An Atg4B Mutant Hampers the Lipidation of LC3 Paralogues and Causes Defects in Autophagosome Closure

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Molecular biology of the cell (Impact Factor: 5.98). 10/2008; 19(11):4651-9. DOI: 10.1091/mbc.E08-03-0312
Source: PubMed

ABSTRACT In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.

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Available from: Takeshi Noda, Jul 25, 2015
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    • "These Gal3-positive Lamp1 puncta dramatically decreased within 10 h, and completely disappeared until 24 h after LLOMe washout. In contrast to control cells, in cells stably expressing an inactive Atg4B mutant, Atg4B C74A , that sequesters LC3 paralogues prior to lipidation and strongly blocks autophagy (Fujita et al, 2008a), GFP-Gal3-positive Lamp1 puncta slightly decreased but were then maintained at a high level even 24 h after LLOMe washout (Figure 2B). These results suggest that disappearance of GFP-Gal3 puncta is due to autophagy. "
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