Temporal dissection of the roles of Atg4 and ESCRT in autophagosome formation in yeast.

Autophagosomes are formed by the enlargement and sealing of phagophores. This is accompanied by the recruitment and release of autophagy-related (Atg) proteins that function therein. Presently, the relationship among factors that act after the initial emergence of the phagophore is unclear. The endosomal sorting complexes required for transport (ESCRT) machinery and Atg4 are known to function in phagophore sealing and Atg8 release, respectively. Here we show that biochemically, both Atg4 and ESCRT promoted phagophore sealing. Intriguingly, Atg4-mediated release of Atg8 from the phagophore promoted phagophore sealing even in the absence of ESCRT. This sealing activity could be reconstituted in vitro using cell lysate and purified Atg4. To elucidate the temporal relationship between Atg4 and ESCRT, we charted a timeline of the autophagosome formation cycle based on the trafficking of Atg proteins and mapped the actions of Atg4 and ESCRT to specific stages. The temporal impact of Atg4-mediated release of Atg8 from phagophore was mapped to the stage after the assembly of phagophore assembly site (PAS) scaffold and phosphatidylinositol-3-kinase (PtdIns-3-K) complex; its retardation only extended the duration of Atg8 release stage, leading to delayed phagophore sealing and accumulation of multiple phagophores. The impacts of ESCRT were mapped to two stages. In addition to promoting phagophore sealing, it also dictates whether PtdIns-3-K recruitment can occur by controlling Atg9 trafficking, thereby determining the incidence of autophagosome formation. Accordingly, ESCRT deficiency led to a combination of reduced autophagosome frequency and extended autophagosome formation duration, manifesting as reduced autophagic flux but normal apparent Atg8 puncta number. Our study thus identifies Atg4-mediated Atg8 shedding as a novel membrane scission mechanism and reveals a new early-stage role for ESCRT in autophagy.