塩田　拓也 (シオタ　タクヤ)

SHIOTA Takuya

写真a

職名

准教授
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  • Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane

    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09月

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    　概要を見る

    © 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.

    DOI PubMed

  • Structure of the mitochondrial import gate reveals distinct preprotein paths

    Araiso Y., Tsutsumi A., Qiu J., Imai K., Shiota T., Song J., Lindau C., Wenz L.S., Sakaue H., Yunoki K., Kawano S., Suzuki J., Wischnewski M., Schütze C., Ariyama H., Ando T., Becker T., Lithgow T., Wiedemann N., Pfanner N., Kikkawa M., Endo T.

    Nature   575 ( 7782 ) 395 - 401   2019年11月

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    © 2019, The Author(s), under exclusive licence to Springer Nature Limited. The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1–4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5–9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1–3. Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

    DOI PubMed

  • Structure of the mitochondrial import gate reveals distinct preprotein paths

    Y. Araiso, A. Tsutsumi, J. Qiu, K. Imai, T. Shiota, J. Song, C. Lindau, L.-S. Wenz, H. Sakaue, K. Yunoki, S. Kawano, J. Suzuki, M. Wischnewski, C. Schütze, H. Ariyama, T. Ando, T. Becker, T. Lithgow, N. Wiedemann, N. Pfanner, M. Kikkawa, T. Endo

    Nature   575   395 - 401   2019年10月

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  • Porin Associates with Tom22 to Regulate the Mitochondrial Protein Gate Assembly

    Sakaue H., Shiota T., Ishizaka N., Kawano S., Tamura Y., Tan K.S., Imai K., Motono C., Hirokawa T., Taki K., Miyata N., Kuge O., Lithgow T., Endo T.

    Molecular Cell   73 ( 5 ) 1044 - 1055.e8   2019年03月

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    © 2019 Elsevier Inc. The mitochondrial protein entry gate, the TOM complex, undergoes a dynamic conversion between the fully assembled trimer and the dimer lacking Tom22. Mitochondrial porin, Por1, regulates the trimer assembly by chaperoning newly imported Tom22 and dimer-trimer dependent functions of the TOM complex together with Tom6.

    DOI PubMed

  • The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane

    Gunasinghe S., Shiota T., Stubenrauch C., Schulze K., Webb C., Fulcher A., Dunstan R., Hay I., Naderer T., Whelan D., Bell T., Elgass K., Strugnell R., Lithgow T.

    Cell Reports   23 ( 9 ) 2782 - 2794   2018年05月

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    © 2018 The Author(s) The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly. Bacteria grow and divide by assembling new material into their surface membranes. Gunasinghe et al. used super-resolution microscopy and in situ crosslinking in live bacterial cells in order to visualize intimate contacts between BAM complexes suggesting a model whereby bacteria use highly organized precincts to drive membrane protein assembly.

    DOI PubMed

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総説・解説記事 【 表示 ／ 非表示 】

  • 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 ( (公社)日本生化学会 )  91回   [2T13a - 07(2P   2018年09月

    総説・解説（大学・研究所紀要）   共著

  • 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 ( 生命科学系学会合同年次大会運営事務局 )  2017年度   [2P - 0271]   2017年12月

    総説・解説（大学・研究所紀要）   共著

  • [Molecular architecture and function of mitochondrial protein entry gate].

    Shiota T

    Seikagaku. The Journal of Japanese Biochemical Society   89 ( 2 ) 282 - 285   2017年04月

    総説・解説（大学・研究所紀要）   単著

    PubMed

  • 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 ( (公社)日本生化学会 )  88回・38回   [2P0021] - [2P0021]   2015年12月

    総説・解説（大学・研究所紀要）   共著

  • Mitochondrial biogenesis: Cell-cycle-dependent investment in making mitochondria

    Shiota T., Traven A., Lithgow T.

    Current Biology ( Current Biology )  25 ( 2 ) R78 - R80   2015年01月

    総説・解説（大学・研究所紀要）   共著

    　概要を見る

    © 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.

    DOI PubMed

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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

    研究活動スタート支援

    研究期間:  2018年  -  2020年03月 

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