title: Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae.
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.
authors: From the Department of Integrated Biosciences, Graduate School of Frontier Sciences, and.
authors: Meipin
authors: M
authors: Ngu
authors: From the Department of Integrated Biosciences, Graduate School of Frontier Sciences, and.
authors: Eri
authors: E
authors: Hirata
authors: From the Department of Integrated Biosciences, Graduate School of Frontier Sciences, and the Bioimaging Center, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan kuninori@k.u-tokyo.ac.jp.
authors: Kuninori
authors: K
authors: Suzuki
mesh_terms: D001343:Autophagy; D000071183:Autophagy-Related Proteins; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000201:enzymology; D010714:Phosphatidylethanolamines; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
chemical_list: D000071183:Autophagy-Related Proteins; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C483858:phosphatidylethanolamine; D044763:Ubiquitin-Conjugating Enzymes; C104235:ATG3 protein, S cerevisiae
keywords: Atg3; Atg8 Lipidation; Autophagosome; Autophagy; Biodegradation; Isolation Membrane; Organelle; Saccharomyces cerevisiae; Yeast
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