LC3 and Autophagy
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 WordsLC3 lipidation ubiquitylation-like reaction autophagosome autolysosome autophagy ATG conjugation system
- 13.Tanida, I., Tanida-Miyake, E., Komatsu, M., Ueno, T., and Kominami, E. (2002) Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p. J. Biol. Chem. 277, 13739–13744.CrossRefPubMedGoogle Scholar
- 18.Umezawa, H., Takeuchi, T., Linuma, H., Suzuki, K., and Ito, M. (1970) A new microbial product, oudenone, inhibiting tyrosine hydroxylase. J. Antibiot. (Tokyo) 23, 514–518.Google Scholar