The role of proteolysis in the modulation of nitrogen metabolite repression in Aspergillus nidulans

Abstract

When Aspergillus nidulans grows with ammonium or glutamine combined with an alternative nitrogen source, the organism preferentially utilises the ammonium or glutamine. The pathways for utilising alternative nitrogen source remains inactive in the presence of ammonium or glutamine and this phenomenon is called nitrogen metabolite repression (NMR). The regulation of NMR in A. nidulans is mediated by multiple mechanisms which include transcription, mRNA stability, translation, posttranslational modification and direct protein-protein interactions. A key feature of NMR in A. nidulans is the interaction between the GATA type transcription activator AreA and the transcription repressor protein NmrA. The transcription repressor NmrA of A. nidulans discriminates between oxidised and reduced dinucleotides; however dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signalling molecules is poorly characterised. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelia grown under nitrogen sufficient conditions but abundant in mycelia starved of nitrogen. Three proteases were identified and two were purified and identified by mass spectroscopy as serine proteases: Q5BGU2_EMENI and Q5BAR4_EMENI, encoded by the genes AN0238.2 and AN2366.2, respectively. Production of the third protease was absent in strain deleted for the areA gene. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. The two proteolytic fragments of NmrA produced by digestion at the C-terminal site remained associated; however the digested NmrA was unable to bind to the AreA zinc finger but retained the ability to bind NAD+. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site showed a resistance to further digestion that was enhanced by the presence of NAD+ and to a lesser extent by NADP+. This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated. The in vitro ordered proteolysis of NmrA reveals a potential new level of regulation relating to nitrogen metabolite repression. The dynamic interplay between the production of NmrA and its subsequent ordered proteolysis facilitates a rapid and finely tuned response to changes in the concentration and nature of the nitrogen source supporting growth.