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Frontier Science Research Center Department of Biological Science Division of RI Research Kiyotake Section |
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Associate Professor |
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Related SDGs |
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SHIOTA Takuya
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Papers 【 display / non-display 】
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Germany E.M., Thewasano N., Imai K., Maruno Y., Bamert R.S., Stubenrauch C.J., Dunstan R.A., Ding Y., Nakajima Y., Lai X.F., Webb C.T., Hidaka K., Tan K.S., Shen H., Lithgow T., Shiota T.
eLife 12 2024.1
Language：English Publishing type：Research paper (scientific journal) Publisher：eLife
Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'β-signal' imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the β-signal, arranging several β-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.
DOI： 10.7554/eLife.90274
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Germany E., Shiota T.
Methods in Enzymology 707 237 - 256 2024.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Methods in Enzymology
The BPA photo-crosslinking method exploits the property of p-benzoyl-L-phenylalanine (pBpa), an amino acid containing a photoreactive side chain, and allows for the crosslinking with nearby proteins upon Ultraviolet irradiation. This feature enables the capture of two proteins within a close proximity with high spatial resolution at the level of amino acid residues. In this chapter, we introduce an example of the employment of the BPA photo-crosslinking method to the Translocase of the Outer Mitochondrial membrane complex of mitochondria in Saccharomyces cerevisiae as a model protein translocase. Here in, we provide three procedures (i) the introduction of pBpa into proteins of interest in living yeast cells by in vivo suppressor tRNA system; (ii) analysis of in vivo subunit-subunit interactions intra-complex; and (iii) analysis of translocase channel-substrate interactions in organello. The use of in vivo and in organello crosslinking tools enable the robust analysis of translocases in a near-to physiological condition.
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Aoki E., Germany E., Shiota T.
Methods in molecular biology (Clifton, N.J.) 2778 65 - 81 2024
Language：English Publishing type：Research paper (scientific journal) Publisher：Methods in molecular biology (Clifton, N.J.)
The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of β-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli (E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented β-barrel membrane proteins via intact β-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled β-barrel membrane proteins by demonstrating four representative β-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.
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Categorization of Escherichia coli Outer Membrane Proteins by Dependence on Accessory Proteins of the β-barrel Assembly Machinery Complex. International journal
Nakajohn Thewasano, Edward M Germany, Yuki Maruno, Yukari Nakajima, Takuya Shiota
The Journal of biological chemistry 299 ( 7 ) 104821 - 104821 2023.7
Language：English Publishing type：Research paper (scientific journal)
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 non-essential 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 (EMM). 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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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 175 ( 4 ) e13957 2023.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Physiologia Plantarum
In floral thermogenesis, sugars play an important role not only as energy providers but also as growth and development facilitators. Yet, the mechanisms underlying sugar translocation and transport in thermogenic plants remain to be studied. Asian skunk cabbage (Symplocarpus renifolius) is a species that can produce durable and intense heat in its reproductive organ, the spadix. Significant morphological and developmental changes in the stamen are well-characterized in this plant. In this study, we focused on the sugar transporters (STPs), SrSTP1 and SrSTP14, whose genes were identified by RNA-seq as the upregulated STPs during thermogenesis. Real-time PCR confirmed that mRNA expression of both STP genes was increased from the pre-thermogenic to the thermogenic stage in the spadix, where it is predominantly expressed in the stamen. SrSTP1 and SrSTP14 complemented the growth defects of a hexose transporter-deficient yeast strain, EBY4000, on media containing 0.02, 0.2, and 2% (w/v) glucose and galactose. Using a recently developed transient expression system in skunk cabbage leaf protoplasts, we revealed that SrSTP1 and SrSTP14-GFP fusion proteins were mainly localized to the plasma membrane. To dig further into the functional analysis of SrSTPs, tissue-specific localization of SrSTPs was investigated by in situ hybridization. Using probes for SrSTP14, mRNA expression was observed in the microspores within the developing anther at the thermogenic female stage. These results indicate that SrSTP1 and SrSTP14 transport hexoses (e.g., glucose and galactose) at the plasma membrane and suggest that SrSTP14 may play a role in pollen development through the uptake of hexoses into pollen precursor cells.
DOI： 10.1111/ppl.13957
Books 【 display / non-display 】
MISC 【 display / non-display 】
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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 2018.9
Language：Japanese Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution) Publisher：(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] 2017.12
Language：Japanese Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution) Publisher：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 2017.4
Language：Japanese Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)
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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] 2015.12
Language：Japanese Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution) Publisher：(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 2015.1
Language：Japanese Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution) Publisher：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.
Presentations 【 display / non-display 】
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BamC, the last uncharacterized subunit of the BAM complex, is essential for the growth of L-form E. coli Invited International coauthorship International conference
NIH seminar 2025.5.29
Event date： 2025.5.29
Language：English Presentation type：Public lecture, seminar, tutorial, course, or other speech
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Assembly mechanisms of β-barrel membrane proteins in mitochondria and Gram-negative bacteria Invited International conference
Takuya Shiota
NIG-JOINT 2025 International Symposium on Molecular Biology and Microbiology: Frontiers in microbial research providing a fundamental insight into conserved and diverse nature of cell duplication system 2026.3.2
Event date： 2026.3.2
Language：English Presentation type：Oral presentation (invited, special)
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甲南大学生物学科の卒業生がβバレル型膜タンパク質の虜になり大学教員になるまで Invited International coauthorship
塩田 拓也
甲南生物学セミナー／統合ニューロバイオロジー研究所セミナー 2026.1.9
Event date： 2026.1.9
Language：Japanese Presentation type：Public lecture, seminar, tutorial, course, or other speech
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β-barrel membrane proteins assembly in bacteria and mitochondria. Invited
Takuya Shiota
Melbourne - Japan workshop Junctions of Mitochondrial and Bacterial sciences 2025.12.17
Event date： 2025.12.17
Language：English Presentation type：Oral presentation (general)
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Bottom-up drug discovery for antimicrobial drug development targeting outer membrane proteins of Gram-negative bacteria
Shiota Takuya, Germany Edward, Maruno Yuki, Thewasano Nakajohn, Imai Kenichiro, Abiru Yuka, Shen Hsin Hui, Lithgow Trevor, Shiomi Daisuke
Supplement of Association of Next Generation Scientists Seminar in The Japanese Pharmacologigal Society 2025 The Japanese Pharmacology Society
Event date： 2025
Language：Japanese Presentation type：Oral presentation (general)
グラム陰性菌は、WHOによる「喫緊に対策が必要な微生物感染症リスト」の実に70%を占めるグループである。グラム陰性菌に対する薬剤治療は、外膜というバリアの存在が問題である。外膜は、内側がリン脂質、外側がLPSからなる非対称膜に、リポタンパク質と外膜タンパク質が存在して形成されている。外膜タンパク質は外膜の主たる構成因子であると同時に、全ての外膜の構成要素の生合成に関与しており、そのバリア機能にとって最も重要な分子である。外膜タンパク質は、膜貫通領域がβバレル構造をとり、この立体構造形成を伴った膜挿入（アセンブリー）が機能を発揮するためには必須のプロセスである。アセンブリーは、グラム陰性菌の外膜に広く存在するBeta-barrel Assembly Machinery (BAM)複合体と呼ばれる分子装置によって行われる。BAM複合体はグラム陰性菌の生育に必須であるため、魅力的な抗菌薬の標的である。事実、BAM複合体への効果的な阻害剤が天然物や化合物ライブラリーから探索するトップダウン創薬により単離されている。しかしながら、これらの化合物は全てBAM複合体のラテラルゲートと呼ばれる触媒部位を標的としている。トップダウン創薬では、最も効果が強い部位にヒットが集中し、耐性菌に対して多様な選択肢を生むための、より広範な部位を標的とする創薬ができない可能性がある。より広範な部位をそれぞれ標的とするためには、詳細な分子機構の解析をもとに標的部位を増やした上で、それらに対して個別に阻害剤を見出していくボトムアップ創薬が求められる。我々は、グラム陰性菌から単離した膜画分を用いたin vitro再構築系として、EMMアセンブリーアッセイを開発した。この方法は検出感度が高く、変異体や阻害剤によるアセンブリーの影響を敏感に捉えることができる。この方法を用いることで、新規の薬剤標的として輸送される外膜タンパク質内に存在する内部シグナルとBAM複合体中の受容体BamDの関係性を明らかにした。さらに、抗菌薬が実際に作用する環境に着目することで、BAM複合体の生体内での必須遺伝子を発見した。これらは、重要な標的部位の拡大、さらには新規抗菌薬の開発に資するものである。
Grant-in-Aid for Scientific Research 【 display / non-display 】
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αヘリカル膜タンパク質のミトコンドリア膜への膜挿入機構の構造生物学的理解
Grant number：25K00101 2025.04 - 2028.03
独立行政法人日本学術振興会 科学研究費基金 基盤研究(B)
Authorship：Coinvestigator(s)
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βバレル型膜タンパク質輸送装置が周辺から受ける影響の解析
Grant number：25KF0261 2025.04 - 2028.03
独立行政法人日本学術振興会 科学研究費基金 特別研究員奨励費
Authorship：Principal investigator
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NR, QCM-D, AFMを駆使したタンパク質輸送装置の「超」複合体の解析
Grant number：24KK0138 2024.04 - 2027.03
独立行政法人日本学術振興会 科学研究費基金 国際共同研究加速基金（海外連携研究）
Authorship：Principal investigator
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Optimization of Neutron Reflectometry for analysis of biological membrane
Grant number：22K12672 2022.04 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Authorship：Principal investigator
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Optimization of multiple method baced on neutron reflectometry for analysis of biogenesis of Gram-negative bacterial membrane.
Grant number：21KK0126 2021.04 - 2024.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
Authorship：Principal investigator