Interaction of Bcl-2 with the autophagy-related GABAA receptor-associated protein (GABARAP): biophysical characterization and functional implications

J Biol Chem. 2013 Dec 27;288(52):37204-15. doi: 10.1074/jbc.M113.528067. Epub 2013 Nov 15.

Abstract

Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.

Keywords: Apoptosis; Autophagy; Bcl-2; GABARAP; LC3; Lipidation; NMR; Protein-Protein Interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Motifs
  • Animals
  • Apoptosis / physiology
  • Autophagy / physiology*
  • Cell Line, Transformed
  • Cytoskeletal Proteins / chemistry
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Lipoylation / physiology*
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Microtubule-Associated Proteins / chemistry
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-bcl-2 / chemistry
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • Rats

Substances

  • Adaptor Proteins, Signal Transducing
  • Cytoskeletal Proteins
  • GABARAP protein, human
  • GABARAP protein, mouse
  • GABARAP protein, rat
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Bcl2 protein, mouse