Lysosomal Turnover, but Not a Cellular Level, of Endogenous LC3 Is a Marker for Autophagy

Autophagy. 2005 Jul;1(2):84-91. doi: 10.4161/auto.1.2.1697. Epub 2005 Jul 31.

Abstract

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / physiology*
  • Biological Transport, Active
  • Biomarkers / metabolism
  • Cell Line
  • Humans
  • Hydrolases / antagonists & inhibitors
  • Leucine / analogs & derivatives
  • Leucine / pharmacology
  • Lysosomes / physiology*
  • Lysosomes / ultrastructure
  • Microscopy, Electron, Transmission
  • Microtubule-Associated Proteins / metabolism*
  • Pepstatins / pharmacology
  • Phagosomes / physiology*
  • Phagosomes / ultrastructure

Substances

  • Biomarkers
  • Microtubule-Associated Proteins
  • Pepstatins
  • light chain 3, human
  • Hydrolases
  • Leucine
  • aloxistatin
  • pepstatin