Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation

Abstract

Summary Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

Fatty Acyl Coenzyme A Synthetase Fat1p Regulates Vacuolar Structure and Stationary-Phase Lipophagy in Saccharomyces cerevisiae

“…S1 in the supplemental material). As a negative control, the atg1 Δ mutant, which fails to form autophagosomes, containing GFP-Atg8 was also tested ( 46 , 47 ). Autophagic flux was low in log-phase cells, as the wild type (WT) and most mutants only incorporated GFP-Atg8 into vacuoles in 5% or fewer cells; however, this was slightly higher in fat1 Δ cells, at 10% of cells ( Fig.…”

“…S1D). Mature autophagosomes are not actually formed in ATG1 deletion strains ( 46 , 47 ), but GFP-Atg8 puncta were still significantly increased in the atg1 Δ strain (Fig. S1D).…”

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

“…pET-28a-6xHis-TEV-3xFLAG-ATP6V1H(1-351) was purchased commercially (GenScript). pFBDM-ATG7-ATG10-ATG12-StrepII 2x -ATG5-ATG16L1 and pFDM-SH-SUMO*-Hrr25 were described previously (Schreiber et al, 2021; Zhang et al, 2023). pcDNA3.1-C-FLAG containing human ATP6V1H transcript variant 1 (NM_015941.4) was purchased commercially (GenScript).…”

Molecular regulation of autophagosome formation

“…The supra-assembly of Atg1/ULK kinase complex, which has only been shown in yeast, is driven by a liquid phase separation mediated by the Atg13's intrinsically disordered region [ 31 ], and the resulting aggregate serves as a scaffold for the subsequent association and orchestration through phosphorylation of the function of the downstream ATG proteins [ 8 , 32 , 33 ]. In yeast, vesicles containing Atg9, the only transmembrane protein between the core ATG proteins, are recruited by the Atg1 kinase complex via the interaction with either Atg17 and the HORMA domain of Atg13 during bulk autophagy, or via Atg11 in selective types of autophagy, to possibly provide the seed membrane for the phagophore nucleation [ 34–37 ].…”