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

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

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.

MeSH terms

  • Adaptor Proteins, Signal Transducing* / genetics
  • Adaptor Proteins, Signal Transducing* / metabolism
  • Amino Acid Motifs
  • Animals
  • Apoptosis Regulatory Proteins
  • Autophagy*
  • Cytoskeletal Proteins* / genetics
  • Cytoskeletal Proteins* / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Membrane Proteins* / genetics
  • Membrane Proteins* / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins* / genetics
  • Microtubule-Associated Proteins* / metabolism
  • Protein Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • Cytoskeletal Proteins
  • GABARAP protein, human
  • GABARAP protein, mouse
  • Map1lc3b protein, mouse
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • light chain 3, human
  • Green Fluorescent Proteins