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

分析蛋白质功能的一种普遍有效的方法是确定其相互作用伙伴。然而，到目前为止，用于分析蛋白质-蛋白质相互作用的工具受到了大量假阳性的影响。因此，需要在耗时且昂贵的过程中验证已确定的交互。就在两年前，一项新的方案被公布，允许在哺乳动物细胞培养中以高灵敏度明确地识别蛋白质-蛋白质相互作用。这项名为QUICK的方案基于13C标记氨基酸对细胞培养的稳定同位素标记、免疫沉淀、RNAi介导的击倒和定量质谱学。我们主要通过引入一种交联步(QUICK-X)来改进QUICK，并成功地将其应用于莱茵衣藻。我们利用这种单细胞藻类作为模式生物来了解i)沉默转基因激活的机制和ii)叶绿体HSP70和HSP90伴侣系统在叶绿体生物发生和信号转导中的作用。我们首先建议进一步改进QUICK-X，使用15NH4Cl的稳定同位素标记。其次，我们希望将衣藻暴露在各种环境条件下，并使用QUICK-X识别新的蛋白质-蛋白质相互作用并监测它们的动态。对这些的了解将为更深入地了解衣藻的转基因激活和叶绿体伴侣功能奠定基础。

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.