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

Abstract

SummaryAutophagy 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 cupshaped 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.

A current perspective of autophagosome biogenesis

“…Recently, Suzuki et al [76] observed that the edge of the isolation membrane was often found next to ER exit sites, the sites of COPII vesicle formation. Interestingly, Atg9 and the Atg2-Atg18 complex accumulated at the edge of the isolation membrane.…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…9,19 During phagophore expansion, the ATG9-ATG2-WIPI1 complex is likely involved in recruiting lipids to the growing phagophore. [20][21][22] In the final step, the trafficking and fusion of autophagosomes with lysosomes is facilitated by PtdIns3P-binding proteins, PIK3C3 complexes, and interacting partners, as well as RAB proteins. 7,16,23 The dynamics of the autophagic process, from the growth and closure of membrane structures to the acquisition of membrane-bound proteins, can be viewed as a membrane trafficking process, reminiscent of the endosomal trafficking pathway.…”

Molecular interactions of theSaccharomyces cerevisiaeAtg1 complex provide insights into assembly and regulatory mechanisms

“…The different Atg proteins are recruited to the PAS in a hierarchical manner. 14,15 The most upstream acting components belong to the Atg1 complex consisting of the Atg1 protein kinase, the regulatory subunit Atg13, and a ternary complex composed of Atg17, Atg31, and Atg29. The Atg1 complex mediates autophagosome formation by initiating phagophore assembly and the localization of the downstream PtdIns3K complex, Atg9, and the ubiquitinlike conjugation systems to this membrane compartment.…”

“…The Atg1 complex mediates autophagosome formation by initiating phagophore assembly and the localization of the downstream PtdIns3K complex, Atg9, and the ubiquitinlike conjugation systems to this membrane compartment. 14,15 Recent biochemical and structural studies have revealed multiple roles of the Atg1 complex in autophagosome formation and autophagy induction.…”

“…Although Atg1 kinase activity is dispensable for the assembly of the Atg1 complex, it is required for the expansion of the phagophore. 14 Furthermore, Atg1 activity is essential to the turnover and recycling of other Atg proteins after autophagosomes are formed. 16 The kinase domain of Atg1 is located at the N-terminal region (residues 1 to 589), and requires an autophosphorylation event at threonine residue 226 for full activity.…”