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Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy
Authors:You-Kyung Lee  Jin-A Lee  You-Kyung Lee  Yong-Woo Jun  Ha-Eun Choi  Yang Hoon Huh  Bong-Kiun Kaang  Deok-Jin Jang  Jin-A Lee
Affiliation:1. Department of Biological Sciences and Biotechnology, College of Life Sciences and Nanotechnology, Hannam University, Daejeon, Korea;2. Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea;3. Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sangju, Korea
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:autophagosome sensor  autophagy  hydrophobic domain  LC3-interacting region motif
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