A VPS33A-binding motif on syntaxin17 controls autophagy completion in mammalian cells

Rebecca Sonia Saleeb, Deirdre M. Kavanagh, Alison R. Dun, Paul A. Dalgarno, Rory R. Duncan

Research output: Contribution to journalArticle

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) heterotrim-erizes 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.

LanguageEnglish
Pages4188-4201
Number of pages14
JournalJournal of Biological Chemistry
Volume294
Issue number11
Early online date17 Jan 2019
DOIs
Publication statusPublished - 15 Mar 2019

Fingerprint

Qa-SNARE Proteins
Autophagy
Fusion reactions
Cells
R-SNARE Proteins
SNARE Proteins
Munc18 Proteins
Membranes
Phosphoserine
Degradation
Peptides
Proteins
Synaptosomes
Optical Imaging
Lysosomes
HeLa Cells
Hydrolysis
Theoretical Models
Fluorescence
Switches

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Saleeb, Rebecca Sonia ; Kavanagh, Deirdre M. ; Dun, Alison R. ; Dalgarno, Paul A. ; Duncan, Rory R. / A VPS33A-binding motif on syntaxin17 controls autophagy completion in mammalian cells. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 11. pp. 4188-4201.
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A VPS33A-binding motif on syntaxin17 controls autophagy completion in mammalian cells. / Saleeb, Rebecca Sonia; Kavanagh, Deirdre M.; Dun, Alison R.; Dalgarno, Paul A.; Duncan, Rory R.

In: Journal of Biological Chemistry, Vol. 294, No. 11, 15.03.2019, p. 4188-4201.

Research output: Contribution to journalArticle

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