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J Biol Chem. 2019 Mar 15;294(11):4188-4201. doi: 10.1074/jbc.RA118.005947. Epub 2019 Jan 17.

A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells.

Author information

1
From the Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, United Kingdom r.saleeb@qmul.ac.uk.
2
From the Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, United Kingdom.
3
From the Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, United Kingdom r.r.duncan@hw.ac.uk.

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.

KEYWORDS:

FLIM-FRET; SNARE proteins; VPS33A; autophagy; fluorescence resonance energy transfer (FRET); imaging; lysosome; membrane; membrane trafficking; phagosome; protein degradation; syntaxin 17

PMID:
30655294
DOI:
10.1074/jbc.RA118.005947
Free PMC Article

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