Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

  1. Hongki Song
  2. Thomas L Torng
  3. Amy S Orr
  4. Axel T Brunger
  5. William T Wickner  Is a corresponding author
  1. Geisel School of Medicine at Dartmouth, United States
  2. Stanford University, United States

Abstract

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/aSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3 4, 5, and 6.

The following previously published data sets were used

Article and author information

Author details

  1. Hongki Song

    Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3761-5434
  2. Thomas L Torng

    Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2295-2777
  3. Amy S Orr

    Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States
    Competing interests
    No competing interests declared.
  4. Axel T Brunger

    Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States
    Competing interests
    Axel T Brunger, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5121-2036
  5. William T Wickner

    Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States
    For correspondence
    William.T.Wickner@dartmouth.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8431-0468

Funding

National Institutes of Health (R35GM118037)

  • William T Wickner

National Institutes of Health (R37MH63105)

  • Axel T Brunger

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Josep Rizo, University of Texas Southwestern Medical Center, United States

Publication history

  1. Received: February 16, 2021
  2. Accepted: April 30, 2021
  3. Accepted Manuscript published: May 4, 2021 (version 1)
  4. Version of Record published: May 24, 2021 (version 2)

Copyright

© 2021, Song et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Hongki Song
  2. Thomas L Torng
  3. Amy S Orr
  4. Axel T Brunger
  5. William T Wickner
(2021)
Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering
eLife 10:e67578.
https://doi.org/10.7554/eLife.67578

Further reading

    1. Biochemistry and Chemical Biology
    Josep Rizo, Klaudia Jaczynska, Karolina P Stepien
    Insight

    Two proteins called Sec17 and Sec18 may have a larger role in membrane fusion than is commonly assumed in textbook models.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Marco Caligaris, Raffaele Nicastro ... Claudio De Virgilio
    Research Advance

    The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallet et. al, 2015) reported that AMPK in yeast, i.e. Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.