start-ver=1.4
cd-journal=joma
no-vol=147
cd-vols=
no-issue=1
article-no=
start-page=107
end-page=124
dt-received=
dt-revised=
dt-accepted=
dt-pub-year=2021
dt-pub=202101
dt-online=
en-article=
kn-article=
en-subject=
kn-subject=
en-title=
kn-title=Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane
en-subtitle=
kn-subtitle=
en-abstract=
kn-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.
en-copyright=
kn-copyright=

en-aut-name=NishiokaKeiji
en-aut-sei=Nishioka
en-aut-mei=Keiji
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=1
ORCID=

en-aut-name=KatoYusuke
en-aut-sei=Kato
en-aut-mei=Yusuke
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=2
ORCID=

en-aut-name=OzawaShin-ichiro
en-aut-sei=Ozawa
en-aut-mei=Shin-ichiro
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=3
ORCID=

en-aut-name=TakahashiYuichiro
en-aut-sei=Takahashi
en-aut-mei=Yuichiro
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=4
ORCID=

en-aut-name=SakamotoWataru
en-aut-sei=Sakamoto
en-aut-mei=Wataru
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=5
ORCID=

affil-num=1
en-affil=Institute of Plant Science and Resources (IPSR), Okayama University
kn-affil=

affil-num=2
en-affil=Institute of Plant Science and Resources (IPSR), Okayama University
kn-affil=

affil-num=3
en-affil=Institute of Plant Science and Resources (IPSR), Okayama University
kn-affil=

affil-num=4
en-affil=Research Institute for Interdisciplinary Science, Okayama University
kn-affil=

affil-num=5
en-affil=Institute of Plant Science and Resources (IPSR), Okayama University
kn-affil=
en-keyword=Chloroplast
kn-keyword=Chloroplast
en-keyword=Phos-tag
kn-keyword=Phos-tag
en-keyword=Protein phosphorylation
kn-keyword=Protein phosphorylation
en-keyword=Thylakoid membrane
kn-keyword=Thylakoid membrane
en-keyword=STN7
kn-keyword=STN7
en-keyword=STN8
kn-keyword=STN8
END