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Development of LC 3/ GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy
Author(s) -
Lee YouKyung,
Jun YongWoo,
Choi HaEun,
Huh Yang Hoon,
Kaang BongKiun,
Jang DeokJin,
Lee JinA
Publication year - 2017
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201696315
Subject(s) - autophagy , autophagosome , green fluorescent protein , microbiology and biotechnology , mitophagy , biology , cytosol , biochemistry , gene , apoptosis , enzyme
Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP / RFP ‐ LC 3/ 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 LC 3/ GABARAP proteins at the autophagosome using an LC 3‐interacting region ( LIR ) and a short hydrophobic domain (HyD). Among HyD‐ LIR ‐ GFP sensors harboring LIR motifs of 34 known LC 3‐binding proteins, HyD‐ LIR ( TP )‐ GFP using the LIR motif from TP 53 INP 2 allowed detection of all LC 3/ GABARAP s‐positive autophagosomes. However, HyD‐ LIR ( TP )‐ GFP preferentially localized to GABARAP / GABARAPL 1‐positive autophagosomes in a LIR ‐dependent manner. In contrast, HyD‐ LIR (Fy)‐ GFP using the LIR motif from FYCO 1 specifically detected LC 3A/B‐positive autophagosomes. HyD‐ LIR ( TP )‐ GFP and HyD‐ LIR (Fy)‐ GFP efficiently localized to autophagosomes in the presence of endogenous LC 3/ 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.