Localization of Atg3 to autophagy-related membranes and its enhancement by the Atg8-family interacting motif to promote expansion of the membranes.

The E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae. In this study, we show that Atg3 localizes to the pre-autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that Atg8-PE conjugates are produced on these structures. We also find that mutations in the Atg8-family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM-mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of Atg8-PE on the membranes.

Sakoh-Nakatogawa M ，Kirisako H ，Nakatogawa H ，Ohsumi Y ... -  《-》

Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae.

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.

Ngu M ，Hirata E ，Suzuki K 《-》

The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation.

Hanada T ，Satomi Y ，Takao T ，Ohsumi Y ... -  《-》

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis.

Nakatogawa H ，Ishii J ，Asai E ，Ohsumi Y ... -  《-》

Atg4 plays an important role in efficient expansion of autophagic isolation membranes by cleaving lipidated Atg8 in Saccharomyces cerevisiae.

Hirata E ，Ohya Y ，Suzuki K 《PLoS One》