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フロンティア科学総合研究センター 実験支援部門ＲＩ分野ＲＩ清武分室 |
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Thewasano N., Germany E.M., Maruno Y., Nakajima Y., Shiota T.
Journal of Biological Chemistry 299 ( 7 ) 104821 2023年7月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
The outer membrane (OM) of gram-negative bacteria is populated by various outer membrane proteins (OMPs) that fold into a unique β-barrel transmembrane domain. Most OMPs are assembled into the OM by the β-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is composed of two essential proteins (BamA and BamD) and three nonessential accessory proteins (BamB, BamC, and BamE). The currently proposed molecular mechanisms of the BAM complex involve only essential subunits, with the function of the accessory proteins remaining largely unknown. Here, we compared the accessory protein requirements for the assembly of seven different OMPs, 8- to 22-stranded, by our in vitro reconstitution assay using an E. coli mid-density membrane. BamE was responsible for the full efficiency of the assembly of all tested OMPs, as it enhanced the stability of essential subunit binding. BamB increased the assembly efficiency of more than 16-stranded OMPs, whereas BamC was not required for the assembly of any tested OMPs. Our categorization of the requirements of BAM complex accessory proteins in the assembly of substrate OMPs enables us to identify potential targets for the development of new antibiotics.
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Molecular characterization of SrSTP14, a sugar transporter from thermogenic skunk cabbage, and its possible role in developing pollen.
Koyamatsu D, Otsubo M, Ohira T, Sato MP, Suzuki-Masuko H, Shiota T, Takano KT, Ozeki M, Otsuka K, Ogura Y, Hayashi T, Watanabe M, Inaba T, Ito-Inaba Y
Physiologia plantarum e13957 2023年6月
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Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila
Ikegawa Y., Combet C., Groussin M., Navratil V., Safar-Remali S., Shiota T., Aouacheria A., Yoo S.K.
EMBO Journal 42 ( 8 ) e110454 2023年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：EMBO Journal
Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3-only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. Here, we identify the first Drosophila BH3-only protein and name it sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonara-mediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. To our knowledge, this is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.
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Chen X., Ding Y., Bamert R.S., Le Brun A.P., Duff A.P., Wu C.M., Hsu H.Y., Shiota T., Lithgow T., Shen H.H.
Biochimica et Biophysica Acta - Biomembranes 1863 ( 9 ) 183587 2021年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Biochimica et Biophysica Acta - Biomembranes
In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex catalyses the assembly of β-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - β-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential β-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane.
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Ding Y., Shiota T., Le Brun A.P., Dunstan R.A., Wang B., Hsu H.Y., Lithgow T., Shen H.H.
Biochimica et Biophysica Acta - Biomembranes 1862 ( 9 ) 183317 - 183317 2020年9月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Biochimica et Biophysica Acta - Biomembranes
© 2020 In Gram–negative bacteria, the multi-protein β-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling β-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl β-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-L-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.
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ミトコンドリアポリンタンパク質Por1による外膜透過装置TOM複合体のアセンブリー制御
阪上 春花, 塩田 拓也, 石坂 直也, 田村 康, 遠藤 斗志也
日本生化学会大会プログラム・講演要旨集 91回 [2T13a - 07(2P 2018年9月
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） 出版者・発行元：(公社)日本生化学会
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ミトコンドリアポリンタンパク質Por1は外膜透過装置TOM複合体のアセンブリー調節因子として機能する
阪上 春花, 石坂 直也, 塩田 拓也, 田村 康, 遠藤 斗志也
生命科学系学会合同年次大会 2017年度 [2P - 0271] 2017年12月
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） 出版者・発行元：生命科学系学会合同年次大会運営事務局
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[Molecular architecture and function of mitochondrial protein entry gate].
Shiota T
Seikagaku. The Journal of Japanese Biochemical Society 89 ( 2 ) 282 - 285 2017年4月
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Tom22のミトコンドリア局在化におけるPor1の役割の解明
石坂 直也, 塩田 拓也, 田村 康, 遠藤 斗志也
日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [2P0021] - [2P0021] 2015年12月
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） 出版者・発行元：(公社)日本生化学会
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Mitochondrial biogenesis: Cell-cycle-dependent investment in making mitochondria
Shiota T., Traven A., Lithgow T.
Current Biology 25 ( 2 ) R78 - R80 2015年1月
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） 出版者・発行元：Current Biology
© 2015 Elsevier Ltd. All rights reserved. Mitochondria cannot be made de novo, so pre-existing mitochondria must be inherited at each cell division. A new study demonstrates cell-cycle-dependent regulation of the activity of the TOM translocase complex to induce mitochondrial biogenesis during the M phase of the cell cycle.
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中性子反射率法による生体膜解析法の開発
研究課題/領域番号：22K12672 2022年04月 - 2025年03月
独立行政法人日本学術振興会 科学研究費補助金 基盤研究(C)
担当区分：研究代表者
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中性子反射率法を中心とした多角的な解析によるグラム陰性菌の膜生合成解析
研究課題/領域番号：21KK0126 2021年04月 - 2024年03月
独立行政法人日本学術振興会 科学研究費補助金 国際共同研究加速基金(国際共同研究強化(B))
担当区分：研究代表者
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EMMアセンブリーアッセイによる大腸菌βバレル型膜タンパク質の膜挿入機構の解析
2018年 - 2020年03月
科学研究費補助金 研究活動スタート支援