Master Thesis
Co-IP of STRIPAK complex proteins in Sordaria macrospora and identification of new SmPP2AA and PRO11 interaction partners
Summary
The “striatin interacting phosphatase and kinase“ (STRIPAK) complex is a conserved multi-protein complex, which is found in mammals and in filamentous fungi but not in prokaryotes and plants. The STRIPAK complex directs divers signalling pathways. Sterile mutants revealed that the STRIPAK complex proteins SmPP2AA, SmMOB3 and PRO11 of Sordaria macrospora are involved in the control of hyphal fusion and fruiting-body development. However, the connection between the STRIPAK proteins is not yet fully investigated. Therefore, Co-IP studies were preformed to clarify the interaction between the SmPP2AA, SmMOB3 and PRO11 proteins in vivo.
The striatin homolog PRO11 is known to form homodimers and to act as scaffold for the binding of other proteins in the STRIPAK complex. PRO11 contains a calmodulin-binding domain, which indicates PRO11 functions in Ca2+ dependent signalling pathways. The strains co-expressing PRO11 and calmodulin for Co-IP interaction studies were generated but because of the limited time of the master thesis Co-IP experiments could not yet be performed but are planned for the future. The SmMOB3 protein is a putative kinase activator which contains a conserved MOB domain, and shares homology with a subunit of the clathrin-adaptor protein complex. In yeast-two hybrid experiments the SmMOB3 protein was not able to form homodimers and this was also confirmed by Co-IP studies. Putative interaction partners of SmMOB3 were identified by GFP-Trap analysis and the results indicate that SmMOB3 might be involved in vesicular trafficking. Up to now, no interaction of SmMOB3 and the scaffolding subunit of the protein phosphatase SmPP2A was detected. The Co-IP studies indicate that SmMOB3 and SmPP2AA might interact with each other but further investigations are required to clarify the findings. GFP-Trap with SmPP2AA might help to investigate the possible SmPP2AA-SmMOB3 interaction but could not be performed because the generation of the required strains was time consuming and needs to be performed in future. The scaffolding subunit of the protein phosphatase PP2A (PP2AA) interacts with regulatory B subunits that associate with the STRIPAK complex for the recruitment of substrates and the targeting to subcellular compartments. In S. macrospora the interaction of SmPP2AA with the B’’’ type regulatory subunit PRO11 was investigated. Although, no conclusive interaction could be proven by Co-IP, GFP-Trap results support a direct interaction between PRO11 and SmPP2AA.