Abstract
In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.
Highlights
Autophagy is a recycling pathway that shuttles surplus and damaged cellular components for degradation in the lysosome under normal and stress conditions
Because we discovered that Vps21, a Rab5 GTPase, plays a role in autophagy in yeast cells (Chen et al, 2014), we decided to determine whether the ESCRT machinery plays a role in this process
In vps21Δ mutant cells, we showed that these GFP-Atg8 structures represent AP clusters and below we show that this is true for ESCRT mutants
Summary
Autophagy is a recycling pathway that shuttles surplus and damaged cellular components for degradation in the lysosome under normal and stress conditions. Defects in this pathway are associated with a myriad of human diseases (Rubinsztein et al, 2012; Choi et al, 2013). Fusion of APs and late endosomes with the lysosome is regulated and mediated by a similar set of factors that include the Ypt GTPase module and the cellular fusion machinery of tethers and SNAREs (Numrich and Ungermann, 2014). AP sealing cannot be catalyzed by the cellular membrane-fusion machinery (e.g., tethers and SNAREs), which catalyzes “pointed” fusion between two membranes. The ESCRT complex can catalyze such a process (Hurley and Hanson, 2010)
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