Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy.
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.
Tanida
Isei
I
Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
Minematsu-Ikeguchi
Naoko
N
Ueno
Takashi
T
Kominami
Eiki
E
D001343:Autophagy / Q000502:physiology*; D001693:Biological Transport, Active; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008247:Lysosomes / Q000502:physiology* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D010588:Phagosomes / Q000502:physiology* / Q000648:ultrastructure
D015415:Biomarkers; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D010436:Pepstatins; D006867:Hydrolases; D007930:Leucine; C108192:aloxistatin; C031375:pepstatin