Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae

Abstract

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 Atg (autophagy-related) 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. Here, 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 dot 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 labeled both the VICS and the cup-shaped IM. We designate this localization the ‘IM’ pattern. The Atg2–Atg18 complex and Atg9 localized at the edge of the IM as two or three dots, in close proximity to the endoplasmic reticulum (ER) via ER exit sites. Thus, we designate these dots as the ‘IM edge’ pattern. These data suggest that Atg proteins play individual roles at spatially distinct localizations during IM expansion. These findings will facilitate detailed investigations of the function of each Atg protein during autophagosome formation.