Analysis of membrane protein clusters using fluorescence lifetime FRET

• Project/Area Number: 22658031
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Applied microbiology
• Research Institution: Aoyama Gakuin University
• Principal Investigator: ABE Fumiyoshi 青山学院大学, 理工学部, 准教授 (30360746)
• Project Period (FY): 2010 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥3,650,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥450,000); Fiscal Year 2011: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2010: ¥1,700,000 (Direct Cost: ¥1,700,000)
• Keywords: 微生物学 / 脂質ラフト / 蛍光寿命FRET / 出芽酵母 / 膜タンパク質 / 蛍光タンパク質 / クラスター形成

Research Abstract

To monitor clustering of the membrane protein using fluorescence lifetime FRET, GFP-or RFP-tagged Tat2, which encodes yeast tryptophan permease Tat2, was expressed in cells. However, the fluorescence signal was too lowto obtain operative photon counting. Revisingthe strategy, native PAGE was performed toelucidateTat2 clusters. The majority of Tat2 proteins appeared to migrate as monomers after the membrane fraction was moderately treated with a non-ionic detergent. By means of high-pressure time-resolved fluorescence anisotropy measurement system, fluidity of the plasma membrane was analyzed in a deep-sea piezophile. The membrane revealed to be rigid as opposed to a general concept that the membrane of deep-seamicrobes is highly fluid.

Research Products

• [Journal Article] New type of pressurized cultivation method providing oxygen for piezotolerant yeast (2012)
  • Author(s): Hiraki, T., Sekiguchi, T., Kato, C. Hatada, Y., Maruyama, T., Abe, F. and Konishi, M.
  • Journal Title: J. Biosci. Bioeng Volume: 113 Issue: 2 Pages: 220-223
  • DOI: 10.1016/j.jbiosc.2011.09.017
  • NAID: 110009418672
  • Peer Reviewed
• [Journal Article] Eicosapentaenoic acid plays a role in stabilizing dynamic membrane structure in the deep-sea piezophile Shewanella violacea: a study employing high-pressure time-resolved fluorescence anisotropy measurement (2011)
  • Author(s): Usui, K., Hiraki, T., Kawamoto, J., Kurihara, T., Nogi, Y., Kato, C., and Abe, F.
  • Journal Title: Biochimica et Biophysica Acta Volume: 1818 Issue: 3 Pages: 574-583
  • DOI: 10.1016/j.bbamem.2011.10.010
  • Peer Reviewed
• [Journal Article] Complete genome sequence of the marine, cellulose and xylan degrading bacterium Glaciecolasp. 4H-3-7+YE-5 (2011)
  • Author(s): Klippel, B., Lochner, A., Bruce, D. C., Davenport, K. W., Detter, C., Goodwin, L. A., Han, J., Han, S., Land, M. L., Mikhailova, N., Nolan, M., Pennacchio, L., Pitluck, S., Tapia, R., Woyke, T., Wiebusch, S., Basner, A., Abe, F., Horikoshi, K., Keller, M., Antranikian, G.
  • Journal Title: J. Bacteriol Volume: 193 Issue: 17 Pages: 4547-4548
  • DOI: 10.1128/jb.05468-11
  • Peer Reviewed
• [Journal Article] Complete Genome Sequences of Krokinobacter sp. 4H-3-7-5 and Lacinutrix sp. 5H-3-7-4, polysaccharide-degrading members of the family Flavobacteriaceae (2011)
  • Author(s): Klippel, B., Lochner, A., Bruce, D. C., Davenport, K. W., Detter, C., Goodwin, L. A., Han, J., Han, S., Land, M. L., Nolan, M., Ovchinnikova, G., Pennacchio, L., Pitluck, S., Tapia, R., Woyke, T., Wiebusch, S., Basner, A., Abe, F., Horikoshi, K., Keller, M., Antranikian, G.
  • Journal Title: J. Bacteriol Volume: 193 Issue: 17 Pages: 4545-4546
  • DOI: 10.1128/jb.05518-11
  • Peer Reviewed
• [Journal Article] Different pressure resistance of lactate dehydrogenases from hagfish is dependent on habitat depth and caused by tetrameric structure dissociation (2011)
  • Author(s): Nishiguchi, Y., Abe, F. and Okada, M.
  • Journal Title: Marine Biotechnol Volume: 13 Issue: 2 Pages: 137-141
  • DOI: 10.1007/s10126-010-9299-6
  • Peer Reviewed
• [Journal Article] Overexpression of EAR1and SSH4 that encode PPxY proteins in the multivesicular body provides stability of tryptophan permease Tat2 allowing yeast cells to grow under high hydrostatic pressure (2010)
  • Author(s): Hiraki, T., Usui, K. and Abe, F.
  • Journal Title: High Pressure Research Volume: 30 Issue: 4 Pages: 514-518
  • DOI: 10.1080/08957959.2010.525512
  • Peer Reviewed
• [Journal Article] Overexpression of SNA3 stabilizes tryptophan permease Tat2, potentially competing for the WW domain of Rsp5 ubiquitin ligase with its binding protein Bul1 (2010)
  • Author(s): Hiraki, T. and Abe, F.
  • Journal Title: FEBS Lett Volume: 584 Issue: 1 Pages: 55-60
  • DOI: 10.1016/j.febslet.2009.11.076
  • Peer Reviewed
• [Journal Article] Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability of tryptophan permease Tat2 allowing yeast cells to grow under high hydrostatic pressure (2010)
  • Author(s): Hiraki, T., Usui, K., Abe, F.
  • Journal Title: High Pressure Research Volume: 30 Pages: 514-518
  • Peer Reviewed
• [Presentation] ランダム変異導入法による出芽酵母の高親和性トリプトファン輸送体Tat2の機能解析 (2012)
  • Author(s): 阿部文快
  • Organizer: 日本農芸化学会
  • Place of Presentation: 京都女子大学(京都)
  • Year and Date: 2012-03-23
• [Presentation] 高圧力下における微生物細胞膜の構造と機能 (2012)
  • Author(s): 阿部文快
  • Organizer: 日本高圧力学会
  • Place of Presentation: 京都、同志社大学
• [Presentation] ランダム変異導入法による出芽酵母の高親和性トリプトファン輸送体Tat2の機能解析 (2012)
  • Author(s): 完田奈緒子、阿部文快
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: 京都女子大学、京都
• [Presentation] ランダム変異導入による高親和性トリプトファン輸送体Tat2の機能解析 (2011)
  • Author(s): 阿部文快
  • Organizer: 酵母遺伝学フォーラム
  • Place of Presentation: 九州大学医学部(福岡)
  • Year and Date: 2011-09-05
• [Presentation] ランダム変異導入による高親和性トリプトファン輸送体Tat2の機能解析 (2011)
  • Author(s): 完田奈緒子、阿部文快
  • Organizer: 酵母遺伝学フォーラム
  • Place of Presentation: 九州大学、福岡福岡
• [Presentation] オルガネラ膜の動的構造解析、酵母遺伝学フォーラム (2010)
  • Author(s): 阿部文快
  • Organizer: ならまちセンター
  • Place of Presentation: 奈良
• [Presentation] Dynamics of microbial membranes under high pressure : a study using time-resolved fluorescence anisotropy measurement (2010)
  • Author(s): Abe, F.
  • Organizer: 6th International Conference on High Pressure Bioscience and Biotechnology(HPBB2010)
  • Place of Presentation: Technische Universitat, Freising, Germany
• [Remarks]
  • URL: http://www.chem.aoyama.ac.jp/Chem/ChemHP/abeflab/