Protocol

Autophagosome and Phagosome

Volume 445 of the series Methods in Molecular Biology™ pp 77-88

LC3 and Autophagy

  • Isei TanidaAffiliated withDepartment of Biochemistry, Juntendo University School of Medicine
  • , Takashi UenoAffiliated withDepartment of Biochemistry, Juntendo University School of Medicine
  • , Eiki KominamiAffiliated withDepartment of Biochemistry, Juntendo University School of Medicine

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Summary

Microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein with a molecular mass of ∼17 kDa that is distributed ubiquitously in mammalian tissues and cultured cells. During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosomal components are degraded by lysosomal hydrolases. At the same time, LC3-II in autolysosomal lumen is degraded. Thus, lysosomal turnover of the autophagosomal marker LC3-II reflects starvation-induced autophagic activity, and detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death. Here we describe basic protocols to assay for endogenous LC3-II by immunoblotting, immunoprecipitation, and immunofluorescence.

Key Words

LC3 lipidation ubiquitylation-like reaction autophagosome autolysosome autophagy ATG conjugation system