Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae

ABSTRACTMacroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS.Abbreviations: ATG: autophagy-related; BARA: the repeated, autophagy-specific Co-IP: co-immunoprecipitation; GFP: green fluorescent protein; IP-MS: immunoprecipitation followed by tandem mass spectrometry; NTD: the N-terminal domain; PAS: phagophore assembly site; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns3K: phosphatidylinositol 3-kinase; SUR: structurally uncharacterized region; Vps34[KD]: Vps34D731N.KEYWORDS: Atg1Atg8Atg18autophagynitrogen starvationSaccharomyces cerevisiaeVps34 AcknowledgmentsWe thank members of the Huh laboratory for helpful discussions. We also thank the Proteomics Core Facility at the School of Biological Sciences in Seoul National University, which is supported by the Center for RNA Research, Institute for Basic Science, for LC-MS/MS analysis. This work was supported by the National Research Foundation of Korea (2020R1A5A1018081 and 2021R1A2C1013718).Disclosure statementNo potential conflict of interest was reported by the author(s).Supplementary materialSupplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2182478Correction StatementThis article has been corrected with minor changes. These changes do not impact the academic content of the article.Additional informationFundingThis work was supported by the National Research Foundation of Korea [2020R1A5A1018081, 2021R1A2C1013718].