2024/02/09 更新

写真a

オザワ シンイチロウ
小澤 真一郎
OZAWA Shinichirou
所属
資源植物科学研究所 助教(特任)
職名
助教(特任)
連絡先
メールアドレス

学位

  • 博士(理学) ( 岡山大学 )

研究キーワード

  • 色素

  • シロイヌナズナ

  • クラミドモナス

  • オオムギ

  • 光化学系

  • プロテオミクス

  • 葉緑体ATP合成酵素

  • 光合成複合体形成

  • 質量分析

  • 構造生物学

  • タンパク質精製

  • 光化学系I

  • 光合成

  • シトクロムb6f複合体

研究分野

  • ライフサイエンス / 生物物理学

  • ライフサイエンス / 分子生物学

  • ライフサイエンス / 構造生物化学

  • ライフサイエンス / 細胞生物学

  • ライフサイエンス / 植物分子、生理科学

学歴

  • 岡山大学   Graduate school of Natural Science and Technology, Doctor Course   Division of Bioscience

    2005年4月 - 2009年9月

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    国名: 日本国

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  • 岡山大学   Graduate school of Natural Science and Technology, Master Course   Division of Molecular and Biologocal Science

    2003年4月 - 2005年3月

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    国名: 日本国

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  • 岡山大学   Faculty of Science   Biology

    1999年4月 - 2003年3月

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    国名: 日本国

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経歴

  • 岡山大学資源植物科学研究所   助教(特任)

    2019年10月 - 現在

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  • 岡山大学   異分野基礎科学研究所 光合成・構造生物学研究コア 機能解析研究分野 高橋裕一郎グループ   助教(特任)

    2016年4月 - 2019年9月

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  • 岡山大学大学院自然科学研究科   理学部生物学科高橋裕一郎研究室   助教(特任)

    2013年12月 - 2016年3月

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  • フランス国立科学センター   UMR7141/マリー・キュリー大学 パリ生物物理化学研究所   博士研究員

    2010年5月 - 2013年11月

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  • 岡山大学大学院自然科学研究科   理学部生物学科高橋裕一郎研究室   博士研究員

    2009年10月 - 2010年4月

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所属学協会

  • The American Society of Plant Biologists

    2022年12月 - 現在

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  • 日本植物生理学会

    2003年1月 - 現在

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  • アメリカ遺伝学会

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  • 日本植物学会

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  • 日本光合成学会

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論文

  • Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts

    Yusuke Kato, Hiroshi Kuroda, Shin-Ichiro Ozawa, Keisuke Saito, Vivek Dogra, Martin Scholz, Guoxian Zhang, Catherine de Vitry, Hiroshi Ishikita, Chanhong Kim, Michael Hippler, Yuichiro Takahashi, Wataru Sakamoto

    eLife   12   2023年11月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:eLife Sciences Publications, Ltd  

    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.

    DOI: 10.7554/elife.88822

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    その他リンク: https://cdn.elifesciences.org/articles/88822/elife-88822-v1.xml

  • Chloroplast ATP synthase biogenesis requires peripheral stalk subunits AtpF and ATPG and stabilization of atpE mRNA by OPR protein MDE1

    Frédéric Chaux, Domitille Jarrige, Marcio Rodrigues‐Azevedo, Sandrine Bujaldon, Oliver D. Caspari, Shin‐Ichiro Ozawa, Dominique Drapier, Olivier Vallon, Yves Choquet, Catherine de Vitry

    The Plant Journal   2023年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    SUMMARY

    Chloroplast ATP synthase contains subunits of plastid and nuclear genetic origin. To investigate the coordinated biogenesis of this complex, we isolated novel ATP synthase mutants in the green alga Chlamydomonas reinhardtii by screening for high light sensitivity. We report here the characterization of mutants affecting the two peripheral stalk subunits b and b′, encoded respectively by the atpF and ATPG genes, and of three independent mutants which identify the nuclear factor MDE1, required to stabilize the chloroplast‐encoded atpE mRNA. Whole‐genome sequencing revealed a transposon insertion in the 3′UTR of ATPG while mass spectrometry shows a small accumulation of functional ATP synthase in this knock‐down ATPG mutant. In contrast, knock‐out ATPG mutants, obtained by CRISPR‐Cas9 gene editing, fully prevent ATP synthase function and accumulation, as also observed in an atpF frame‐shift mutant. Crossing ATP synthase mutants with the ftsh1‐1 mutant of the major thylakoid protease identifies AtpH as an FTSH substrate, and shows that FTSH significantly contributes to the concerted accumulation of ATP synthase subunits. In mde1 mutants, the absence of atpE transcript fully prevents ATP synthase biogenesis and photosynthesis. Using chimeric atpE genes to rescue atpE transcript accumulation, we demonstrate that MDE1, a novel octotricopeptide repeat (OPR) protein, genetically targets the atpE 5′UTR. In the perspective of the primary endosymbiosis (~1.5 Gy), the recruitment of MDE1 to its atpE target exemplifies a nucleus/chloroplast interplay that evolved rather recently, in the ancestor of the CS clade of Chlorophyceae, ~300 My ago.

    DOI: 10.1111/tpj.16448

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  • Two specific domains of the γ subunit of chloroplast F o F 1 provide redox regulation of the ATP synthesis through conformational changes 国際誌

    Kentaro Akiyama, Shin-Ichiro Ozawa, Yuichiro Takahashi, Keisuke Yoshida, Toshiharu Suzuki, Kumiko Kondo, Ken-ichi Wakabayashi, Toru Hisabori

    Proceedings of the National Academy of Sciences   120 ( 6 )   e2218187120   2023年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences  

    <jats:p>
    Chloroplast F
    <jats:sub>o</jats:sub>
    F
    <jats:sub>1</jats:sub>
    -ATP synthase (CF
    <jats:sub>o</jats:sub>
    CF
    <jats:sub>1</jats:sub>
    ) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CF
    <jats:sub>o</jats:sub>
    CF
    <jats:sub>1</jats:sub>
    complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga
    <jats:italic>Chlamydomonas reinhardtii</jats:italic>
    , we analyzed the ATP synthesis reaction and its regulation in CF
    <jats:sub>o</jats:sub>
    CF
    <jats:sub>1</jats:sub>
    . The domains in the γ subunit involved in the redox regulation of CF
    <jats:sub>o</jats:sub>
    CF
    <jats:sub>1</jats:sub>
    were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CF
    <jats:sub>o</jats:sub>
    CF
    <jats:sub>1</jats:sub>
    complex from
    <jats:italic>C. reinhardtii</jats:italic>
    and subsequently examined ATP synthesis activity by the acid–base transition method. We found that truncation of the β-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the β-hairpin and the redox loop domains specific to CF
    <jats:sub>o</jats:sub>
    CF
    <jats:sub>1</jats:sub>
    .
    </jats:p>

    DOI: 10.1073/pnas.2218187120

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  • Algal PETC-Pro171-Leu suppresses electron transfer in cytochrome b6f under acidic lumenal conditions

    Shin-Ichiro Ozawa, Felix Buchert, Ruby Reuys, Michael Hippler, Yuichiro Takahashi

    Plant Physiology   191 ( 3 )   1803 - 1817   2022年12月

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    担当区分:筆頭著者, 責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press (OUP)  

    Abstract

    Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

    DOI: 10.1093/plphys/kiac575

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  • Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana

    Hanaki Maeda, Koharu Takahashi, Yoshifumi Ueno, Kei Sakata, Akari Yokoyama, Kozue Yarimizu, Fumiyoshi Myouga, Kazuo Shinozaki, Shin-Ichiro Ozawa, Yuichiro Takahashi, Ayumi Tanaka, Hisashi Ito, Seiji Akimoto, Atsushi Takabayashi, Ryouichi Tanaka

    Journal of Plant Research   135 ( 2 )   361 - 376   2022年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media LLC  

    The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b559. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.

    DOI: 10.1007/s10265-022-01376-x

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    その他リンク: https://link.springer.com/article/10.1007/s10265-022-01376-x/fulltext.html

  • Assembly Apparatus of Light-Harvesting Complexes: Identification of Alb3.1–cpSRP–LHCP Complexes in the Green Alga Chlamydomonas reinhardtii

    Mithun Kumar Rathod, Sreedhar Nellaepalli, Shin-Ichiro Ozawa, Hiroshi Kuroda, Natsumi Kodama, Sandrine Bujaldon, Francis-André Wollman, Yuichiro Takahashi

    63 ( 1 )   70 - 81   2021年10月

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  • Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane. 国際誌

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

    Photosynthesis research   147 ( 1 )   107 - 124   2021年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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.

    DOI: 10.1007/s11120-020-00803-1

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  • Red-shifted chlorophyll a bands allow uphill energy transfer to photosystem II reaction centers in an aerial green alga, Prasiola crispa, harvested in Antarctica 査読

    Kosugi M, Ozawa SI, Takahashi Y, Kamei Y, Itoh S, Kudoh S, Kashino Y, Koike H

    Biochim Biophys Acta Bioenerg   1861 ( 2 )   148139 - 148147   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/J.BBABIO.2019.148139

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    その他リンク: http://orcid.org/0000-0001-7698-5350

  • The OPR Protein MTHI1 Controls the Expression of Two Different Subunits of ATP Synthase CFo in Chlamydomonas reinhardtii

    Shin-Ichiro Ozawa, Marina Cavaiuolo, Domitille Jarrige, Richard Kuras, Mark Rutgers, Stephan Eberhard, Dominique Drapier, Francis-André Wollman, Yves Choque

    32 ( 4 )   1179 - 1203   2020年1月

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    担当区分:筆頭著者   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1105/tpc.19.00770

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  • The BF4 and p71 antenna mutants from Chlamydomonas reinhardtii. 査読

    Bujaldon S, Kodama N, Rathod MK, Tourasse N, Ozawa SI, Sellés J, Vallon O, Takahashi Y, Wollman FA

    Biochimica et biophysica acta. Bioenergetics   1861 ( 4 )   148085 - 148085   2019年10月

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bbabio.2019.148085

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  • Structure of the green algal photosystem I supercomplex with a decameric light-harvesting complex I

    Michihiro Suga, Shin-Ichiro Ozawa, Kaori Yoshida-Motomura, Fusamichi Akita, Naoyuki Miyazaki, Yuichiro Takahashi

    5 ( 6 )   626 - 636   2019年6月

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41477-019-0438-4

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    その他リンク: https://www.nature.com/articles/s41477-019-0438-4

  • The photosystem I assembly apparatus consisting of Ycf3–Y3IP1 and Ycf4 modules 査読

    Nellaepalli Sreedhar, Ozawa Shin-Ichiro, Kuroda Hiroshi, Takahashi Yuichiro

    Nature Communications   9 ( 1 )   2439   2018年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41467-018-04823-3

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  • Configuration of Ten Light-Harvesting Chlorophyll a/b Complex I Subunits in Chlamydomonas reinhardtii Photosystem I 査読

    Ozawa Shin-Ichiro, Bald Till, Onishi Takahito, Xue Huidan, Matsumura Takunori, Kubo Ryota, Takahashi Hiroko, Hippler Michael, Takahashi Yuichiro

    Plant Physiology   178 ( 2 )   583 - 595   2018年

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1104/pp.18.00749

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  • Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization 査読

    Shogo Takatani, Shinichiro Ozawa, Noriyoshi Yagi, Takashi Hotta, Takashi Hashimoto, Yuichiro Takahashi, Taku Takahashi, Hiroyasu Motose

    SCIENTIFIC REPORTS   7   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    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 beta-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant beta-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of beta-tubulin and the resulting destabilization of cortical microtubules.

    DOI: 10.1038/s41598-017-08453-5

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  • Chloroplast-mediated regulation of CO2-concentrating mechanism by Ca2+-binding protein CAS in the green alga Chlamydomonas reinhardtii 査読

    Lianyong Wang, Takashi Yamano, Shunsuke Takane, Yuki Niikawa, Chihana Toyokawa, Shin-ichiro Ozawa, Ryutaro Tokutsu, Yuichiro Takahashi, Jun Minagawa, Yu Kanesaki, Hirofumi Yoshikawa, Hideya Fukuzawa

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   113 ( 44 )   12586 - 12591   2016年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    Aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic activity in CO2-limiting conditions by sensing environmental CO2 and light availability. Previously, a novel high-CO2-requiring mutant, H82, defective in the induction of the CCM, was isolated. A homolog of calcium (Ca2+)-binding protein CAS, originally found in Arabidopsis thaliana, was disrupted in H82 cells. Although Arabidopsis CAS is reported to be associated with stomatal closure or immune responses via a chloroplast-mediated retrograde signal, the relationship between a Ca2+ signal and the CCM associated with the function of CAS in an aquatic environment is still unclear. In this study, the introduction of an intact CAS gene into H82 cells restored photosynthetic affinity for inorganic carbon, and RNA-seq analyses revealed that CAS could function in maintaining the expression levels of nuclear-encoded CO2-limiting-inducible genes, including the HCO(3)(-)transporters high-light activated 3 (HLA3) and low-CO2-inducible gene A (LCIA). CAS changed its localization from dispersed across the thylakoid membrane in high-CO2 conditions or in the dark to being associated with tubule-like structures in the pyrenoid in CO2-limiting conditions, along with a significant increase of the fluorescent signals of the Ca2+ indicator in the pyrenoid. Chlamydomonas CAS had Ca2+-binding activity, and the perturbation of intracellular Ca2+ homeostasis by a Ca2+-chelator or calmodulin antagonist impaired the accumulation of HLA3 and LCIA. These results suggest that Chlamydomonas CAS is a Ca2+-mediated regulator of CCM-related genes via a retrograde signal from the pyrenoid in the chloroplast to the nucleus.

    DOI: 10.1073/pnas.1606519113

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  • D1 fragmentation in photosystem II repair caused by photo-damage of a two-step model 査読

    Yusuke Kato, Shin-ichiro Ozawa, Yuichiro Takahashi, Wataru Sakamoto

    PHOTOSYNTHESIS RESEARCH   126 ( 2-3 )   409 - 416   2015年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Light energy drives photosynthesis, but it simultaneously inactivates photosynthetic mechanisms. A major target site of photo-damage is photosystem II (PSII). It further targets one reaction center protein, D1, which is maintained efficiently by the PSII repair cycle. Two proteases, FtsH and Deg, are known to contribute to this process, respectively, by efficient degradation of photo-damaged D1 protein processively and endoproteolytically. This study tested whether the D1 cleavage accomplished by these proteases is affected by different monochromic lights such as blue and red light-emitting-diode light sources, remaining mindful that the use of these lights distinguishes the current models for photoinhibition: the excess-energy model and the two-step model. It is noteworthy that in the two-step model, primary damage results from the absorption of light energy in the Mn-cluster, which can be enhanced by a blue rather than a red light source. Results showed that blue and red lights affect D1 degradation differently. One prominent finding was that D1 fragmentation that is specifically generated by luminal Deg proteases was enhanced by blue light but not by red light in the mutant lacking FtsH2. Although circumstantial, this evidence supports a two-step model of PSII photo-damage. We infer that enhanced D1 fragmentation by luminal Deg proteases is a response to primary damage at the Mn-cluster.

    DOI: 10.1007/s11120-015-0144-7

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  • Requirement for Asn298 on D1 Protein for Oxygen Evolution: Analyses by Exhaustive Amino Acid Substitution in the Green Alga Chlamydomonas reinhardtii 査読

    Hiroshi Kuroda, Natsumi Kodama, Xiao-Yu Sun, Shin-ichiro Ozawa, Yuichiro Takahashi

    PLANT AND CELL PHYSIOLOGY   55 ( 7 )   1266 - 1275   2014年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    PSII generates strong oxidants used for water oxidation. The secondary electron donor, Y-Z, is Tyr161 on PSII reaction center D1 protein and mediates electron transfer from the oxygen-evolving Mn4CaO5\ cluster to the primary electron donor, P680. The latest PSII crystal structure revealed the presence of a hydrogen bond network around Y-Z, which is anticipated to play important roles in the electron and proton transfer reactions. Y-Z forms a hydrogen bond with His190 which in turn forms a hydrogen bond with Asn298 on D1 protein. Although functional roles of Y-Z and His190 have already been characterized, little is known about the functional role of Asn298. Here we have generated 19 mutants from a green alga Chlamydomonas reinhardtii, in which the Asn298 has been substituted by each of the other 19 amino acid residues. All mutants showed significantly impaired or no photosynthetic growth. Seven mutants capable of photosynthetic growth showed oxygen-evolving activity although at a significantly reduced rate. Interestingly the oxygen-evolving activity of these mutants was markedly photosensitive. The 19 mutants accumulated PSII at variable levels and showed a light-induced electron transfer reaction from 1,5-diphenylcarbazide (DPC) to 2,6-dichlorophenolin-dophenol (DCIP), suggesting that Asn298 is important for the function and photoprotection of the Mn4CaO5 cluster.

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  • Proton gradient regulation 5-mediated cyclic electron flow under ATP- or redox-limited conditions: a study of ΔATpase pgr5 and ΔrbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii. 査読

    Johnson X, Steinbeck J, Dent RM, Takahashi H, Richaud P, Ozawa S, Houille-Vernes L, Petroutsos D, Rappaport F, Grossman AR, Niyogi KK, Hippler M, Alric J

    Plant physiology   165 ( 1 )   438 - 452   2014年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • 5'-Monohydroxyphylloquinone is the Dominant Naphthoquinone of PSI in the Green Alga Chlamydomonas reinhardtii 査読

    Shin-ichiro Ozawa, Makiko Kosugi, Yasuhiro Kashino, Takashi Sugimura, Yuichiro Takahashi

    PLANT AND CELL PHYSIOLOGY   53 ( 1 )   237 - 243   2012年1月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Thylakoid membranes contain two types of quinones, benzoquinone (plastoquinone) and naphthoquinone, which are involved in photosynthetic electron transfer. Unlike the benzoquinone, the chemical species of naphthoquinone present (phylloquinone, menaquinone-4 and 5'-monohydroxyphylloquinone) varies depending on the oxygenic photosynthetic organisms. The green alga Chlamydomonas reinhardtii has been used as a model organism to study the function of the naphthoquinone bound to PSI. However, the level of phylloquinone and the presence of other naphthoquinones in this organism remain unknown. In the present study, we found that 5'-monohydroxyphylloquinone is the predominant naphthoquinone in cell and thylakoid extracts based on the retention time during reverse phase HPLC, absorption and mass spectrometry measurements. It was shown that 5'-monohydroxyphylloquinone is enriched 2.5-fold in the PSI complex as compared with thylakoid membranes but that it is absent from PSI-deficient mutant cells. We also found a small amount of phylloquinone in the cells and in the PSI complex and estimated that accumulated 5'-monohydroxyphylloquinone and phylloquinone account for approximately 90 and 10%, respectively, of the total naphthoquinone content. The ratio of these two naphthoquinones remained nearly constant in the cells and in the PSI complexes from logarithmic and stationary cell growth stages. We conclude that both 5'-monohydroxyphylloquinone and phylloquinone stably co-exist as major and minor naphthoquinones in Chlamydomonas PSI.

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  • Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system 査読

    Yuhei Chadani, Katsuhiko Ono, Shin-ichiro Ozawa, Yuichiro Takahashi, Kazuyuki Takai, Hideaki Nanamiya, Yuzuru Tozawa, Kazuhiro Kutsukake, Tatsuhiko Abo

    MOLECULAR MICROBIOLOGY   78 ( 4 )   796 - 808   2010年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    P&gt;Although SsrA(tmRNA)-mediated trans-translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in-frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome-rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA-arfA synthetic lethality was alleviated by SsrADD, an SsrA variant that adds a proteolysis-refractory tag through trans-translation, indicating that ArfA-deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the Delta arfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon-independent mimic of aminoacyl-tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non-stop mRNA, which was otherwise SsrA-tagged and degraded. In vitro, purified ArfA acted on a ribosome-nascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3' end of a non-stop mRNA without involving trans-translation.

    DOI: 10.1111/j.1365-2958.2010.07375.x

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  • Light and Low-CO2-Dependent LCIB-LCIC Complex Localization in the Chloroplast Supports the Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii 査読

    Takashi Yamano, Tomoki Tsujikawa, Kyoko Hatano, Shin-ichiro Ozawa, Yuichiro Takahashi, Hideya Fukuzawa

    PLANT AND CELL PHYSIOLOGY   51 ( 9 )   1453 - 1468   2010年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    The carbon-concentrating mechanism (CCM) is essential to support photosynthesis under CO2-limiting conditions in aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii. The CCM is assumed to be comprised of inorganic carbon transport systems that, in conjunction with carbonic anhydrases, maintain high levels of CO2 around ribulose-1, 5-bisphosphate carboxylase/oxygenase in a specific compartment called the pyrenoid. A set of transcripts up-regulated during the induction of the CCM was identified previously and designated as low-CO2 (LC)-inducible genes. Although the functional importance of one of these LC-inducible genes, LciB, has been shown recently, the biochemical properties and detailed subcellular localization of its product LCIB remain to be elucidated. Here, using yeast two-hybrid, immunoprecipitation and mass spectrometry analyses we provide evidence to demonstrate that LCIB interacts with the LCIB homologous protein LCIC in yeast and invivo. We also show that LCIB and LCIC are co-localized in the vicinity of the pyrenoid under LC conditions in the light, forming a hexamer complex of approximately 350kDa, as estimated by gel filtration chromatography. LCIB localization around the pyrenoid was dependent on light illumination and LC conditions during active operation of the CCM. In contrast, in the dark or under high-CO2 conditions when the CCM was inactive, LCIB immediately diffused away from the pyrenoid. Based on these observations, we discuss possible functions of the LCIBLCIC complex in the CCM.

    DOI: 10.1093/pcp/pcq105

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  • Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri 査読

    Wesley D. Swingley, Masakazu Iwai, Yang Chen, Shin-ichiro Ozawa, Kenji Takizawa, Yuichiro Takahashi, Jun Minagawa

    BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS   1797 ( 8 )   1458 - 1464   2010年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins. LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed. (C) 2010 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.bbabio.2010.04.017

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  • Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii 査読

    Shin-ichiro Ozawa, Takahito Onishi, Yuichiro Takahashi

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 26 )   20072 - 20079   2010年6月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12-15 core and 4-9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.

    DOI: 10.1074/jbc.M109.098954

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  • Biochemical and Structural Studies of the Large Ycf4-Photosystem I Assembly Complex of the Green Alga Chlamydomonas reinhardtii 査読

    Shin-ichiro Ozawa, Jon Nield, Akihiro Terao, Einar J. Stauber, Michael Hippler, Hiroyuki Koike, Jean-David Rochaix, Yuichiro Takahashi

    PLANT CELL   21 ( 8 )   2424 - 2442   2009年8月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC PLANT BIOLOGISTS  

    Ycf4 is a thylakoid protein essential for the accumulation of photosystem I (PSI) in Chlamydomonas reinhardtii. Here, a tandem affinity purification tagged Ycf4 was used to purify a stable Ycf4-containing complex of &gt; 1500 kD. This complex also contained the opsin-related COP2 and the PSI subunits PsaA, PsaB, PsaC, PsaD, PsaE, and PsaF, as identified by mass spectrometry (liquid chromatography-tandem mass spectrometry) and immunoblotting. Almost all Ycf4 and COP2 in wildtype cells copurified by sucrose gradient ultracentrifugation and subsequent ion exchange column chromatography, indicating the intimate and exclusive association of Ycf4 and COP2. Electron microscopy revealed that the largest structures in the purified preparation measure 285 x 185 angstrom; these particles may represent several large oligomeric states. Pulse-chase protein labeling revealed that the PSI polypeptides associated with the Ycf4-containing complex are newly synthesized and partially assembled as a pigment-containing subcomplex. These results indicate that the Ycf4 complex may act as a scaffold for PSI assembly. A decrease in COP2 to 10% of wild-type levels by RNA interference increased the salt sensitivity of the Ycf4 complex stability but did not affect the accumulation of PSI, suggesting that COP2 is not essential for PSI assembly.

    DOI: 10.1105/tpc.108.063313

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  • Photosystem II Complex in Vivo Is a Monomer 査読

    Takeshi Takahashi, Natsuko Inoue-Kashino, Shin-ichiro Ozawa, Yuichiro Takahashi, Yasuhiro Kashino, Kazuhiko Satoh

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 23 )   15598 - 15606   2009年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Photosystem II (PS II) complexes are membrane protein complexes that are composed of &gt;20 distinct subunit proteins. Similar to many other membrane protein complexes, two PS II complexes are believed to form a homo-dimer whose molecular mass is similar to 650 kDa. Contrary to this well known concept, we propose that the functional form of PS II in vivo is a monomer, based on the following observations. Deprivation of lipids caused the conversion of PS II from a monomeric form to a dimeric form. Only a monomeric PS II was detected in solubilized cyanobacterial and red algal thylakoids using blue-native polyacrylamide gel electrophoresis. Furthermore, energy transfer between PS II units, which was observed in the purified dimeric PS II, was not detected in vivo. Our proposal will lead to a re-evaluation of many crystallographic models of membrane protein complexes in terms of their oligomerization status.

    DOI: 10.1074/jbc.M109.000372

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  • Chloroplast-encoded polypeptide PsbT is involved in the repair of primary electron acceptor Q(A) of photosystem II during photoinhibition in Chlamydomonas reinhardtii 査読

    Norikazu Ohnishi, Yasuhiro Kashino, Kazuhiko Satoh, Shin-ichiro Ozawa, Yuichiro Takahashi

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 10 )   7107 - 7115   2007年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the D1/D2 heterodimer of the photosystem II (PSII) reaction center and is required for the efficient posttranslational repair of photodamaged PSII. Here we addressed that role in detail in Chlamydomonas reinhardtii wild type and Delta psbT cells by analyzing the activities of PSII, the assembly of PSII proteins, and the redox components of PSII during photoinhibition and repair. Strong illumination of cells for 15 min decreased the activities of electron transfer through PSII and Q, photoreduction by 50%, and it reduced the amount of atomic manganese by 20%, but it did not affect the steadystate level of PSII proteins, photoreduction of pheophytin (pheo(D1)), and the amount of bound plastoquinone (Q,), indicating that the decrease in PSII activity resulted mainly from inhibition of the electron transfer from pheo(D1) to Q(A). In wild type cells, we observed parallel recovery of electron transfer activity through PSII and Q, photoreduction, suggesting that the recovery of Q, activity is one of the rate-limiting steps of PSII repair. In Delta psbT cells, the repairs of electron transfer activity through PSII and of QA photoreduction activity were both impaired, but PSII protein turnover was unaffected. Moreover, about half the QA was lost from the PSI1 core complex during purification. Since PsbT is intimately associated with the QA-binding region on D2, we propose that this polypeptide enhances the efficient recovery of QA photoreduction by stabilizing the structure of the Q.A-binding region.

    DOI: 10.1074/jbc.M606763200

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MISC

  • 光化学系I複合体の分子集合機構の解明の進展 査読

    ネレパリスリーダー, 小澤 真一郎, 高橋裕一郎

    光合成研究   28 ( 3 )   148 - 156   2018年12月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(その他)  

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  • Identification of β-Cryptoxanthin in Oxygen-Evolving Photosystem II from Thermosynechococcus vulcanus 査読

    Keisuke Kawakami, Ritsuko Fujii, Yasufumi Umena, Shin-ichiro Ozawa, Yuichiro Takahashi, Hideki Hashimoto, Jian-Ren Shen, Nobuo Kamiya

    CAROTENOID SCIENCE   19   48 - 51   2015年

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    記述言語:英語   掲載種別:記事・総説・解説・論説等(その他)  

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  • 緑藻クラミドモナスの無機炭素濃縮機構を制御する因子CCM1の相互作用因子および翻訳後修飾基の同定

    佐々木優, 中野博文, 山原洋佑, 小澤真一郎, 高橋裕一郎, 福澤秀哉

    日本植物生理学会年会要旨集   51st   2010年

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  • STRUCTURE AND FUNCTION OF CCM1 COMPLEX CONTROLLING CO2-CONCENTRATING MECHANISM IN A GREEN ALGA, CHLAMYDOMONAS REINHARTII 査読

    H. Fukuzawa, Y. Yamahara, H. Nakano, S. Ozawa, Y. Takahashi

    PHYCOLOGIA   48 ( 4 )   33 - 33   2009年7月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:INT PHYCOLOGICAL SOC  

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  • タンパク質の質量分析 招待 査読

    小澤 真一郎, 井上-菓子野, 名津子, 高橋 裕一郎

    低温科学   67   387 - 395   2009年3月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(大学・研究所紀要)  

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  • 界面活性剤 招待 査読

    小澤 真一郎, 高橋 裕一郎

    Low Temperature Science   67   409 - 413   2009年3月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(大学・研究所紀要)  

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  • カラムクロマトグラフィー 招待 査読

    小澤 真一郎, 岡室 彰, 高橋 裕一郎

    低温科学   67   397 - 407   2009年3月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(大学・研究所紀要)  

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  • CO2濃縮機構を制御するCCM1複合体の構成因子P80の解析

    中野博文, 山原洋佑, 小澤真一郎, 高橋裕一郎, 福澤秀哉

    生化学   2009年

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  • 無機炭素濃縮機構を制御する因子CCM1複合体の構成因子P80の解析

    中野博文, 山原洋佑, 小澤真一郎, 高橋裕一郎, 福澤秀哉

    日本植物生理学会年会要旨集   50th   2009年

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  • 緑藻クラミドモナスにおける無機炭素濃縮機構を制御するCCM1複合体の質量分析法を用いた解析

    山原洋佑, 中野博文, 小澤真一郎, 高橋裕一郎, 福澤秀哉

    日本植物生理学会年会要旨集   49th   2008年

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  • Molecular dynamics of photosynthetic reaction center complexes using chloroplast transformation in the green alga Chlamydomonas reinhardtii 査読

    Yuichiro Takahashi, Shin-ichiro Ozawa, Takahito Onishi, Mei Funakawa

    GENES & GENETIC SYSTEMS   82 ( 6 )   514 - 514   2007年12月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:GENETICS SOC JAPAN  

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  • Probing the structure of a Ycf4-containing protein complex involved in green algal Photosystem I assembly 査読

    M. Cullen, S. Ozawa, Y. Takahashi, J. Nield

    PHOTOSYNTHESIS RESEARCH   91 ( 2-3 )   207 - 207   2007年2月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SPRINGER  

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  • Identification and characterization of a semi-stable assembly intermediate of photosystem I complex 査読

    Y. Takahashi, S. Ozawa, T. Onishi

    PHOTOSYNTHESIS RESEARCH   91 ( 2-3 )   205 - 206   2007年2月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:SPRINGER  

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  • Identification and characterization of an assembly intermediate of photosystem I complex in Chlamlydomonas reinhardtii 査読

    Shin-ichiro Ozawa, Takahito Onishi, Yuichiro Takahashi

    PLANT AND CELL PHYSIOLOGY   48   S127 - S127   2007年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:OXFORD UNIV PRESS  

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  • Structure and assembly of photosystem I complex

    Y Takahashi, S Ozawa, T Ohnishi

    PLANT AND CELL PHYSIOLOGY   47   S10 - S10   2006年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:OXFORD UNIV PRESS  

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  • Purification and biochemical characterization of the assembly apparatus of photosystem I complex

    S Ozawa, Y Takahashi

    PLANT AND CELL PHYSIOLOGY   46   S178 - S178   2005年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:OXFORD UNIV PRESS  

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  • Purification of assembly apparatus of photosystem I complex in the green alga Chlamydomonas reinhardtii

    S Ozawa, H Koike, Y Takahashi

    PLANT AND CELL PHYSIOLOGY   45   S42 - S42   2004年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:OXFORD UNIV PRESS  

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  • Purification of assembly apparatus of photosystem I complex in the green alga Chlamydomonas reinhardtii

    S Ozawa, A Terao, Y Takahashi

    PLANT AND CELL PHYSIOLOGY   44   S155 - S155   2003年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:OXFORD UNIV PRESS  

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受賞

  • Poster recognition

    2006年6月   The 12th International Conference on the Cell and Molecular Biology of Chlamydomonas  

    小澤 真一郎

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  • 岡山大学自然科学研究科研究科長賞

    2005年3月   岡山大学自然科学研究科  

    小澤 真一郎

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共同研究・競争的資金等の研究

  • チラコイド膜リモデリング因子の構造に基づく光合成環境適応原理

    研究課題/領域番号:23H04959  2023年04月 - 2028年03月

    日本学術振興会  科学研究費助成事業  学術変革領域研究(A)

    坂本 亘, 小澤 真一郎

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    配分額:111540000円 ( 直接経費:85800000円 、 間接経費:25740000円 )

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  • 光合成における電子伝達と光捕集システムの相互制御の検証

    研究課題/領域番号:21K06217  2021年04月 - 2024年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    小澤 真一郎

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    配分額:4160000円 ( 直接経費:3200000円 、 間接経費:960000円 )

    光合成明反応では電子伝達と集光性アンテナによる光エネルギー利用効率が適切に制御されることが重要となる。本研究では両者の相関を明らかにすることをめざす。広い範囲の光強度で適切に電子伝達を制御し光合成を行ない、遺伝子情報が整備され遺伝子改変も可能な緑藻クラミドモナスを材料とする。
    強光下でチラコイド膜ルーメンが過度に酸性化するとPhotosynthetic controlが誘導され電子伝達活性を抑制し光合成器官の損傷を防ぐと考えられている。シロイヌナズナの pgr1 変異株は、より弱いルーメン酸性化で電子伝達速度が抑制されるため、プロトン駆動力の形成が不十分となり、NPQの誘導が低下する。緑藻クラミドモナスにシロイヌナズナpgr1変異に相当する変異を導入したPETC-P171L株を作出し解析した。PETC-P171L株はシロイヌナズナよりも低い光強度下でPhotosynthetic controlが誘導された。またPETC-P171L株は強光下で光合成的生育速度が低下し、NPQ誘導が低下することを見いだした。
    緑藻クラミドモナスの光化学系I複合体(PSI)に結合するアンテナタンパク質複合体(LHCI)は高い光エネルギー利用調節機能をもつことが構造から示唆された。緑藻クラミドモナスのLHCIを構成するサブユニットをコードする9種類の遺伝子のうち7種類の遺伝子について、それぞれの単一遺伝子欠損株を解析した。細胞の蛍光発光スペクトルを液体窒素温度で測定すると、LHCA3またはLHCA7欠損株において700 nm近傍の成分が野生株と比較して短波長側へシフトしシグナル強度も大きかった。さらにこれらの欠損株からPSIを精製すると、LHCIの構造が不安定となっていた。よってLHCA3またはLHCA7欠損はLHCIの構造を不安定化しLHCIからPSIへのエネルギー移動効率が低下すると考えられる。

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  • Purification and Biochemical characterization of the Assembly apparatus of PhotosystemⅠ complex

    2005年 - 2007年

    Grant-in-Aid for Scientific Research 

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    資金種別:競争的資金

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  • 光化学系Ⅰ複合体の分子集合装置の精製ならびにその機能と構造の解析

    2005年 - 2007年

    その他の研究制度 

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    資金種別:競争的資金

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  • 光化学系I複合体の分子集合装置の精製ならびにその機能と構造の解析

    研究課題/領域番号:05J03605  2005年 - 2007年

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    小澤 真一郎

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    配分額:2700000円 ( 直接経費:2700000円 )

    系I複合体の分子集合メカニズムには未知の部分が多い。本研究では、緑藻クラミドモナスを材料として、系I複合体の分子集合に必須であるYcf4を含む大きな複合体(以下、分子集合装置と略記)の構造・機能解析を進める。アフィニティーカラムで精製した分子集合装置をショ糖密度勾配超遠心分離で精製後、ゲル濾過カラムクロマトグラフィーと電子顕微鏡を用いた単粒子解析を行い、1500kDa以上の大きな構造体であることを示した。
    系I複合体が分子集合する過程を明確に示した研究はこれまで殆どなされていない。本研究では、パルスラベル・チェイス実験と、カラムクロマトグラフィー、SDS-PAGEを駆使し、研究を進めた。その結果、系I複合体の周辺部に位置しているPsaG、Kサブユニットが結合することにより、LHCIとPSIコアとの結合が安定化し、系I複合体の分子集合が完結するという過程を示した。
    より詳細に系I複合体の分子集合過程を研究するためには、PSI-LHCIの構造を知る必要がある。そこで本研究では、PSI-LHCIのアンテナザイズ、LHCIタンパク質のコピー数を生化学的に求めた。
    クラミドモナスにおけるPSI-LHCIのアンテナザイズを、P700の光酸化とHPLCにより求めた。これらの結果により、アンテナサイズを270-300クロロフィル程度であると決定した。次に、PSI-LHCIにおける、PSIコアに対するLHCIタンパク質のコピー数を求めた。High-TrisとMES-Trisを組み合わせた二種類の電気泳動システムによって、各LHCIタンパク質をシングルスポットとして高度に分離したのち、蛍光色素によって染色し定量した。PSIコアに対して8-9のLHCIタンパク質が結合しており、これはアンテナサイズの結果とも良く一致している。

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