Screening of autophagosomal outer membrane proteins required for membrane fusion

2017 Fiscal Year Final Research Report

• 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
• Keywords: オートファジー / 膜融合 / SNARE

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.

Free Research Field

生化学