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

  • Curated by eLife

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    Evaluation Summary:

    This is a very important paper that challenges the generally accepted dogma that full zippering of SNARE complexes is essential for intracellular membrane fusion. Previous work had already shown that C-terminal truncation of one SNARE arrested liposome fusion mediated by the yeast vacuolar SNARE complex and that Sec17/Sec18 could rescue fusion, but it was argued that such rescue could arise because Sec17/Sec18 restored C-terminal zippering. This paper now shows that Sec17/Sec18 rescue fusion even when three SNAREs are crippled -by truncation or mutation- to definitively prevent zippering, thus showing that Sec17/18 have a direct, positive role in membrane fusion.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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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/αSNAP 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 can 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 that 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.

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  1. Version published to 10.7554/elife.67578 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    The energy released upon zippering of SNARE complexes from the N-terminus to the membrane-proximal C-terminus is widely believed to provide the driving force for membrane fusion, and the cis-SNARE complexes resulting after fusion are disassembled by formation of a 20S complex with Sec17 and Sec18, followed by ATP-hydrolysis by Sec18. This paper now shows that membrane fusion still occurs when the hydrophobic interactions that drive C-terminal zippering of the yeast vacuolar SNARE complex is completely prevented by C-terminal truncation of two of the SNAREs and replacement of the hydrophobic residues at the C-terminus of the SNARE domain of a third SNARE with polar residues, and that such fusion requires Sec17, Sec18 and non-hydrolyzable ATP homologues, in addition to the HOPS tethering complex, which mediates SNARE complex assembly. The results also show that Sec17 plays a key role in fusion through hydrophobic residues in an N-terminal loop that are known to interact with membranes. These results suggest that the core membrane fusion machinery is formed by the SNAREs, Sec17 and Sec18 rather than by the SNAREs alone, and that fusion is driven by a combination of SNARE C-terminal zippering and perturbation of the lipid bilayers by the hydrophobic loops of Sec17. These conclusions are strongly supported by a variety of membrane fusion experiments. FRET assays to SNARE complex assembly also support the conclusions but are less convincing.

    Thank you. We feel that the FRET assays of HOPS-dependent, Sec17-driven zippering are important, as they indicate one of the 3 vital Sec17 functions: (1) promote SNARE zippering which, when it can occur, makes an important contribution to fusion. This is the first demonstration of Sec17-induced zippering in the context of HOPS. (2) promote fusion directly even when zippering contributes no energy. and (3) support Sec18 in disassembly of SNARE complexes.

    Reviewer #2 (Public Review):

    This manuscript shows that the Sec17/18 machine can do more than we might have expected, and places new constraints on models for how this works. As the field expects from the Wickner lab, the work is creative and beautifully executed. I do still have some reservations, however, about whether the manuscript ultimately forwards our mechanistic understanding enough to merit publication in eLife. Some of the outstanding mechanistic questions articulated by the authors include:

    1. Why is HOPS required for Sec17/18/ATPγS activity? The authors suggest that HOPS and Sec17 bind to one another, but the assay (Figure 4) is rather non-physiological and the result does not really answer the question.

    We have not established why HOPS can work with Sec17/Sec18 while other nonspecific tethers cannot, but future explorations of this question will be founded on a knowledge of which components bind directly to the others, including HOPS:Sec17. Each demonstration of binding between two purified proteins is of course non-physiological, yet contributes to our understanding. Still, we "hear you", and we've moved this to a supplemental figure.

    1. What is the mechanistic role of Sec18? An intricate inhibitor experiment (Figure 9) suggests that Sec18 acts later than Vps33. This is consistent with current thinking on the early role of SM proteins, but does not further delineate the mechanistic role of Sec18.

    Exactly. This is a fascinating question which is reinforced, but not answered, by our current work. We note that Sec17 is now seen to perform 3 functions (working with Sec18 for ATP-driven SNARE complex disassembly, driving completion of SNARE zippering, and supporting fusion per se) and Sec18 performs 2 functions (SNARE complex disassembly and supporting Sec17 for fusion per se).

    1. Does "entropic confinement" explain the role of Sec17? This very interesting question was not, so far as I could tell, directly addressed. My understanding is that the concept of entropic confinement comes from studies of chaperonins such as GroEL/ES, which entirely enclose their substrates in what Paul Sigler memorably described as "a temple for protein folding". Here, it's much less clear that Sec17 could sufficiently constrain the presumably-unfolded juxtamembrane regions of the truncated and/or mutant SNAREs to drive membrane fusion. Indeed, Schwartz et al. (2017) noted "open portals" between adjacent Sec17 molecules that would "allow SNARE residues spanning the partially-zipped helical bundle and the transmembrane anchors to pass cleanly between pairs of adjacent Sec17 subunits".

    We have removed the term "entropic confinement", and in deference to Schwartz et al. (which we cite) we refer to the Sec17 open cage and the folding environment it may create for SNARE complex assembly.

    1. What is the mechanistic role of the "hydrophobic loop" at the N-terminus of Sec17? Previous work from the Wickner lab (Song et al., 2017) concluded that its main function under normal circumstances was to promote Sec17 membrane association, but when zippering was incomplete it might act as a wedge to perturb the bilayers. These experiments made use of artificially membrane-anchored Sec17, either wild-type or the "FSMS" hydrophobic loop mutant. This approach was extended here (Figure 8) but did not, so far as I could tell, greatly advance our mechanistic understanding.

    Agreed. Each of your points 2-4 reinforce central questions which our lab, and others, will strive to answer: What does Sec18 do? How does Sec17 oligomerization around the SNAREs relate to those SNAREs? What is the role of the Sec17 apolar loop? We do find though that Sec17 and Sec18, however they act, are so important as contributors toward driving fusion that they can compensate for only partial zippering when tested to do so.

  3. eLife

    Reviewer #3 (Public Review):

    In this work, the authors used in vitro binding and liposome fusion assays to study how Sec17 and Sec18 regulate SNARE-driven fusion. In previous studies, it was found that deletion of the C-terminal layers of the Qc SNARE involved in yeast vacuole fusion blocks fusion but the inhibition can be partially bypassed by addition of Sec17 and Sec18. This work extended the finding and showed that Sec17 and Sec18 can even restore fusion when two Q SNAREs are C-terminally truncated and the third chain bears point mutations. The authors conclude that HOPS and membrane anchored Rabs first promote the tethering of vacuole membranes. Subsequently, HOPS promotes membrane docking - the initial assembly of the SNAREs, likely through the SM protein in the HOPS complex. Then Sec17 and Sec18 kick in to activate the zippering of membrane-proximal regions of SNAREs. This function seems to require interactions of Sec17 with HOPS. The findings are unexpected and raise important questions.

  4. eLife

    Reviewer #2 (Public Review):

    This manuscript shows that the Sec17/18 machine can do more than we might have expected, and places new constraints on models for how this works. As the field expects from the Wickner lab, the work is creative and beautifully executed. I do still have some reservations, however, about whether the manuscript ultimately forwards our mechanistic understanding enough to merit publication in eLife. Some of the outstanding mechanistic questions articulated by the authors include:

    1. Why is HOPS required for Sec17/18/ATPγS activity? The authors suggest that HOPS and Sec17 bind to one another, but the assay (Figure 4) is rather non-physiological and the result does not really answer the question.

    2. What is the mechanistic role of Sec18? An intricate inhibitor experiment (Figure 9) suggests that Sec18 acts later than Vps33. This is consistent with current thinking on the early role of SM proteins, but does not further delineate the mechanistic role of Sec18.

    3. Does "entropic confinement" explain the role of Sec17? This very interesting question was not, so far as I could tell, directly addressed. My understanding is that the concept of entropic confinement comes from studies of chaperonins such as GroEL/ES, which entirely enclose their substrates in what Paul Sigler memorably described as "a temple for protein folding". Here, it's much less clear that Sec17 could sufficiently constrain the presumably-unfolded juxtamembrane regions of the truncated and/or mutant SNAREs to drive membrane fusion. Indeed, Schwartz et al. (2017) noted "open portals" between adjacent Sec17 molecules that would "allow SNARE residues spanning the partially-zipped helical bundle and the transmembrane anchors to pass cleanly between pairs of adjacent Sec17 subunits".

    4. What is the mechanistic role of the "hydrophobic loop" at the N-terminus of Sec17? Previous work from the Wickner lab (Song et al., 2017) concluded that its main function under normal circumstances was to promote Sec17 membrane association, but when zippering was incomplete it might act as a wedge to perturb the bilayers. These experiments made use of artificially membrane-anchored Sec17, either wild-type or the "FSMS" hydrophobic loop mutant. This approach was extended here (Figure 8) but did not, so far as I could tell, greatly advance our mechanistic understanding.

  5. eLife

    Reviewer #1 (Public Review):

    The energy released upon zippering of SNARE complexes from the N-terminus to the membrane-proximal C-terminus is widely believed to provide the driving force for membrane fusion, and the cis-SNARE complexes resulting after fusion are disassembled by formation of a 20S complex with Sec17 and Sec18, followed by ATP-hydrolysis by Sec18. This paper now shows that membrane fusion still occurs when the hydrophobic interactions that drive C-terminal zippering of the yeast vacuolar SNARE complex is completely prevented by C-terminal truncation of two of the SNAREs and replacement of the hydrophobic residues at the C-terminus of the SNARE domain of a third SNARE with polar residues, and that such fusion requires Sec17, Sec18 and non-hydrolyzable ATP homologues, in addition to the HOPS tethering complex, which mediates SNARE complex assembly. The results also show that Sec17 plays a key role in fusion through hydrophobic residues in an N-terminal loop that are known to interact with membranes. These results suggest that the core membrane fusion machinery is formed by the SNAREs, Sec17 and Sec18 rather than by the SNAREs alone, and that fusion is driven by a combination of SNARE C-terminal zippering and perturbation of the lipid bilayers by the hydrophobic loops of Sec17. These conclusions are strongly supported by a variety of membrane fusion experiments. FRET assays to SNARE complex assembly also support the conclusions but are less convincing.

  6. eLife

    Evaluation Summary:

    This is a very important paper that challenges the generally accepted dogma that full zippering of SNARE complexes is essential for intracellular membrane fusion. Previous work had already shown that C-terminal truncation of one SNARE arrested liposome fusion mediated by the yeast vacuolar SNARE complex and that Sec17/Sec18 could rescue fusion, but it was argued that such rescue could arise because Sec17/Sec18 restored C-terminal zippering. This paper now shows that Sec17/Sec18 rescue fusion even when three SNAREs are crippled -by truncation or mutation- to definitively prevent zippering, thus showing that Sec17/18 have a direct, positive role in membrane fusion.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  7. Version published to 10.1101/2021.02.16.431494v1 on bioRxiv