, transcription of the yeast glucose transporters, HXT2 and HXT4, is prevented by Rgt1p, and at high levels of glucose, Mig1p represses the expression of HXT2 and HXT4. Only at low glucose concentrations are both repressors inactive, leading to a 10- to 20-fold derepression of gene expression. The repressor function of Rgt1p is inhibited by Grr1p, a subunit of ubiquitin ligase complex (Barral et al., 1995). The SCFGrr complex targets Gis4p, a factor involved in carbon signalling, and over-expression of GIS4 suppresses the snf1Δ, at least on raffinose (de Rue et al., 2005).
To date, little is known about the molecular events that allow CreA function to result in carbon catabolite repression. Key unresolved questions are investigated here. Firstly, we have investigated whether the auto-regulation of creA transcription (Arst et al., 1990, Shroff et al., 1996) is essential for repression and derepression of genes subject to CreA-mediated repression. The second question is whether differential stability of CreA plays a crucial role in carbon catabolite repression, and thus we investigated whether there is a reduction in the overall amount of CreA in the cell in conditions that are carbon catabolite derepressing compared to conditions that are repressing when creA expression is deregulated and over-expressed. The third question is whether altered intracellular localisation of CreA plays a key role in carbon catabolite repression, and to address this we determined the localisation of CreA in cells grown in repressing and in derepressing conditions, and undertook preliminary experiments to locate the sequences required for nuclear localisation. We have also investigated whether the amount or intracellular localisation of CreA was affected by the absence of CreB, CreD, or AcrB. We were also able to investigate, in cells where CreA is over-expressed and present in the nucleus, whether genes subject to carbon catabolite repression can be activated appropriately when repression is removed.
Section snippets
Strains
The full genotypes of strains used in this research are outlined in Table 1.
Plasmids constructed for the production of tagged versions of CreA
Various plasmids were constructed to express tagged versions of full length CreA (Fig. 1). In order to detect the CreA protein in A. nidulans, a PCR product containing the coding region of the creA gene was inserted into an expression plasmid, such that it was driven by the gpdA promotor sequence and contained sequences to produce a HIS tag at the 3′ end, to produce pGPD::CreA:HIS. A Myc-epitope tag was then inserted in
Constitutive over-expression of creA in A. nidulans does not affect normal repression or derepression
In preliminary experiments, a plasmid was constructed to express a GFP tagged version of the CreA protein from the native creA promoter. Transformants were generated that phenotypically complemented the creA deletion mutants, but the level of CreA expression was such that no fluorescence could be detected in mycelia grown in any media tested (results not shown). The native creA transcript is auto-regulated, and in the wild-type creA mRNA is present in higher amounts in mycelia grown in carbon
Discussion
In order to examine whether the CreA protein acts without modification as a transcriptional repressor, we have sought to determine whether degradation of CreA plays a key role, and whether CreA is regulated by nuclear localisation. If CreA is present in the nucleus in conditions that are not carbon catabolite repressing, and genes subject to carbon catabolite repression can still be activated, then we can conclude that CreA requires modification or protein interaction to become a functional
Acknowledgments
We thank Dr. Stephen Gregory for his help with microscopy. This research was supported by an Australian Research Council Discovery Project [DP0557699] to J.M.K.
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