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Deformation of vacuolar membranes mediated by endosomal sorting complex required for transport (ESCRT) is necessary for microautophagy. Target of rapamycin complex 1 (TORC1) protein kinase negatively regulates ESCRT-0 (Vps27–Hse1) recruitment onto vacuolar membranes and microautophagy induction. However, whether and how protein phosphatase regulates these events is unknown. Here, we show that the TORC1-downstream protein phosphatase PP2A–Cdc55 is important for these events after TORC1 inactivation in budding yeast. Loss of PP2A–Cdc55 compromised vacuolar localization of Hse1, but not Vps27. This study revealed that the orchestrated action of PP2A induces microautophagy upon TORC1 inactivation. • PP2A–Cdc55 promotes microautophagy induction after TORC1 inactivation. • PP2A–Cdc55 promotes vacuolar localization of Hse1 after TORC1 inactivation. • PP2A–Cdc55 promotes ESCRT-0 complex formation on vacuolar membranes. [ABSTRACT FROM AUTHOR]

Microautophagy is promoted after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) kinase. Invagination of vacuolar membranes by endosomal sorting complex required for transport (ESCRT) is required for microautophagy. Vps27, a subunit of ESCRT-0, is recruited onto vacuolar membranes via dephosphorylation after TORC1 inactivation. Here, we showed that Hse1, another ESCRT-0 subunit, is also recruited onto vacuolar membranes after TORC1 inactivation, promoting formation of ESCRT-0 complex on vacuolar membranes. Hse1 recruitment was dependent on Vps27, whereas Vps27 recruitment was independent of Hse1. Not only Vps27 but also Hse1 was required for ESCRT-III recruitment onto vacuolar membranes and microautophagy induction after TORC1 inactivation. This study revealed that ESCRT-0 (Vps27–Hse1) complex formation on vacuolar membranes is important for microautophagy inactivation after TORC1 inactivation. • TORC1 inactivation promotes ESCRT-0 complex formation on vacuolar membranes. • Hse1 recruitment onto vacuolar membranes is dependent on Vps27, but not vise versa. • Both Vps27 and Hse1 are required for TORC1 inactivation-induced microautophagy. [ABSTRACT FROM AUTHOR]