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EMBO J. 2017 Apr 13;36(8):1100-1116. doi: 10.15252/embj.201696315. Epub 2017 Mar 20.

Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy.

Author information

1
Department of Biological Sciences and Biotechnology, College of Life Sciences and Nanotechnology, Hannam University, Daejeon, Korea.
2
Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sangju, Korea.
3
Center for Electron Microscopy Research, Korea Basic Science Institute, Daejeon, Korea.
4
Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea kaang@snu.ac.kr jangdj@knu.ac.kr leeja@hnu.kr.
5
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.
6
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.

Abstract

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.

KEYWORDS:

LC3‐interacting region motif; autophagosome sensor; autophagy; hydrophobic domain

PMID:
28320742
PMCID:
PMC5391143
DOI:
10.15252/embj.201696315
[Indexed for MEDLINE]
Free PMC Article

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