Abstract
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.
Highlights
Atg3 is an E2-like enzyme in the Autophagy-related 8 (Atg8) system, an autophagy-related ubiquitin-like conjugation system
We found that Atg3-GFP is localized to the isolation membrane (IM) by fine-localization analysis
We visualized the IM under a fluorescence microscope and found that Atg3-GFP is localized to the IM, supporting the idea that Atg3 is involved in autophagosome formation at the IM
Summary
Atg is an E2-like enzyme in the Atg system, an autophagy-related ubiquitin-like conjugation system. Conclusion: Atg is likely to play an important role in autophagosome formation at the isolation membrane. Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane This Atg3-GFP dot colocalized with 2؋ mCherry-tagged Atg, demonstrating that Atg is localized to autophagic structures. Taking advantage of the known protein structure of S. cerevisiae Atg3 [20], we inserted the GFP sequence just after a unique long ␣-helix domain of Atg that interacts with Atg, termed the handle region. This construct could complement the autophagic defect in atg3⌬ cells. We visualized the IM under a fluorescence microscope and found that Atg3-GFP is localized to the IM, supporting the idea that Atg is involved in autophagosome formation at the IM
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