In this study, we showed that 1) the amount of Atg8 regulates the level of autophagy by specifically modulating the size of the autophagosomes, whereas the number of autophagosomes is unaffected, 2) each round of autophagosome formation involves a cycle of Atg8 trafficking in which Atg8 is first recruited to an expanding structure and later released from it, and 3) the release of Atg8 is essential for the completion of autophagosome formation and is mediated by deconjugation. In mammalian cells, the autophagosomes produced in response to group A Streptococcus
invasion are larger than those seen in starvation conditions (Nakagawa et al., 2004
). According to those data, where the signal is not saturated, the level of Atg8 induced by group A Streptococcus
invasion is higher than that induced by starvation, suggesting that the regulation of autophagosome size achieved by controlling the amount of Atg8 may be a conserved mechanism.
For the first time, our data allowed temporal dissection of the autophagosome formation process based on real-time observations ( and ). Here we propose a five-stage model. In the first stage, cargos and factors required for the PAS recruitment of Atg8 arrive at the PAS; in stage 2, Atg8 arrives at the PAS and the Atg8-containing structure expands; stage 3 is a transitional step, allowing the completion of expansion and initiating release of Atg8 through deconjugation; in stage 4, additional Atg8 molecules are gradually released from the PAS; and in stage 5, the phagophore matures into an autophagosome, and some Atg8 molecules are trapped inside. Previously, the lack of a data-derived multistage model restricted most studies on autophagosome formation to the PAS recruitment-dependency of autophagy-related proteins. Our model provides the foundation for reanalysis of the functions of known autophagy-related proteins and for screening new genes whose products act at each stage. In addition, this model serves as a reference point to coordinate the dynamics of other autophagy-related genes. For instance, it would be interesting to find at which stage Atg9, a protein known to cycle between the PAS and peripheral sites, departs from the PAS (Reggiori et al., 2004
; Young et al., 2006
Our results suggest that the expansion and deformation of the phagophore happens concurrently or slightly after the recruitment of Atg8 to the PAS, given that Atg8 is a causal factor in determination of autophagosome size. When Atg8 is released, the Atg8-containing structures retained their sizes (Figure S2), indicating that at this moment the expansion of the phagophore should be near completion, but not fully closed so that Atg8 molecules attached to the concave side of the phagophore (that will become the inner membrane of the autophagosome) can leave the membrane.
At present, how Atg8 modifies the phagophore to produce different-sized autophagosomes is not clear; however, our data indicate that Atg8 is essential in autophagosome formation. In rare cases (in ~1% of the cells), small vesicular structures can be observed in atg8
Δ cells (Supplementary Figure S3). Previously, small autophagosome-like structures have also been detected in atg8
Δ cells (Abeliovich et al., 2000
). Currently, the identity of these structures cannot be determined in the absence of an autophagic membrane marker other than Atg8. In addition, we note that similar rare structures can also be detected in atg1
Δ cells, which are defective in autophagosome formation (Supplementary Figure S3). Further experiments are therefore needed to elucidate the nature of these vesicles.