start-ver=1.4 cd-journal=joma no-vol=12 cd-vols= no-issue= article-no= start-page=RP88822 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20231121 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts en-subtitle= kn-subtitle= en-abstract= kn-abstract=Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH. en-copyright= kn-copyright= en-aut-name=KatoYusuke en-aut-sei=Kato en-aut-mei=Yusuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KurodaHiroshi en-aut-sei=Kuroda en-aut-mei=Hiroshi 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=SaitoKeisuke en-aut-sei=Saito en-aut-mei=Keisuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=DograVivek en-aut-sei=Dogra en-aut-mei=Vivek kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ScholzMartin en-aut-sei=Scholz en-aut-mei=Martin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=ZhangGuoxian en-aut-sei=Zhang en-aut-mei=Guoxian kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=de VitryCatherine en-aut-sei=de Vitry en-aut-mei=Catherine kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=IshikitaHiroshi en-aut-sei=Ishikita en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=KimChanhong en-aut-sei=Kim en-aut-mei=Chanhong kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=HipplerMichael en-aut-sei=Hippler en-aut-mei=Michael kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 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=12 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=13 ORCID= affil-num=1 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=2 en-affil=Research Institute for Interdisciplinary Science, 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 Center for Advanced Science and Technology, The University of Tokyo kn-affil= affil-num=5 en-affil=Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences kn-affil= affil-num=6 en-affil=Institute of Plant Biology and Biotechnology, University of M?nster kn-affil= affil-num=7 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=8 en-affil=Institut de Biologie Physico-Chimique, Unit? Mixte de Recherche 7141, Centre National de la Recherche Scientifique and Sorbonne Universit? Pierre et Marie Curie kn-affil= affil-num=9 en-affil=Research Center for Advanced Science and Technology, The University of Tokyo kn-affil= affil-num=10 en-affil=Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences kn-affil= affil-num=11 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= affil-num=12 en-affil=Research Institute for Interdisciplinary Science, Okayama University kn-affil= affil-num=13 en-affil=Institute of Plant Science and Resources (IPSR), Okayama University kn-affil= en-keyword=post-translational modification kn-keyword=post-translational modification en-keyword=Arabidopsis thaliana kn-keyword=Arabidopsis thaliana en-keyword=protein degradation kn-keyword=protein degradation en-keyword=photosystem II kn-keyword=photosystem II en-keyword=photo-oxidative damage kn-keyword=photo-oxidative damage en-keyword=tryptophan oxidation kn-keyword=tryptophan oxidation en-keyword=Chlamydomonas reinhardtii kn-keyword=Chlamydomonas reinhardtii END 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 start-ver=1.4 cd-journal=joma no-vol=7 cd-vols= no-issue= article-no= start-page=7826 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2017 dt-pub=20170810 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization; en-subtitle= kn-subtitle= en-abstract= kn-abstract=@Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in -tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant -tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of -tubulin and the resulting destabilization of cortical microtubules. en-copyright= kn-copyright= en-aut-name=TakataniShogo en-aut-sei=Takatani en-aut-mei=Shogo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=OzawaShinichiro en-aut-sei=Ozawa en-aut-mei=Shinichiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=YagiNoriyoshi en-aut-sei=Yagi en-aut-mei=Noriyoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HottaTakashi en-aut-sei=Hotta en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HashimotoTakashi en-aut-sei=Hashimoto en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 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=6 ORCID= en-aut-name=TakahashiTaku en-aut-sei=Takahashi en-aut-mei=Taku kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=MotoseHiroyasu en-aut-sei=Motose en-aut-mei=Hiroyasu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=2 en-affil=Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Biological Science, Nara Institute of Science and Technology kn-affil= affil-num=4 en-affil=Graduate School of Biological Science, Nara Institute of Science and Technology kn-affil= affil-num=5 en-affil=Graduate School of Biological Science, Nara Institute of Science and Technology kn-affil= affil-num=6 en-affil=Graduate School of Natural Science and Technology/Faculty of Science, Okayama University kn-affil= affil-num=7 en-affil=Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University kn-affil= affil-num=8 en-affil=Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University kn-affil= en-keyword=Cell growth kn-keyword=Cell growth en-keyword=Microtubules kn-keyword=Microtubules en-keyword=Plant cytoskeleton kn-keyword=Plant cytoskeleton END start-ver=1.4 cd-journal=joma no-vol=1797 cd-vols= no-issue=2 article-no= start-page=278 end-page=284 dt-received= dt-revised= dt-accepted= dt-pub-year=2010 dt-pub=201002 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium en-subtitle= kn-subtitle= en-abstract= kn-abstract=Ycf12 (Psb30) and PsbZ are two low molecular weight subunits of photosystem II (PSII), with one and two trans-membrane helices, respectively. In order to study the functions of these two subunits from a structural point of view, we constructed deletion mutants lacking either Ycf12 or PsbZ from Thermosynechococcus elongatus, and purified, crystallized and analyzed the structure of PSII dimer from the two mutants. Our results showed that Ycf12 is located in the periphery of PSII, close to PsbK, PsbZ and PsbJ, and corresponded to the unassigned helix X1 reported previously, in agreement with the recent structure at 2.9 ? resolution (A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 ? resolution: role of quinones, lipids, channels and chloride, Nat. Struct. Mol. Biol. 16 (2009) 334?342). On the other hand, crystals of PsbZ-deleted PSII showed a remarkably different unit cell constants from those of wild-type PSII, indicating a role of PsbZ in the interactions between PSII dimers within the crystal. This is the first example for a different arrangement of PSII dimers within the cyanobacterial PSII crystals. PSII dimers had a lower oxygen-evolving activity from both mutants than that from the wild type. In consistent with this, the relative content of PSII in the thylakoid membranes was lower in the two mutants than that in the wild type. These results suggested that deletion of both subunits affected the PSII activity, thereby destabilized PSII, leading to a decrease in the PSII content in vivo. While PsbZ was present in PSII purified from the Ycf12-deletion mutant, Ycf12 was present in crude PSII but absent in the finally purified PSII from the PsbZ-deletion mutant, indicating a preferential, stabilizing role of PsbZ for the binding of Ycf12 to PSII. These results were discussed in terms of the PSII crystal structure currently available en-copyright= kn-copyright= en-aut-name=TakasakaKenji en-aut-sei=Takasaka en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=IwaiMasako en-aut-sei=Iwai en-aut-mei=Masako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=UmenaYasufumi en-aut-sei=Umena en-aut-mei=Yasufumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KawakamiKeisuke en-aut-sei=Kawakami en-aut-mei=Keisuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=OhmoriYukari en-aut-sei=Ohmori en-aut-mei=Yukari kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=IkeuchiMasahiko en-aut-sei=Ikeuchi en-aut-mei=Masahiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 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=7 ORCID= en-aut-name=KamiyaNobuo en-aut-sei=Kamiya en-aut-mei=Nobuo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=ShenJian-Ren en-aut-sei=Shen en-aut-mei=Jian-Ren kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil= kn-affil=Division of Bioscience, Graduate School of Natural Science and Technology/Faculty of Science; Okayama University affil-num=2 en-affil= kn-affil=Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science affil-num=3 en-affil= kn-affil=Department of Chemistry, Graduate School of Science, Osaka City University affil-num=4 en-affil= kn-affil=Division of Bioscience, Graduate School of Natural Science and Technology/Faculty of Science; Okayama University affil-num=5 en-affil= kn-affil=Division of Bioscience, Graduate School of Natural Science and Technology/Faculty of Science; Okayama University affil-num=6 en-affil= kn-affil=Department of Life Sciences (Biology), Graduate School of Arts and Science, The University of Tokyo affil-num=7 en-affil= kn-affil=Division of Bioscience, Graduate School of Natural Science and Technology/Faculty of Science; Okayama University affil-num=8 en-affil= kn-affil=Department of Chemistry, Graduate School of Science, Osaka City University affil-num=9 en-affil= kn-affil=Division of Bioscience, Graduate School of Natural Science and Technology/Faculty of Science; Okayama University en-keyword=Photosystem II kn-keyword=Photosystem II en-keyword=Mutant kn-keyword=Mutant en-keyword=Crystal structure kn-keyword=Crystal structure en-keyword=Ycf12 kn-keyword=Ycf12 en-keyword=PsbZ kn-keyword=PsbZ en-keyword=Oxygen evolution kn-keyword=Oxygen evolution END