Fig. 4

Atg1 and Atg4 interact at the PAS. a Atg1–Atg4 interaction at the PAS was visualized by BiFC. WT (JSY185), atg2Δ (JSY190), and atg2Δ atg13Δ (JSY215) cells expressing both endogenous Atg1–VN and Atg4–VC, and carrying a pCumCherryV5ATG8 plasmid were grown in SMD before being nitrogen starved in SD-N medium for 1 h. Fluorescence images were taken before and after nitrogen starvation. Differential interference contrast (DIC). Scale bar, 5 µm. b Mechanistic model for the regulation of Atg4 during autophagy. Newly synthesized Atg8 is constitutively processed by the cysteine protease Atg4 in the cytoplasm, where it is not inhibited by Atg1. After the cleavage of the C-terminal arginine, a glycine residue is exposed allowing Atg8 to be conjugated to the PE on autophagosomal membranes at the PAS. During the phagophore expansion, the Atg4 adjacent or coming in proximity of autophagosomal membranes, is locally inhibited by the action of the Atg1 kinase complex. Upon autophagosome completion, the release of Atg1 form autophagosomal membranes and/or its local inactivation allows Atg4 to act on Atg8–PE and release Atg8 from the PE anchor. This molecular mechanism, possibly together with other unknown regulatory events, would drive the dissociation of other Atg proteins from the surface of autophagosomes allowing their subsequent fusion with vacuoles