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SNARE priming is essential for maturation of autophagosomes but not for their formation

  1. Zvulun Elazara,1
  1. aDepartment of Biomolecular Sciences, Weizmann Institute of Science, 76100 Rehovot, Israel;
  2. bDepartment of Chemical Research Support, Weizmann Institute of Science, 76100 Rehovot, Israel;
  3. cLife Sciences Core Facilities, Weizmann Institute of Science, 76100 Rehovot, Israel
  1. Edited by Sharon Anne Tooze, Francis Crick Institute, London, United Kingdom, and accepted by Editorial Board Member Pietro De Camilli October 17, 2017 (received for review April 6, 2017)

Significance

The contribution of SNARE-mediated membrane fusion to the different stages along the autophagic process is not well understood. In this work we demonstrate that inhibition of SNARE priming leads to impairment of the autophagic flux manifested by the accumulation of mature autophagosomes that do not fuse with the lysosomes. Our results suggest that SNARE priming is essential for the fusion of the autophagosome with the lysosome but not for autophagosome formation.

Abstract

Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. αSNAP, the adaptor molecule for the SNARE-priming enzyme N-ethylmaleimide-sensitive factor (NSF) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of αSNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of αSNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal–lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss.

Footnotes

  • ?1To whom correspondence should be addressed. Email: zvulun.elazar{at}weizmann.ac.il.
  • Author contributions: A.A. and Z.E. designed research; A.A., S.L.-Z., Z.P., and T.D. performed research; A.A., T.D., and Z.E. analyzed data; and A.A. and Z.E. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. S.A.T. is a guest editor invited by the Editorial Board.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1705572114/-/DCSupplemental.

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