The filamentous fungus Aspergillus nidulans is used as a reference organism to study hydrolytic enzyme production. The presence of glucose, exerts a repressive effect on the expression of enzyme-encoding genes, that are involved in the use of alternative carbon sources. This process is called carbon catabolite repression (CCR) and is regulated co-ordinately by an array of different proteins, with the Cys2His2 transcription factor CreA being the major regulator of CCR in A. nidulans. In the presence of glucose, CreA moves to the nucleus where it represses target genes required for the use of alternative carbon sources. There is strong evidence that CreA is regulated by phosphorylation. The homologue of CreA in Saccharomyces cerevisiae is termed Mig1p, and is phosphorylated by the protein kinase Snf1p. Phosphorylation of Mig1p causes translocation to the cytoplasm, therefore releasing CCR. Protein kinases transfer phosphoryl groups onto target proteins (phosphorylation) and work in an opposite manner than protein phosphatases (de-phosphorylation), with both types of proteins regulating important cellular processes. Although some protein kinases and phosphatases have been described as being important for CCR, they have not been characterised for a role in this signalling pathway. This project therefore aims at investigating the interactome dynamics of protein kinases and phosphatases involved in CCR regulation. A preliminary screening of a A. nidulans protein kinase with 103 deletion strains and phosphatase deletion library in the presence of the CCR-defect indicators identified several candidates that showed disturbed CCR phenotypes. This project therefore proposes to tag potential candidates include MAPKs (mitogen activated protein kinase) into HOG (high osmolarity glycerol) pathway regulating osmoregulation with GFP or TAP and carry out immuno-precipitation during CC-repressing and CC-de-repressing conditions. Subsequently, mass spectrometry will be carried out to identify putative interacting partners and a protein interactome map will be established. This study is expected to provide novel insights into sensing signalling pathways underlying CCR with the ultimate aim to genetically manipulate biotechnological-relevant microorganisms.
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