Functional Genomics of Protein Phosphorylation in Arabidopsis Chloroplasts

蛋白质磷酸化是新陈代谢、基因表达和细胞结构调节的主要方式。在拟南芥中预测或已经发现152个蛋白激酶(OPKs，细胞器蛋白激酶)的叶绿体或线粒体定位。本项目的目标是通过一种组合的方法系统地确定OPKs介导的磷酸化的靶标和生理作用，包括(I)通过瞬时表达RFP-Fusion来定位具有有争议细胞器靶向的50个OPKs的细胞内定位，(Ii)在多种细胞器相关条件下OPKs的mRNA表达谱，(Iii)鉴定75个OPKs的敲除突变体及其在角斗型方法中关于关键细胞器功能的特征，(Iv)差异蛋白质组学鉴定OPKs的底物(32P标记的细胞器蛋白的二维凝胶分析结合野生型[WT]与OPK敲除的质谱分析)。(V)对于选定的OPKs，将产生转基因植物(过表达和/或获得功能突变体)，并通过系统的双杂交筛选来鉴定互作因子。

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.