In this study, we demonstrate that deconjugation of Atg8−PE by Atg4 plays dual roles in the autophagosome formation process (). At the beginning stage of autophagosome formation, the release of Atg8 from non-PAS transient reservoirs by Atg4-dependent deconjugation was critical in maintaining a normal supply of Atg8 to the PAS. In the absence of deconjugation, the amount of Atg8 reaching the PAS was reduced. At a late stage of autophagosome formation, the release of Atg8 from the phagophore membrane facilitated the maturation of the phagophore into fusion-capable autophagosomes. In the absence of deconjugation, the defect at this stage could not be overcome by overexpressing Atg8ΔR. Targeting Atg4 to the vacuolar membrane in deconjugation-defective cells fully restored the supply of Atg8 to the PAS, although the defect in the later stage was only partially alleviated. Targeting additional Atg8 to the vacuolar membrane in wild-type cells did not affect autophagy significantly.
Our current data indicate that prior to reaching the PAS, some Atg8 molecules transiently associate with other endomembranes, including the vacuolar membrane. In the absence of deconjugation, Atg8−PE molecules from other compartments eventually reach the vacuole through a Vps4-dependent pathway (). The transient nature of the association with non-PAS structures suggest that the balance of conjugation vs. deconjugation is regulated differently than that at the PAS. One notable dissimilarity here is that the association with the vacuole is not mediated by the E3 complex (), which might give Atg4 an upper hand in shifting the balance toward deconjugation. Likewise, the instability of Atg8−PE at non-PAS sites may stem from the absence of other core machinery proteins, some of which may help protect Atg8−PE.27
Considering that the surface area of the vacuole is approximately two orders of magnitude larger than that of the phagophore, the total amount Atg8 that transits through the vacuolar membrane would be significant. However, the physiological importance of this transient association is unclear. The simplest interpretation would be that the association of Atg8 with other endomembranes is “off target,” and that the deconjugation by Atg4 salvages those mislocalized Atg8 molecules. Alternatively, these non-PAS Atg8 molecules may be participating in other intracellular trafficking pathways. Consistent with such a possibility, two mammalian homologs of Atg8, GABARAP and LC3, have been reported to participate in the transport of GABAA
receptors and in Toll-like receptor-mediated phagocytosis, respectively.28,29
In contrast to the role of deconjugation in supplying Atg8 to the PAS, the role of deconjugation in the maturation of the phagophore into fusion-capable autophagosomes cannot be bypassed by overexpressing Atg8ΔR (). In a certain sense, this result is similar to what has been recently shown for Doa4, a deubiquitination enzyme functioning in the multivesicular body (MVB) pathway.23,30
Doa4 deubiquitinates cargo proteins prior to their sorting into the forming intralumenal vesicles of late endosomes. Accordingly, the lack of Doa4 results in decreased availability of ubiquitin monomers. Restoring the level of ubiquitin by overexpresssion does not rescue the sorting defect of ubiquitinated proteins in doa4
Δ cells; instead, these proteins remain on the surface of endosomes and subsequently reach the vacuolar membrane.30
It is well appreciated that modification by ubiquitin and Ubls constitutes important signals dictating the fate of target molecules. In many cases, the significance of the reverse process, that is, deconjugation, has been downplayed to be a matter of recycling. Together with the aforementioned Doa4 reports, our data suggest that the act of deconjugation can also serve as an important signal for the proper progression of the respective processes.
It is worth noting that aside from a common involvement of deconjugation, the formation processes of autophagosomes and MVBs have distinct differences. In the MVB pathway, it is a subset of cargo proteins that rely on the reversible ubiquitination for their entry into intralunemal vesicles. The formation of the intralumenal vesicles per se is not significantly affected by the lack of deconjugation.31
In autophagosome formation, however, the target of Atg8 conjugation is PE in the membrane.12
Unlike ubiquitin, Atg8 has a direct role in the formation of autophagosomes.5,7
In the absence of deconjugation, the overall process of autophagy is notably reduced ().15
Furthermore, there is no indication from existing publications that Atg8−PE is preferentially sorted to the inner vesicles of autophagosomes (which topologically correspond to the intralumenal vesicles of the MVB). Therefore the mechanism underlying the involvement of Atg4 in the late stage of autophagosome formation may differ significantly from that of Doa4 in the MVB pathway. In both pathways, further studies are needed to elucidate the precise details.