Screening of autophagosomal outer membrane proteins required for membrane fusion

• Project/Area Number: 16K14720
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Cell biology
• Research Institution: The University of Tokyo
• Principal Investigator: Yamamoto Hayashi 東京大学, 大学院医学系研究科(医学部), 講師 (80551283)
• Co-Investigator(Renkei-kenkyūsha): MIZUSHIMA Noboru 東京大学, 大学院医学系研究科, 教授 (10353434)
• Research Collaborator: UEMATSU Masaaki 東京大学, 大学院医学系研究科, 大学院生
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: オートファジー / 膜融合 / SNARE / プロテオーム / 膜動態

Outline of Final Research Achievements

Autophagy is a fundamental degradation system conserved in eukaryotes. Upon induction of autophagy, a double-membrane structure, called an autophagosome, is generated and fuses with lysosomes to degrade its contents. Although many ATG proteins have been identified, it remains unclear how autophagosome-lysosome fusion is regulated. In this study, we tried to develop a biochemical method to purify autophagosomes and to identify autophagosomal outer membrane proteins involved in the membrane fusion. For this purpose, we prepared GFP-STX17DN cells to accumulate autophagosomes, harvested an autophagosome-enriched fraction by OptiPrep flotation, and purified autophagosomes using 3xFLAG-LC3. Finally, outer membrane proteins were labeled by a membrane-impermeable biotinylation reagent. By mass spectrometry of the biotinylated proteins, we obtained several candidates of outer membrane proteins. We prepared KO cells (CRISPR) or KD cells (siRNA), however, significant phenotypes were not observed.

Research Products

• [Journal Article] Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets (2017)
  • Author(s): Tamura Norito、Nishimura Taki、Sakamaki Yuriko、Koyama-Honda Ikuko、Yamamoto Hayashi、Mizushima Noboru
  • Journal Title: FEBS Letters Volume: 591 Issue: 23 Pages: 3819-3830
  • DOI: 10.1002/1873-3468.12901
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants (2017)
  • Author(s): Uematsu Masaaki、Nishimura Taki、Sakamaki Yuriko、Yamamoto Hayashi、Mizushima Noboru
  • Journal Title: Autophagy Volume: 13 Issue: 8 Pages: 1452-1464
  • DOI: 10.1080/15548627.2017.1327940
  • Peer Reviewed
• [Journal Article] Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains (2017)
  • Author(s): Nishimura, T., Tamura, N., Kono, N., Shimanaka, Y., Arai, H., Yamamoto, H., Mizushima, N.
  • Journal Title: The EMBO Journal Volume: 印刷中 Issue: 12 Pages: 1719-1735
  • DOI: 10.15252/embj.201695189
  • Peer Reviewed
• [Journal Article] The Intrinsically Disordered Protein Atg13 Mediates Supramolecular Assembly of Autophagy Initiation Complexes. (2016)
  • Author(s): Yamamoto H, Fujioka Y, Suzuki SW, Noshiro D, Suzuki H, Kondo-Kakuta C, Kimura Y, Hirano H, Ando T, Noda NN, Ohsumi Y
  • Journal Title: Developmental Cell Volume: 38 Issue: 1 Pages: 86-99
  • DOI: 10.1016/j.devcel.2016.06.015
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] オートファジータンパク質群の動的相互作用と分子集合形態の解析 (2016)
  • Author(s): 山本 林
  • Organizer: 第54回 日本生物物理学会年会
  • Place of Presentation: つくば国際会議場（筑波）
  • Year and Date: 2016-11-25
  • Invited
• [Presentation] 天然変性タンパク質Atg13によるオートファジー始動複合体の高次集積機構の解明 (2016)
  • Author(s): 山本 林，藤岡 優子，鈴木 翔，野田 展生，大隅 良典
  • Organizer: 第89回 日本生化学会大会
  • Place of Presentation: 仙台国際センター（仙台）
  • Year and Date: 2016-09-25