Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy.
Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR(TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.
Department of Biological Sciences and Biotechnology, College of Life Sciences and Nanotechnology, Hannam University, Daejeon, Korea.
You-Kyung
YK
Lee
Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sangju, Korea.
Yong-Woo
YW
Jun
Department of Biological Sciences and Biotechnology, College of Life Sciences and Nanotechnology, Hannam University, Daejeon, Korea.
Ha-Eun
HE
Choi
Center for Electron Microscopy Research, Korea Basic Science Institute, Daejeon, Korea.
Yang Hoon
YH
Huh
Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea kaang@snu.ac.kr jangdj@knu.ac.kr leeja@hnu.kr.
Bong-Kiun
BK
Kaang
Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sangju, Korea kaang@snu.ac.kr jangdj@knu.ac.kr leeja@hnu.kr.
Deok-Jin
DJ
Jang
Department of Biological Sciences and Biotechnology, College of Life Sciences and Nanotechnology, Hannam University, Daejeon, Korea kaang@snu.ac.kr jangdj@knu.ac.kr leeja@hnu.kr.
Jin-A
0000-0002-6757-8371
JA
Lee
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D003598:Cytoskeletal Proteins; C116741:GABARAP protein, human; C480861:GABARAP protein, mouse; C477012:MAP1LC3A protein, human; C480860:Map1lc3b protein, mouse; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins
LC3‐interacting region motif; autophagosome sensor; autophagy; hydrophobic domain