Analysis of dynamic protein-protein interactions in Chlamydomonas using QUICK-X, a novel quantitative mass spectrometry-based approach

• Applicant: Professor Dr. Michael Schroda
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2009 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 135884105

A generally fruitful approach towards the analysis of a protein’s function is the identification of its interaction partners. However, tools employed until now for analysing protein-protein interactions suffer from the high number of false positives. Hence, interactions identified need to be verified in a time-consuming, costly process. Only two years ago, a new protocol was published allowing for the unequivocal identification of protein-protein interactions at high sensitivity in mammalian cell cultures. This protocol - termed QUICK - is based on stable isotope labeling of cell cultures with 13C-labeled amino acids, immunoprecipitation, RNAi-mediated knock-down, and quantitative mass spectrometry. We have improved QUICK mainly by introducing a crosslinking step (QUICK-X) and have successfully applied it to Chlamydomonas reinhardtii. We use this unicellular alga as model organism to understand i) the mechanisms leading to the activation of silenced transgenes and ii) the roles of the chloroplast HSP70 and HSP90 chaperone systems in chloroplast biogenesis and signalling. We propose first to further improve QUICK-X by using stable isotope labeling with 15NH4Cl. Second, we want to expose Chlamydomonas to various environmental conditions and, using QUICK-X, identify novel protein-protein interactions and monitor their dynamics. Knowledge of these will provide the fundament for a deeper understanding of transgene activation and chloroplast chaperone function in Chlamydomonas.

• DFG Programme: Research Grants