Functional Genomics of Protein Phosphorylation in Arabidopsis Chloroplasts

蛋白质磷酸化是调节代谢、基因表达和细胞结构的主要模式。叶绿体或线粒体定位的预测或已经发现的152蛋白激酶（OPKs，细胞器蛋白激酶）在拟南芥。本项目的目标是通过一种组合方法系统地定义OPKs介导的磷酸化的靶点和生理作用，包括（i）通过RFP融合物的瞬时表达对50种具有有争议的细胞器靶向的OPKs进行细胞内定位，（ii）OPKs在许多细胞器相关条件下的mRNA表达谱，（iii）鉴定75种OPK的敲除突变体，并以手套型方法表征其关键细胞器功能，（iv）通过差异蛋白质组学鉴定OPK的底物（32 P标记的细胞器蛋白的2-D凝胶分析结合质谱法用于野生型[WT]与OPK敲除）。(v)对于选定的OPK，将产生转基因植物（过表达子和/或获得性功能突变体），并通过系统性双杂交筛选鉴定互作子。

Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cell architecture. Chloroplast or mitochondrial localization is predicted or has been found for 152 protein kinases (OPKs, organellar protein kinases) in Arabidopsis thaliana. The goal of this project is to define systematically targets and physiological role of phosphorylation mediated by OPKs by a combined approach, including (i) intracellular localization of the 50 OPKs with disputable organelle targeting by transient expression of RFP-fusions, (ii) mRNA expression profiling of OPKs for a number of organelle-relevant conditions, (iii) identification of knock-out mutants for 75 OPKs and their characterization with respect to key organellar functions in a gauntlet-type approach, (iv) identification of substrates of OPKs by differential proteomics (2-D gel analyses of 32P-labelled organellar proteins combined with mass spectrometry for wild-type [WT] versus OPK knock-outs). (v) For selected OPKs, transgenic plants (overexpressors and/or gain-offunction mutants) will be generated, and interactors will be identified by systematic two-hybrid screens.