Summary
Autophagy is a bulk degradation system mediated by biogenesis of autophagosomes under starvation conditions. In Saccharomyces cerevisiae, a membrane sac called the isolation membrane (IM) is generated from the pre-autophagosomal structure (PAS); ultimately, the IM expands to become a mature autophagosome. Eighteen autophagy-related (Atg) proteins are engaged in autophagosome formation at the PAS. However, the cup-shaped IM was visualized just as a dot by fluorescence microscopy, posing a challenge to further understanding the detailed functions of Atg proteins during IM expansion. In this study, we visualized expanding IMs as cup-shaped structures using fluorescence microscopy by enlarging a selective cargo of autophagosomes, and finely mapped the localizations of Atg proteins. The PAS scaffold proteins (Atg13 and Atg17) and phosphatidylinositol 3-kinase complex I were localized to a position at the junction between the IM and the vacuolar membrane, termed the vacuole–IM contact site (VICS). By contrast, Atg1, Atg8 and the Atg16–Atg12–Atg5 complex were present at both the VICS and the cup-shaped IM. We designate this localization the ‘IM’ pattern. The Atg2–Atg18 complex and Atg9 localized to the edge of the IM, appearing as two or three dots, in close proximity to the endoplasmic reticulum exit sites. Thus, we designate these dots as the ‘IM edge’ pattern. These data suggest that Atg proteins play individual roles at spatially distinct locations during IM expansion. These findings will facilitate detailed investigations of the function of each Atg protein during autophagosome formation.
Footnotes
Author contributions
K.S. designed the project, worked on experiments using a fluorescence microscope and wrote the paper. M.A., C.K. and H.Y. constructed plasmids and strains. H.Y. and Y.O. contributed to writing the paper. Y.O. supervised the entire project.
Funding
This work was supported by the Hamaguchi Foundation for the Advancement of Biochemistry [grant number H23-3 to K.S.]; the NOVARTIS Foundation (Japan) for the Promotion of Science [grant number 11-128 to K.S.]; and by Grants-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [grant numbers 24121707, 24657083, 25291040 to K.S.; 24770182 to H.Y.; 23000015 to Y.O.]. Deposited in PMC for immediate release.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122960/-/DC1
- Accepted March 18, 2013.
- © 2013. Published by The Company of Biologists Ltd
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