Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

蛋白质磷酸化是所有生物体中基本的翻译后修饰。在光合自养生物中，蛋白质磷酸化对于光合作用的微调至关重要。光系统 II (PSII) 核心的可逆磷酸化和 PSII (LHCII) 的光捕获复合物有助于调节光合活动。除了这些主要蛋白质的磷酸化外，最近的磷酸化蛋白质组学分析表明，有几种蛋白质在类囊体膜中被磷酸化。在这项研究中，我们利用 Phos-tag 技术对拟南芥类囊体膜中的蛋白质磷酸化进行了综合评估. Phos-tag SDS-PAGE 可实现磷酸化蛋白质与其非磷酸化异构体相比的迁移率变化，从而将磷酸化蛋白质与其非磷酸化异构体区分开来。我们将此技术外推到二维 (2D) SDS-PAGE 以检测类囊体膜中的蛋白质磷酸化。通过常规 SDS-PAGE 在第一维分离类囊体蛋白，通过 Phos-tag SDS-PAGE 在第二维分离。除了主要磷酸化光合作用相关蛋白的分离外，2D Phos-tag SDS-PAGE 还可以检测类囊体膜中的几种次要磷酸化蛋白。类囊体激酶突变体的分析表明，光依赖性蛋白磷酸化主要限于 PSII 核心和 LHCII 蛋白的磷酸化。此外，我们评估了类囊体膜、颗粒核心、颗粒边缘和基质层结构域的磷酸化状态。总体而言，这些结果表明 Phos-tag SDS-PAGE 是一种有用的生化工具，可用于研究类囊体膜蛋白中的体内蛋白质磷酸化。