Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane
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ORIGINAL ARTICLE
Phos‑tag‑based approach to study protein phosphorylation in the thylakoid membrane Keiji Nishioka1 · Yusuke Kato1,3 · Shin‑ichiro Ozawa1,2 · Yuichiro Takahashi2 · Wataru Sakamoto1 Received: 6 August 2020 / Accepted: 19 November 2020 / Published online: 2 December 2020 © The Author(s) 2020
Abstract 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. Keywords Chloroplast · Phos-tag · Protein phosphorylation · Thylakoid membrane · STN7 · STN8 Abbreviations GC Grana core GM Grana margin Supplementary Information The online version of this article (https://doi.org/10.1007/s11120-020-00803-1) contains supplementary material, which is available to authorized users. * Yusuke Kato [email protected] * Wataru Sakamoto [email protected]‑u.ac.jp 1
Institute of Plant Science and Resources (IPSR), Okayama University, 2‑20‑1 Chuo, Kurashiki, Okayama 710‑0046, Japan
2
Research Institute for Interdisciplinary Science, Okayama University, 3‑1‑1 Tsushima‑naka, Kita‑ku, Okayama City, Okayama 700‑8530, Japan
3
Present Address: Faculty of Agriculture, Setsunan University, 45‑1 Nagaotoge‑cho, Hirakata, Osaka 573‑0101, Japan
LHCI Light-harvesting complex of photosystem I LHCII Light-harvesting complex of photosystem II
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