eLife Sciences Publications Acta Medica Okayama 2050-084X 12 2023 Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts RP88822 EN Yusuke Kato Institute of Plant Science and Resources (IPSR), Okayama University Hiroshi Kuroda Research Institute for Interdisciplinary Science, Okayama University Shin-Ichiro Ozawa Institute of Plant Science and Resources (IPSR), Okayama University Keisuke Saito Research Center for Advanced Science and Technology, The University of Tokyo Vivek Dogra Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences Martin Scholz Institute of Plant Biology and Biotechnology, University of Münster Guoxian Zhang Institute of Plant Science and Resources (IPSR), Okayama University Catherine de Vitry Institut de Biologie Physico-Chimique, Unité Mixte de Recherche 7141, Centre National de la Recherche Scientifique and Sorbonne Université Pierre et Marie Curie Hiroshi Ishikita Research Center for Advanced Science and Technology, The University of Tokyo Chanhong Kim Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences Michael Hippler Institute of Plant Science and Resources (IPSR), Okayama University Yuichiro Takahashi Research Institute for Interdisciplinary Science, Okayama University Wataru Sakamoto Institute of Plant Science and Resources (IPSR), Okayama University 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. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0166-8595 147 1 2021 Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane 107 124 EN Keiji Nishioka Institute of Plant Science and Resources (IPSR), Okayama University Yusuke Kato Institute of Plant Science and Resources (IPSR), Okayama University Shin-ichiro Ozawa Institute of Plant Science and Resources (IPSR), Okayama University Yuichiro Takahashi Research Institute for Interdisciplinary Science, Okayama University Wataru Sakamoto Institute of Plant Science and Resources (IPSR), Okayama University 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. No potential conflict of interest relevant to this article was reported.

Nature Publishing Group Acta Medica Okayama 20452322 7 2017 Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization; 7826 EN Shogo Takatani Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Shinichiro Ozawa Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Noriyoshi Yagi Graduate School of Biological Science, Nara Institute of Science and Technology Takashi Hotta Graduate School of Biological Science, Nara Institute of Science and Technology Takashi Hashimoto Graduate School of Biological Science, Nara Institute of Science and Technology Yuichiro Takahashi Graduate School of Natural Science and Technology/Faculty of Science, Okayama University Taku Takahashi Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University Hiroyasu Motose Department of Biological Science, Graduate School of Natural Science and Technology, Okayama University 　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 β-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant β-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of β-tubulin and the resulting destabilization of cortical microtubules. No potential conflict of interest relevant to this article was reported.

Elsevier Science BV. Acta Medica Okayama 0005-2728 1797 2 2010 Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium 278 284 EN Kenji Takasaka Masako Iwai Yasufumi Umena Keisuke Kawakami Yukari Ohmori Masahiko Ikeuchi Yuichiro Takahashi Nobuo Kamiya Jian-Ren Shen 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 Å resolution (A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å 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 No potential conflict of interest relevant to this article was reported.