Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Keiji Nishioka,Yuichiro Takahashi,Wataru Sakamoto,Shin-Ichiro Ozawa,Yusuke Kato

doi:10.1007/s11120-020-00803-1

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.

Highlights

Protein phosphorylation is the most common post-translational modification that regulates and/or fine tunes protein functions
Phosphorylated light-harvesting complex of PSII (LHCII) migrates from the grana core, in which the photosystem II (PSII) is mostly localized, to the grana margin and stroma lamella, in which the photosystem I (PSI) is rich during state transition (Dumas et al 2016)
Thylakoid membrane proteins isolated from Arabidopsis ecotype Columbia (Col) were separated in the first dimension by conventional SDS-PAGE and followed by Phos-tag SDSPAGE in the second dimension (Fig. 1)

Summary

Introduction

Protein phosphorylation is the most common post-translational modification that regulates and/or fine tunes protein functions. STN8 kinase regulates the phosphorylation of D1, which is dephosphorylated by PSII core phosphatase (PBCP) (Bonardi et al 2005; Samol et al 2012). These phosphorylation states of protein complexes are expected to change the structural domains of the thylakoid membrane, grana core (appressed region), grana margin, end membrane, and stroma lamella (non-appressed region). Phosphorylated LHCII migrates from the grana core, in which the PSII is mostly localized, to the grana margin and stroma lamella, in which the PSI is rich during state transition (Dumas et al 2016). The phosphorylation state of most of the thylakoid membrane proteins, according to their spatial distribution within the thylakoid membrane, remains to be elucidated

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Results

Conclusion