Blocking autophagosome closure manifests the roles of mammalian Atg8-family proteins in phagophore formation and expansion during nutrient starvation

Macroautophagy/autophagy, an evolutionarily conserved cellular degradation pathway, involves phagophores that sequester cytoplasmic constituents and mature into autophagosomes for subsequent lysosomal delivery. The <i>ATG8</i> gene family, comprising the <i>MAP1LC3/LC3</i> and <i>GABARAP/GBR</i> subfamilies in mammals, encodes ubiquitin-like proteins that are conjugated to phagophore membranes during autophagosome biogenesis. A central question in the field is how Atg8-family proteins are precisely involved in autophagosome formation, which remains controversial and challenging, at least in part due to the short lifespan of phagophores. In this study, we depleted the autophagosome closure regulator VPS37A to arrest autophagy at the vesicle completion step and determined the roles of mammalian Atg8-family proteins (mATG8s) in nutrient starvation-induced autophagosome biogenesis. Our investigation revealed that <i>LC3</i> loss hinders phagophore formation, while <i>GBR</i> loss impedes both phagophore formation and expansion. The defect in membrane expansion by <i>GBR</i> loss appears to be attributed to compromised recruitment of ATG proteins containing an LC3-interacting region (LIR), including ULK1 and ATG3. Moreover, a combined deficiency of both <i>LC3</i> and <i>GBR</i> subfamilies nearly completely inhibits phagophore formation, highlighting their redundant regulation of this process. Consequently, cells lacking all <i>mATG8</i> members exhibit defects in downstream events such as ESCRT recruitment and autophagic flux. Collectively, these findings underscore the critical roles of mammalian Atg8-family proteins in phagophore formation and expansion during autophagy.<b>Abbreviation</b>: AIM: Atg8-family interacting motif; ADS: Atg8-interacting motif docking site; ATG: autophagy related; BafA1: bafilomycin A₁; CL: control; ESCRT: endosomal sorting complex required for transport; FACS: fluorescence activated cell sorting; GBR: GABARAP; GBRL1: GABARAPL1; GBRL2: GABARAPL2; GBRL3: GABARAPL3; HKO: hexa-knockout; IP: immunoprecipitation; KO: knockout; LDS: LC3-interacting-region docking site; LIR: LC3-interacting region; mATG8: mammalian Atg8-family protein; MIL: membrane-impermeable ligands; MPL: membrane-permeable ligands; RT: room temperature; Stv: starved; TKO: triple-knockout; TMR: tetramethylrhodamine; UEVL: ubiquitin E2 variant-like; WCLs: whole cell lysates; WT: wild-type.