Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy |
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| 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 |
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| 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 |
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| 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. |
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| Keywords: | autophagosome sensor autophagy hydrophobic domain LC3-interacting region motif |
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