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

TLDR

During starvation‑induced autophagy, autophagosomes fuse with lysosomes to degrade contents, and LC3 is lipidated to LC3‑II on these structures; although LC3‑II accumulation is commonly used as an autophagy marker, its lysosomal degradation has not been examined. The study aimed to investigate the lysosomal turnover of endogenous LC3‑II during starvation‑induced autophagy. Using lysosomal protease inhibitors E64d and pepstatin A, the authors measured LC3‑II accumulation in HEK293 and HeLa cells under starvation, observing 3.5‑fold and 6.7‑fold increases, respectively. Morphological analysis showed increased LC3‑positive puncta and autolysosomes, indicating that lysosomal degradation of LC3‑II after autolysosome formation reflects starvation‑induced autophagic activity.

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.