Abstract
Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis. It is completed by SNARE-mediated fusion of the autophagosome and endolysosome membranes. This process must be carefully regulated to maintain the organization of the membrane system and prevent mistargeted degradation. As yet, models of autophagosomal fusion have not been verified within a cellular context because of difficulties with assessing protein interactions in situ. Here, we used high-resolution fluorescence lifetime imaging (FLIM)-FRET of HeLa cells to identify protein interactions within the spatiotemporal framework of the cell. We show that autophagosomal syntaxin 17 (Stx17) heterotrimerizes with synaptosome-associated protein 29 (SNAP29) and vesicle-associated membrane protein 7 (VAMP7) in situ, highlighting a functional role for VAMP7 in autophagosome clearance that has previously been sidelined in favor of a role for VAMP8. Additionally, we identified multimodal regulation of SNARE assembly by the Sec1/Munc18 (SM) protein VPS33A, mirroring other syntaxin–SM interactions and therefore suggesting a unified model of SM regulation. Contrary to current theoretical models, we found that the Stx17 N-peptide appears to interact in a positionally conserved, but mechanistically divergent manner with VPS33A, providing a late “go, no-go” step for autophagic fusion via a phosphoserine master-switch. Our findings suggest that Stx17 fusion competency is regulated by a phosphosite in its N-peptide, representing a previously unknown regulatory step in mammalian autophagy.
Highlights
Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis
We show that autophagosomal syntaxin 17 (Stx17) heterotrimerizes with synaptosome-associated protein 29 (SNAP29) and vesicle-associated membrane protein 7 (VAMP7) in situ, highlighting a functional role for VAMP7 in autophagosome clearance that has previously been sidelined in favor of a role for VAMP8
The correlation between mCherry-Stx17 and EGFP-LC3, the latter of which alone is quenched by the lysosomal environment [29], increases when fusion is chemically blocked with bafilomycin A1 (Fig. S1, d and e), suggesting that these are fusion-competent autophagosomes
Summary
Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis It is completed by SNARE-mediated fusion of the autophagosome and endolysosome membranes. This process must be carefully regulated to maintain the organization of the membrane system and prevent mistargeted degradation. Autophagy sequesters cargo by the growth of an open-ended double-membrane vesicle, named a phagophore, which forms de novo [2] upon nucleation at endoplasmic reticulum– mitochondrial contact sites [3]. Closure of this structure forms an autophagosome, which travels to the endolysosome and deposits its contents by membrane fusion. Mammalian Stx has been shown to associate in vitro with the soluble Qbc-SNARE, SNAP29, and the endolysosomal R-SNARE, VAMP8 [7]
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