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Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

  1. Eric A Prinslow
  2. Karolina P Stepien
  3. Yun-Zu Pan
  4. Junjie Xu
  5. Josep Rizo  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
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Cite this article as: eLife 2019;8:e38880 doi: 10.7554/eLife.38880

Abstract

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.

https://doi.org/10.7554/eLife.38880.001

Introduction

The release of neurotransmitters by Ca2+-triggered synaptic vesicle exocytosis is a central event for interneuronal communication and involves multiple steps. Synaptic vesicles first dock at specialized sites on the plasma membrane called active zones, undergo one or more priming reactions that leave the vesicles ready for release, and fuse with the plasma membrane upon Ca2+ influx evoked by an action potential (Südhof, 2013). Extensive research has shown that these steps are exquisitely regulated by a sophisticated protein machinery and has led to defined models for the functions of key components from this machinery (Brunger et al., 2018; Jahn and Fasshauer, 2012; Rizo, 2018; Südhof and Rothman, 2009). The soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) synaptobrevin, syntaxin-1 and SNAP-25 form a parallel four-helix bundle called the SNARE complex that brings the synaptic vesicle and plasma membranes together and is key for membrane fusion (Hanson et al., 1997; Poirier et al., 1998; Söllner et al., 1993; Sutton et al., 1998). N-ethylmaleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs; no relation to SNAP-25) disassemble SNARE complexes after release to recycle the SNAREs for another round of fusion (Banerjee et al., 1996; Mayer et al., 1996; Söllner et al., 1993). Munc18-1 and Munc13s orchestrate SNARE complex assembly in an NSF-SNAP-resistant manner (Ma et al., 2013) that improves the fidelity of parallel assembly (Lai et al., 2017). The underlying mechanism involves binding of Munc18-1 to a self-inhibited ‘closed conformation’ of syntaxin-1 (Dulubova et al., 1999; Misura et al., 2000) and to synaptobrevin, thus forming a template to assemble the SNARE complex (Baker et al., 2015; Parisotto et al., 2014; Sitarska et al., 2017), while Munc13s bridge the vesicle and plasma membranes (Liu et al., 2016) and help to open syntaxin-1 (Ma et al., 2011; Richmond et al., 2001; Yang et al., 2015). Synaptotagmin-1 acts as the major Ca2+ sensor that triggers release through interactions with phospholipids (Fernández-Chacón et al., 2001) and the SNARE complex (Brewer et al., 2015; Zhou et al., 2015; Zhou et al., 2017), in a tight interplay with complexins (Giraudo et al., 2006; Schaub et al., 2006; Tang et al., 2006).

Despite these and other crucial advances, fundamental questions remain about how trans-SNARE complexes that bridge the vesicle and plasma membranes are formed, about the interplay between Munc18-1, Munc13-1, NSF and αSNAP in promoting trans-SNARE complex assembly or disassembly, and about the nature of the primed state(s) of the release machinery in the readily-releasable pool (RRP) of vesicles. The primed state is believed to include trans-SNARE complexes that are partially formed, with the N-terminal half assembled and at least part of the C-terminal, membrane proximal portion unassembled (e.g. Sørensen et al., 2006; Walter et al., 2010), but it is unclear which other components are bound to the SNAREs in this state. Reconstitution assays showing that the fusion between synaptobrevin-containing liposomes and syntaxin-1-SNAP-25-containing liposomes observed in the presence of synaptotagmin-1 and Ca2+ is abolished by NSF-αSNAP, but occurs efficiently upon further addition of Munc18-1 and a Munc13-1 fragment, led to the notion that Munc18-1 and Munc13-1 mediate a pathway for trans-SNARE complex assembly that is resistant to NSF-αSNAP (Liu et al., 2016; Ma et al., 2013), explaining the essential nature of Munc18-1 and Munc13s for vesicle priming (Aravamudan et al., 1999; Richmond et al., 1999; Varoqueaux et al., 2002; Verhage et al., 2000). This interpretation arose in part because NSF-αSNAP disassemble not only cis-SNARE complexes but also syntaxin-1-SNAP-25 heterodimers (Hayashi et al., 1995), thus preventing trans-SNARE complex formation by the SNAREs alone, and because of evidence suggesting that NSF-αSNAP cannot disassemble trans-SNARE complexes (Weber et al., 2000). However, studies of yeast vacuolar fusion showed that the NSF-αSNAP homologues Sec18-Sec17 disassemble trans-SNARE complexes and that disassembly is prevented by HOPS, a tethering complex that includes the Munc18-1 homologue Vps33 and coordinates SNARE complex formation (Mima et al., 2008; Xu et al., 2010). Moreover, recent reports showed that at least a fraction of neuronal trans-SNARE complexes can be disassembled by NSF-αSNAP in vitro (Yavuz et al., 2018) and that Munc18-1 and Munc13-1 are critical to prevent de-priming of readily-releasable synaptic vesicles in neurons, but such requirement can be bypassed by the NSF inactivating agent N-ethylmaleimide (He et al., 2017).

These findings suggest that the cytoplasmic environment of a presynaptic terminal favors disassembly of all kinds of SNARE complexes and hence that the trans-SNARE complexes formed after priming must be protected against disassembly by NSF-αSNAP. However, the mechanisms underlying such protection are unknown. The results of He et al. (2017) indicate that Munc18-1 and Munc13-1 play key roles in such protection, in addition to mediating an NSF-αSNAP-resistant pathway of trans-SNARE complex assembly, but this hypothesis has not been tested, and Munc18-1 and Munc13-1 are often assumed to be dispensable after mediating assembly. Moreover, it is plausible that protection against disassembly by NSF-αSNAP depends also on other proteins such as synaptotagmin-1 and complexins, which bind to SNARE complexes and have also been proposed to facilitate trans-SNARE complex formation (Diao et al., 2013; Li et al., 2017). In this context, while initial studies suggested that synaptic vesicle priming is not altered in neurons from synaptotagmin-1 knockout (KO) mice (Geppert et al., 1994) and in complexin-1/2 double knockout (DKO) mice (Reim et al., 2001), subsequent analyses revealed that absence of these proteins does decrease the RRP of vesicles (Bacaj et al., 2015; Chang et al., 2018; Xue et al., 2010; Yang et al., 2010). Such decreases were not as dramatic as those observed in Munc18-1 KO mice (Verhage et al., 2000) and Munc13-1/2 DKO mice (Varoqueaux et al., 2002), where priming is totally abrogated, but do suggest that synaptotagmin-1 and complexins are involved in priming or perhaps in maintenance of the RRP. Thus, while it is known that the existence of an RRP of vesicles depends on Munc18-1, Munc13-1, synaptotagmin-1 and complexins, it is still unclear to what extent the roles of these various factors arise because they mediate priming by facilitating trans-SNARE complex assembly and/or because they stabilize primed vesicles by protecting against trans-SNARE complex disassembly by NSF-SNAPs.

The study presented herein was designed to address these questions and understand the interplay between these proteins in trans-SNARE complex assembly and disassembly, using a fluorescence resonance energy transfer (FRET) assay. Our data show that trans-SNARE complex assembly in the presence of NSF-αSNAP requires Munc18-1 and Munc13-1, as expected from our previous reconstitution experiments (Ma et al., 2013), but does not require complexin-1. Moreover, we find that Munc18-1 and Munc13-1 synergistically help to maintain assembled trans-SNARE complexes in the presence of NSF-αSNAP, which is strongly enhanced by Ca2+, and that trans-SNARE complexes are protected against disassembly by complexin-1. Synaptotagmin-1 facilitates NSF-αSNAP-resistant trans-SNARE complex assembly and may contribute to stabilizing trans-SNARE complexes, but its effects are less marked. We propose a model whereby Munc18-1 and Munc13-1 play key roles not only in priming synaptic vesicles to a readily-releasable state but also in protecting them against de-priming by NSF-SNAPs, while synaptotagmin-1 plays a less critical role in both priming and maintenance of the RRP, and complexin-1 does not mediate priming but stabilizes primed vesicles.

Results

A sensitive assay to monitor trans-SNARE complex assembly and disassembly

In order to investigate the factors that influence trans-SNARE complex assembly and disassembly, membrane fusion must be prevented to avoid the conversion of trans-SNARE complexes into cis complexes that are well known to be disassembled by NSF-αSNAP (Söllner et al., 1993). To set up a trans-SNARE complex assembly assay without interference from membrane fusion, we used a similar approach to that described recently by Yavuz et al. (2018), which was published during the course of this work and used a mutation at the C-terminus of the synaptobrevin SNARE motif to prevent C-terminal assembly of the SNARE complex. For our assay, we designed a SNAP-25 mutant bearing two single residue substitutions (M71D,L78D) that replace buried hydrophobic residues with negatively charged residues at the C-terminus of the SNARE four-helix bundle (Figure 1A), and thus are also expected to strongly hinder C-terminal zippering of the SNARE complex. To verify this expectation, we used a membrane fusion assay that simultaneously measures lipid and content mixing between synaptobrevin-containing liposomes and syntaxin-1-SNAP-25-containing liposomes in the presence of NSF, αSNAP, Munc18-1 and a fragment containing the C1, C2B, MUN and C2C domains of Munc13-1 (Liu et al., 2016; Liu et al., 2017). This fragment, which we refer to as C1C2BMUNC2C, spans the entire highly conserved C-terminal region of Munc13-1 and is sufficient to efficiently rescue neurotransmitter release in Munc13-1/2 DKO neurons (Liu et al., 2016). As expected, we observed highly efficient, Ca2+-dependent membrane fusion in experiments performed with wild type (WT) SNAP-25; however, content mixing was abolished and lipid mixing was very inefficient when SNAP-25m was used in the assays instead of WT SNAP-25 (Figure 1B,C), demonstrating that the M71D,L78D mutation in SNAP-25 indeed prevents membrane fusion. Note that the small amount of lipid mixing that we observed might arise from lipid transfer without membrane merger (Rizo, 2018) and that some lipid mixing was observed in reconstitution assays even when long flexible linkers were introduced between the synaptobrevin SNARE motif and transmembrane region (McNew et al., 1999).

Design of a SNAP-25 mutation that abrogates its ability to support membrane fusion.

(A) Ribbon diagram of the crystal structure of the SNARE complex (PDB accession code 1SFC) (Sutton et al., 1998). Synaptobrevin is red, syntaxin-1 yellow and SNAP-25 green, with the side chains of the two residues that were mutated to aspartate (M71 and L78) shown as pink spheres. Note that the side chains are pointing toward the hydrophobic interior of the four-helix bundle. Hence, mutating these residues to aspartate is expected to prevent C-terminal zippering of the SNARE complex. The residue numbers of the two mutated residues and of the N-termini of synaptobrevin and syntaxin-1 SNARE motifs are indicated. (B,C) The SNAP-25 M71D,L78D mutation abrogates membrane fusion in reconstitution assays. Lipid mixing (B) between V- and T-liposomes was monitored from the fluorescence de-quenching of Marina Blue lipids and content mixing (C) was monitored from the increase in the fluorescence signal of Cy5-streptavidin trapped in the V-liposomes caused by FRET with PhycoE-biotin trapped in the T-liposomes upon liposome fusion. The assays were performed in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C, NSF and αSNAP with T-liposomes that contained syntaxin-1 and wild type (WT) SNAP-25 or SNAP-25 M71D,L78D mutant (SNAP-25m). Experiments were started in the presence of 100 μM EGTA and 5 μM streptavidin, and Ca2+ (600 μM) was added at 300 s.

https://doi.org/10.7554/eLife.38880.002

To test for formation of trans-SNARE complexes, we developed a FRET assay based on attachment of donor (Alexa488) and acceptor (tetramethylrhodamine, TMR) fluorescent probes on single-cysteine mutants of full-length synaptobrevin (L26C) and syntaxin-1 (S186C), respectively (all native cysteines were mutated to serine or hydrophobic residues). Residues L26 of synaptobrevin and S186 of syntaxin-1 were chosen to place the fluorescent probes because they closely precede the N-termini of the SNARE motifs in the four-helix bundle (Sutton et al., 1998), that is residue 29 of synaptobrevin and 190 of syntaxin-1 (Figure 1A). Hence, SNARE complex assembly is not expected to be perturbed by attachment of fluorescent probes to these residues but should bring the fluorescent probes into close proximity for efficient FRET, while disassembly by NSF-αSNAP should eliminate the FRET (Figure 2A). Attachment of fluorescent probes to residue 26 of synaptobrevin and 186 of syntaxin-1 is also expected to have no effect on binding of both proteins to NSF-αSNAP, complexin-1, synaptotagmin-1 or Munc18-1 based on the three-dimensional structural information available on the 20S complex formed by NSF, αSNAP and the SNAREs (Zhao et al., 2015), on the complexin-SNARE complex (Chen et al., 2002), on three synaptotagmin-1-SNARE complexes (Brewer et al., 2015; Zhou et al., 2015; Zhou et al., 2017), on the Munc18-1-closed syntaxin-1 complex (Misura et al., 2000), and on the vacuolar Vps33-Nyv1 complex (Baker et al., 2015), which most likely provides a reliable model for the homologous Munc18-1-synaptobrevin complex (Sitarska et al., 2017). Since in the experiments described below we relied on the donor fluorescence emission to monitor FRET, we tested whether the emission spectrum of liposomes containing Alexa488-synaptobrevin is affected by various proteins used in this study, including Munc18-1, Munc13-1 C1C2BMUNC2C, NSF, αSNAP, complexin-1 and a soluble fragment of synaptotagmin-1 spanning the two C2 domains that form most of its cytoplasmic region (C2AB) and include its Ca2+-binding sites (Fernandez et al., 2001; Sutton et al., 1995; Ubach et al., 1998). None of these proteins substantially affected the fluorescence spectrum except for a slight increase in fluorescence caused by Munc13-1 C1C2BMUNC2C (Figure 2—figure supplement 1) that did not affect the conclusions derived from our data.

Figure 2 with 4 supplements see all
An assay to measure assembly of trans-SNARE complexes and disassembly by NSF-αSNAP.

(A) Diagram illustrating the assay used to monitor trans-SNARE complex assembly and disassembly. V-liposomes containing synaptobrevin labeled with a FRET donor (Alexa488, green star) at residue 26 are mixed with T-liposomes containing SNAP-25m and syntaxin-1 labeled at residue 186 with a FRET acceptor (TMR, red star) in the presence of different factors. After monitoring the decrease in donor fluorescence intensity resulting from trans-SNARE complex formation under diverse conditions, NSF and αSNAP are added to test for disassembly of trans SNARE complexes. Synaptobrevin is red, SNAP-25m green and syntaxin-1 orange (N-terminal Habc domain) and yellow (SNARE motif). Although an excess of SNAP-25m was used in preparing the syntaxin-1-SNAP-25m liposomes, the majority of syntaxin-1-SNAP-25m complexes are expected to have a 2:1 stoichiometry such that the second syntaxin-1 SNARE molecule occupies the position of the synaptobevin SNARE motif in the SNARE four-helix bundle (bottom left diagram), hindering SNARE complex formation (reviewed in Rizo and Südhof, 2012). In some of the experiments, trans-SNARE complex assembly was facilitated by inclusion of the Syb49-93 peptide, which spans the C-terminal part of the synaptobrevin SNARE motif and displaces the second syntaxin-1 molecule from the syntaxin-1-SNAP-25m heterodimer, yielding the intermediate shown between brackets. Because Syb49-93 lacks the N-terminal half of the synaptobrevin SNARE motif, it can readily be displaced by full-length synaptobrevin to form trans-SNARE complexes (Pobbati et al., 2006). In other experiments, NSF-αSNAP were added from the beginning to investigate trans-SNARE complex assembly in their presence. (B) Fluorescence emission spectra (excitation at 468 nm) of a mixture of V-liposomes containing Alexa488-synaptobrevin and T-liposomes containing TMR-syntaxin-1-SNAP-25m (1:4 V- to T-liposome ratio) that had been incubated for five hours with Syb49-93 (black trace), and of the same sample after adding NSF-αSNAP plus ATP and Mg2+ (red trace). The blue curve shows a control spectrum obtained by adding spectra acquired separately for V- and T-liposomes at the same concentrations. (C) Fluorescence emission spectra acquired under conditions similar to those of (B), with pre-incubated mixtures of Syb49-93 with V- and T-liposomes before (black curve) or after addition of NSF-αSNAP plus ATP and EDTA (green curve) or NSF-αSNAP plus ATPγS and Mg2+ (red curve). (D,E) Fluorescence emission spectra acquired under similar conditions to those of (B), except that for the green curve WT αSNAP was replaced with the αSNAP FS (D) or KE (E) mutant. The red, black and blue curves are the same as in panel (B). All spectra were corrected for dilution caused by addition of reagents.

https://doi.org/10.7554/eLife.38880.003

A potential problem with FRET assays to monitor trans-SNARE complex formation is that only a small subset of the SNAREs may form these complexes, leading to low FRET efficiency and hindering quantification of the degree of SNARE complex assembly (or disassembly). To maximize the amount of observable FRET based on the decrease in donor fluorescence emission intensity, we employed liposomes containing Alexa488-labeled synaptobrevin at a low protein-to-lipid (P/L) ratio (1:10,000) (V-liposomes) and used a large excess of liposomes containing TMR-syntaxin-1 and SNAP-25m at higher P/L ratio (1:800) (T-liposomes) in our FRET assays. Mixing the V- and T-liposomes at a 1:4 ratio led to a very slow decrease in donor fluorescence intensity (Figure 2—figure supplement 2A) that shows that trans-SNARE complex assembly is very inefficient under these conditions, most likely because SNARE complex assembly is hindered by formation of syntaxin-1-SNAP-25m heterodimers where synaptobrevin is replaced by a second syntaxin-1 molecule, leading to a 2:1 stoichiometry (Figure 2A). To overcome this problem, we used a synaptobrevin peptide spanning residues 49–93 (Syb49-93), which is expected to facilitate SNARE complex formation by displacing the second syntaxin-1 molecule (Pobbati et al., 2006). Indeed, inclusion of Syb49-93 strongly accelerated the rate of decrease in donor fluorescence intensity upon mixing V- and T-liposomes (Figure 2—figure supplement 2A). No further decrease in FRET was observed after five hours of incubation at 37°C, indicating that this time was sufficient to maximize the formation of trans-SNARE complexes. Cryo-electron microscopy (cryo-EM) images obtained for samples prepared under these conditions revealed well-dispersed liposomes that often exhibited close contacts with one or two other liposomes but did not form large clusters (Figure 2—figure supplement 3), as expected because of the use of low synaptobrevin-to-lipid ratios in the V-liposomes.

Comparison of the fluorescence emission spectrum acquired after incubating V- and T-liposomes with Syb49-93 for five hours with a control spectrum obtained by adding the spectra of separate samples of V- and T-liposomes confirmed a clear decrease in donor fluorescence, showing that efficient FRET developed as a result of trans-SNARE complex formation (Figure 2B, black and blue curves, respectively). The efficient FRET suggests that most of the accessible synaptobrevin molecules were incorporated into SNARE complexes, as only half of the Alexa488-labeled synaptobrevin molecules are expected to be accessible on the surface of the liposomes. Further addition of NSF-αSNAP led to a substantial but not complete recovery of the donor fluorescence (Figure 2B, red curve). These results show that NSF-αSNAP disassembled a fraction of the trans-SNARE complexes (estimated to be about 50%) while the remaining complexes were resistant to NSF-αSNAP, in agreement with the results of Yavuz et al. (2018). It is worth noting that in these experiments there was a small degree of direct excitation of the large excess of acceptor probes used (see Figure 2—figure supplement 4A), even though the excitation wavelength corresponded to the donor. As expected, the acceptor fluorescence increased with respect to the V + T control upon incubating V- and T-liposomes for five hours, due to trans-SNARE complex formation (Figure 2B, black and blue curves). However, the acceptor fluorescence exhibited only a small increase upon addition of NSF-αSNAP (Figure 2B, red curve). This finding arises because the acceptor fluorescence is considerably affected by NSF-αSNAP, in contrast to the donor fluorescence (see below and Figure 5—figure supplements 2 and 3). Hence, the donor fluorescence provides a more reliable parameter than the acceptor fluorescence to assess the degree of trans-SNARE complex disassembly by NSF-αSNAP.

Disassembly of trans-SNARE complexes required ATP hydrolysis by NSF, as no disassembly was observed when Mg2+ was replaced by EDTA in the reaction, or NSF was bound to ATPγS rather than ATP (Figure 2C). A similar amount of disassembly was observed in parallel assays performed with WT SNAP-25 instead of SNAP-25m in the presence of ATP and Mg2+ (Figure 2—figure supplement 4B), showing that the reaction is not affected by the M71D,L78D mutation. We also examined whether trans-SNARE complex disassembly is affected by a K122E,K163E (KE) mutation in αSNAP that impairs SNARE binding (Zhao et al., 2015) and by an F27S,F28S (FS) mutation in an N-terminal loop of αSNAP that impairs disassembly of membrane-anchored cis-SNARE complexes because it disrupts binding of αSNAP to membranes (Winter et al., 2009). Both αSNAP mutations strongly impaired the recovery of donor fluorescence observed when trans-SNARE complexes were disassembled by NSF in the presence of wild type (WT) αSNAP (compare green and red curves in Figure 2D,E), showing that interactions of αSNAP with both the membranes and the SNAREs are critical for trans-SNARE complex disassembly.

Interplay between NSF, αSNAP, Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 in trans-SNARE complex assembly-disassembly

Our previous reconstitution studies showing that fusion between synaptobrevin-liposomes and syntaxin-1-SNAP-25-liposomes in the presence of NSF-αSNAP requires Munc18-1 and Munc13-1 led us to propose that Munc18-1 and Munc13-1 organize trans-SNARE complex assembly in an NSF-αSNAP resistant manner (Liu et al., 2016; Ma et al., 2013). However, trans-SNARE complex assembly was not directly monitored in these studies and it was unclear whether Munc18-1 and Munc13-1 were dispensable after trans-SNARE complex assembly. The finding that trans-SNARE complexes can be disassembled by NSF-αSNAP raises the question as to whether, in addition to providing an NSF-αSNAP-resistant pathway for trans-SNARE complex assembly, Munc18-1 and Munc13-1 protect assembled trans-SNARE complexes from disassembly by NSF-αSNAP. To address this question and also investigate the roles of synaptotagmin-1 and complexin-1 in trans-SNARE complex assembly and protection against disassembly, we performed kinetic experiments where we used our FRET assay, monitoring the decrease in donor emission fluorescence associated with trans-SNARE complex assembly in the presence of NSF-αSNAP and various combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, the synaptotagmin-1 C2AB fragment and complexin-1. Ca2+ was added after 750 s to test its effects on assembly.

In the presence of Munc18-1 and Munc13-1 C1C2BMUNC2C, we observed some slow trans-SNARE complex assembly before Ca2+ addition and assembly was dramatically enhanced by Ca2+, while there was almost no assembly in reactions with Munc18-1 alone or Munc13-1 C1C2BMUNC2C alone (Figure 3A). Complexin-1 and synaptotagmin-1 C2AB were unable to support trans-SNARE complex assembly in the presence of NSF-αSNAP even after Ca2+ addition, and did not appear to enhance the rate of trans-SNARE complex assembly supported by Munc18-1 and Munc13-1 C1C2BMUNC2C (Figure 3B). These results confirm our proposal that Munc18-1 and Munc13-1 organize trans-SNARE complex assembly in an NSF-αSNAP resistant manner based on liposome fusion assays (Liu et al., 2016; Ma et al., 2013) but note that, in those assays, fusion might ensue quickly, in a concerted fashion, upon trans-SNARE complex formation without a chance for disassembly. Because in our FRET assays of trans-SNARE complex formation fusion is prevented by the mutation in SNAP-25m, the efficient decrease in donor fluorescence observed in the presence of Ca2+, Munc18-1 and Munc13-1 C1C2BMUNC2C (Figure 3A) suggests that these factors prevent disassembly of trans-SNARE complexes in addition to mediating NSF-αSNAP-resistant assembly. Note however that we cannot completely rule out the possibility that, instead of physically preventing disassembly, Munc18-1 and Munc13-1 C1C2BMUNC2C mediate fast re-assembly of trans-SNARE complexes after they are disassembled. For simplicity, below we use terms like ‘prevent’ or ‘protect against disassembly’ to reflect the observation that a particular factor(s) increases the amount of assembled trans-SNARE complexes observed in the presence of NSF-αSNAP, but it is important to keep in mind both possible interpretations (see discussion).

Figure 3 with 2 supplements see all
Influence of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 on trans-SNARE complex assembly-disassembly in the presence of NSF-αSNAP.

(A,B) Kinetic assays monitoring trans-SNARE complex assembly between V- and T-liposomes (1:4 ratio) in the presence of NSF-αSNAP from the decrease in the donor fluorescence emission intensity. The experiments were performed in the absence of other proteins (T + V) or in the presence of different combinations of Munc18-1 (M18), Munc13-1 C1C2BMUNC2C (M13), complexin-1 (Cpx), synaptotagmin-1 C2AB and Syb49-93, as indicated by the colors. Experiments were started in 100 μM EGTA and Ca2+ (600 μM) was added after 750 s. (C) Analogous kinetic assays performed in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C, NSF-αSNAP and 100 μM EGTA, but adding 240 μM Ca2+ at 2 min to stimulate trans-SNARE complex assembly and adding 500 μM EGTA at different times to chelate the Ca2+ and interrogate whether there is trans-SNARE complex disassembly. An experiment that was also started in 100 μM EGTA but without addition of Ca2+ or EGTA at later times (gray trace) is shown for comparison. (D) Experiments analogous to those of (C), with addition of 240 μM Ca2+ at 2 min and 500 μM EGTA at 17 min, performed in the absence or presence of complexin-1 and/or synaptotagmin-1 C2AB. (E) Kinetic assays monitoring trans-SNARE complex assembly between VSyt1- and T-liposomes (1:4 ratio) in the presence of NSF-αSNAP and different combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and Syb49-93, as indicated by the colors. Experiments were started in 100 μM EGTA and Ca2+ (600 μM) was added after 750 s. (F) Kinetic assays analogous to those of (E) performed in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C, NSF-αSNAP and 100 μM EGTA, but adding 240 μM Ca2+ at 2 min to stimulate trans-SNARE complex assembly and adding 500 μM EGTA at different times to chelate the Ca2+ and interrogate whether there is trans-SNARE complex disassembly. An experiment that was also started in 100 μM EGTA but without addition of Ca2+ or EGTA at later times (gray trace) is shown for comparison. The light blue trace shows an additional experiment started in 100 μM EGTA in the presence of complexin-1, with addition of 240 μM Ca2+ at 2 min and 500 μM EGTA at 17 min. All experiments were performed in the presence of Mg2+ and ATP. For all traces shown in (A–F), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents.

https://doi.org/10.7554/eLife.38880.008

Munc18-1 and Munc13-1 C1C2BMUNC2C mediate some Ca2+-independent assembly of trans-SNARE complexes (Figure 3A), suggesting that these proteins also protect against disassembly in the absence of Ca2+. However, it is unclear whether such protection is as efficient as that occurring in the presence of Ca2+ because of the low level of Ca2+-independent assembly observed in the time scale of these assays. To overcome this problem, we performed additional assays where Ca2+ was added early to promote efficient trans-SNARE complex assembly, and EGTA was added afterwards to test whether, in the absence of Ca2+, Munc18-1 and Munc13-1 C1C2BMUNC2C could keep the trans-SNARE complexes assembled. EGTA caused some recovery of the donor fluorescence intensity, but the recovery leveled off with time, and the later EGTA was added, the lower was the donor fluorescence at the latest time point, showing that we did not reach equilibrium in these assays (Figure 3C). Nevertheless, the observation that the donor fluorescence intensity at the end is markedly lower than the initial intensity shows that a population of trans-SNARE complexes remained assembled, suggesting that Munc18-1 and Munc13-1 indeed protect trans-SNARE complexes from disassembly by NSF-αSNAP in the absence of Ca2+. Using a similar approach, we tested whether the synaptotagmin-1 C2AB or complexin-1 protect against the disassembly of trans-SNARE complexes observed upon addition of EGTA. Including complexin-1 completely prevented such disassembly, whereas synaptotagmin-1 C2AB had no effect (Figure 3D). Note that complexin-1 seemed to enhance the assembly rate in these assays, in contrast to those of Figure 3B; thus, it is unclear from these data whether complexin-1 assists in assembly.

The use of the soluble synaptotagmin-1 C2AB fragment allowed us to directly compare the assembly and disassembly of trans-SNARE complexes in the absence and presence of the synaptotagmin-1 C2 domains with the same V-liposomes, but in vivo synaptotagmin-1 is anchored on synaptic vesicles. To investigate how membrane anchoring of synaptotagmin-1 influences trans-SNARE complex assembly-disassembly, we performed FRET experiments analogous to those described above but using liposomes that contained the same low P/L ratio of Alexa488-labeled synaptobrevin (1:10,000) and synaptotagmin-1 incorporated at a 1:1,000 P/L [comparable to physiological ratios for sybaptotagmin-1; (Takamori et al., 2006) (referred to below as VSyt1-liposomes). Trans-SNARE complex formation between VSyt1- and T-liposomes was again stimulated strongly by the Syb49-93 peptide (Figure 2—figure supplement 2B) and fluorescence spectra acquired after a long incubation with Syb49-93 revealed efficient formation of trans-SNARE complexes, while addition of NSF-αSNAP disassembled about 55% of these complexes (Figure 2—figure supplement 4C), similar to the results obtained with V-liposomes (Figure 2B).

No trans-SNARE complex assembly between VSyt1- and T-liposomes was observed in kinetic experiments performed in the presence of NSF-αSNAP together with Syb49-93, Syb49-93 plus complexin-1, Munc18-1 alone or Munc13-1 alone (Figure 3E). However, considerable trans-SNARE complex assembly was observed in the presence of Munc18-1 and Munc13-1 C1C2BMUNC2C, which was strongly accelerated by Ca2+ (Figure 3E, red trace). These results show again that Munc18-1 and Munc13-1 C1C2BMUNC2C are critical for trans-SNARE complex assembly in the presence of NSF-αSNAP, as observed in the experiments performed with V- and T-liposomes (Figure 3A,B). Interestingly, Ca2+-independent assembly was more efficient with the VSyt1-liposomes than with V-liposomes, suggesting that membrane-anchored synaptotagmin-1 facilitates the NSF-αSNAP-resistant assembly mediated by Munc18-1 and Munc13-1 C1C2BMUNC2C. To test whether, in addition, membrane anchored synaptotagmin-1 helps to protect trans-SNARE complexes once they are formed, we again performed kinetic assays where we added Ca2+ shortly after mixing the VSyt1- and T-liposomes, and EGTA was added afterwards at different time points. We again observed partial recovery of the donor fluorescence intensity upon EGTA addition (Figure 3F), but the overall amount of trans-SNARE complexes that remained assembled was higher than in the experiments performed with V- and T-liposomes (Figure 3C). These results indicate that membrane-anchored synaptotagmin-1 may help to protect trans-SNARE complexes against disassembly by NSF-αSNAP once they are formed.

In parallel experiments including complexin-1, no donor fluorescence recovery was observed when EGTA was added, showing again that complexin-1 protects against disassembly, and the overall efficiency of assembly was higher (Figure 3F). It is also worth noting that, in our standard assembly assays where Ca2+ was added at 750 s, Ca2+-independent assembly was slower in the presence of complexin-1 than in its absence, but did not appear to level off at this time, as did the reaction without complexin-1 (Figure 3E, red and blue curves). Indeed, at longer time periods Ca2+-independent assembly was more efficient in the presence of complexin-1 even though it was lower in the beginning (Figure 3—figure supplement 1). These results suggest that, in the absence of Ca2+, complexin-1 partially inhibits assembly of trans-SNARE complexes between VSyt1- and T-liposomes but increases the overall assembly efficiency because it protects trans-SNARE complexes against disassembly by NSF-αSNAP, which was further supported by additional experiments described below.

The substantial amount of Ca2+-independent trans-SNARE complex assembly between VSyt1- and T-liposomes observed in our FRET assays in the presence of NSF-αSNAP, Munc18-1 and Munc13-1 C1C2BMUNC2C contrasts with the absence of content mixing that we commonly observed in fusion assays performed with V- or VSyt1-liposomes using a synaptotabrevin-to-lipid ratio of 1:500 and incorporating WT SNAP-25 in the T-liposomes (Figure 1C and Liu et al., 2016). To verify the latter result with the same synaptobrevin density used for the trans-SNARE complex assembly assays, we performed fusion assays using VSyt1-liposomes with the same synaptobrevin-to-lipid ratio (1:10,000). We did not observe any fusion in the absence of Ca2+ while lipid and content mixing were efficient but slow upon Ca2+ addition (Figure 3—figure supplement 2), in contrast with both the substantial Ca2+-independent trans-SNARE complex assembly and the fast Ca2+-dependent assembly observed in our FRET assays (Figure 3E). These data illustrate that trans-SNARE complex assembly does not necessarily lead to membrane fusion under these conditions, as observed previously in other reconstitution assays (e.g. Zick and Wickner, 2014; reviewed in Rizo, 2018).

Multiple factors stabilize trans-SNARE complexes against disassembly by NSF-αSNAP

The kinetic assays of Figure 3 show how different factors influence trans-SNARE complex assembly in the presence of NSF-αSNAP and provide some information on which of these factors protect trans-SNARE complexes against disassembly. However, since only a fraction of trans-SNARE complexes formed by SNAREs alone are disassembled by NSF-αSNAP (Figure 2B), it is plausible that the absence of disassembly during our kinetic assays arises from formation of NSF-αSNAP-resistant trans-SNARE complexes, rather than because the various proteins actively protect against disassembly. To gain further insights into whether Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 can prevent disassembly of trans-SNARE complexes by NSF-αSNAP, we performed similar kinetic assays where trans-SNARE complex assembly between V- and T-liposomes was monitored by FRET in the presence of various combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, synaptotagmin-1 C2AB and complexin-1, but adding NSF-αSNAP at the end of the reaction, rather than the beginning. For experiments with complexin-1 and synaptotagmin-1 C2AB, we included the Syb49-93 peptide to facilitate trans-SNARE complex assembly, but the peptide was not included for experiments with Munc18-1 and Munc13-1 C1C2BMUNC2C because these proteins presumably can overcome at least in part the inhibition arising from formation of 2:1 syntaxin-1-SNAP-25 heterodimers (Ma et al., 2011; Ma et al., 2013). We note that we did not attempt to monitor the kinetics of disassembly upon addition of NSF-αSNAP, because disassembly generally occurred rapidly while reagents were added to multiple parallel reactions (see Materials and methods).

The rate of trans-SNARE complex assembly was similar in assays started in the absence of Ca2+ with or without synaptotagmin-1 C2AB, and the recovery of donor fluorescence upon addition of NSF-αSNAP was also comparable (Figure 4A, green and black curves), showing that C2AB does not alter assembly or disassembly in the absence of Ca2+. However, assembly was dramatically accelerated by C2AB in the presence of Ca2+ (Figure 4A, blue curve), likely because C2AB can bridge two membranes together (Araç et al., 2006), and addition of NSF-αSNAP led to only a small amount of donor fluorescence recovery, suggesting that Ca2+-bound C2AB markedly protected trans-SNARE complexes against disassembly (but see below). Complexin-1 accelerated trans-SNARE complex assembly (Figure 4A, red curve), consistent with previous results (Diao et al., 2013), and appeared to partially prevent disassembly by NSF-αSNAP. The contrast of these results with those of Figure 3B most likely arises because syntaxin-1-SNAP-25m heterodimers constitute the starting point for trans-SNARE complex assembly facilitated by complexin-1 and synaptotagmin-1 C2AB, but this pathway is blocked in the presence of NSF-αSNAP because they disassemble the heterodimers.

Figure 4 with 1 supplement see all
Influence of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 on trans-SNARE complex assembly in the absence of NSF-αSNAP and on protection against disassembly upon addition of NSF-αSNAP.

(A) Kinetic assays monitoring trans-SNARE complex assembly upon mixing V- and T-liposomes (1:4 ratio) in the presence of Syb49-93 and disassembly upon addition of NSF-αSNAP (indicated by the arrows), from the changes in the donor fluorescence emission intensity. The experiments included Syb49-93 alone (black trace) or together with complexin-1 (Cpx) (red trace), synaptotagmin-1 C2AB (green trace) and synaptotagmin-1 plus Ca2+ (blue trace). (B) Kinetic assays analogous to those of (A) but performed in the absence of Syb49-93 and the presence of Munc18-1 (M18), Munc13-1 C1C2BMUNC2C (M13) or both (blue, green and red traces, respectively). Experiments were started in 100 μM EGTA and Ca2+ (600 μM) was added after 700 s. (C) Analogous kinetic assays monitoring trans-SNARE complex assembly between VSyt1- and T-liposomes (1:4 ratio) in the presence of Syb49-93 alone (black trace) or together with Ca2+ (blue trace) or complexin-1 (Cpx) (red trace), and addition of NSF-αSNAP at the end (black arrow). In the experiments shown in (A–C), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces shown in (A–C), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents.

https://doi.org/10.7554/eLife.38880.011

Munc13-1 C1C2BMUNC2C alone or Munc18-1 alone were unable to promote trans-SNARE complex assembly, but together they did mediate trans-SNARE complex assembly that was slow in the absence of Ca2+ and was strongly accelerated upon Ca2+ addition (Figure 4B). Addition of NSF-αSNAP consistently led to a slight further decrease in donor fluorescence intensity (Figure 4B, red curve), supporting the notion that Munc18-1 and Munc13-1 C1C2BMUNC2C protect against trans-SNARE complex disassembly by NSF-αSNAP, and suggesting that they in fact cooperate with NSF-αSNAP in formation of trans-SNARE complexes. This notion is further supported by the observation that trans-SNARE complex assembly was more efficient in the experiments performed with Munc18-1 and Munc13-1 C1C2BMUNC2C when NSF-αSNAP were present from the beginning (Figure 3A, red curve). These results most likely arise because, in the former experiments, Munc18-1 must displace the SNAP-25m bound to syntaxin-1 in the T-liposomes to initiate the Munc18-1-closed syntaxin-1 pathway. Such displacement is slow and is accelerated when NSF-αSNAP are added from the beginning because they disassemble the syntaxin-1-SNAP-25m heterodimers, facilitating binding of Munc18-1 to closed syntaxin-1 and initiating the NSF-αSNAP-resistant pathway of trans-SNARE complex assembly.

We also performed assays where we monitored formation of trans-SNARE complexes between VSyt1- and T-liposomes, including Syb49-93 to facilitate assembly and adding NSF-αSNAP at the end to test for disassembly. We observed similar rates of assembly and similar amounts of disassembly in the absence and presence of Ca2+ (Figure 4C), which indicates that synaptotagmin-1 by itself does not protect against disassembly and contrasts with the results obtained with V- and T-liposomes in the presence of synaptotagmin-1 C2AB (Figure 4A; see discussion). Including complexin-1 decreased the assembly rate but enhanced the overall efficiency of assembly and strongly hindered disassembly of trans-SNARE complexes by NSF-αSNAP (Figure 4C, red curve), in correlation with the results obtained in kinetic experiments performed with NSF-αSNAP from the beginning (Figure 3E,F, Figure 3—figure supplement 1). We did not pursue these kinetic experiments further because, although they suggested that Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 have differential abilities to protect trans-SNARE complexes against disassembly NSF-αSNAP, it is difficult to quantify these abilities from these assays because of the different extent of trans-SNARE complex assembly under the various conditions, because of a small amount of photobleaching occurring during the experiments, and because it is unclear to what extent trans-SNARE complexes that are intrinsically resistant to NSF-αSNAP were formed under the various conditions.

To overcome these problems and have a common benchmark that can give a quantitative idea of the protecting activity of the various proteins, we again followed the approach of pre-forming trans-SNARE complexes by incubation of V- and T-liposomes (1:4 ratio) in the presence of Syb49-93 for five hours, after which there are no further changes in the fluorescence spectrum [note that Syb49-93 is released from the syntaxin-1-SNAP-25 complexes upon trans-SNARE complex assembly (Yavuz et al., 2018) and hence should not interfere in the measurement of protection against disassembly]. Different aliquots of the same reaction mixture where then incubated with synaptotagmin-1 C2AB, complexin-1, Munc18-1 and Munc13-1 C1C2BMUNC2C in different combinations, with or without Ca2+ whenever C2AB and/or C1C2BMUNC2C were present. Fluorescence emission spectra of the resulting samples were acquired before and after addition of NSF-αSNAP to quantify the changes in FRET caused by NSF-αSNAP (Figure 5A and Figure 5—figure supplement 1). We also acquired control fluorescence spectra of separate samples where we preformed trans-SNARE complexes between V-liposomes that contained the donor probe and T-liposomes lacking the acceptor probe (referred to as V*+T), as well as analogous trans-SNARE complexes that contained the acceptor probe but not the donor probe (V + T*); both sets of liposomes were also incubated with various proteins and fluorescence spectra were acquired before and after addition of NSF-αSNAP (Figure 5—figure supplements 2 and 3). The control spectra showed that none of the proteins substantially affect the donor fluorescence, except for a slight but consistent increase caused by Munc13-1 C1C2BMUNC2C, while NSF-αSNAP did cause a considerable decrease of the acceptor fluorescence in the V + T* controls that was prevented by Munc18-1. Hence, we focused on the donor fluorescence to quantitate the protection against disassembly.

Figure 5 with 5 supplements see all
Quantitative analysis of how Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP.

(A) Fluorescence emission spectra of mixtures of V-liposomes containing Alexa488-synaptobrevin and T-liposomes containing TMR-syntaxin-1-SNAP-25m (1:4 V- to T-liposome ratio) that were incubated for five hours with Syb49-93; Munc18-1 (M18), Munc13-1 C1C2BMUNC2C (M13), complexin-1 (Cpx), synaptotagmin-1 C2AB (C2AB) and Ca2+ were then added and, after an additional incubation for five minutes, spectra were acquired before (black trace) or after (red trace) addition of NSF-αSNAP. (B) Bar diagram illustrating the ability of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1, synaptotagmin-1 C2AB and Ca2+ to protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP. As in (A), V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1, synaptotagmin-1 C2AB and Ca2+. Fluorescence emission spectra were acquired before and after addition of NSF-αSNAP and the ratio r between the donor fluorescence intensities at 518 nm measured after and before NSF-αSNAP addition was calculated. Representative examples of the spectra acquired under different conditions are shown in Figure 5—figure supplement 1. (C) Bar diagram illustrating the ability of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and Ca2+ to protect pre-formed trans-SNARE complexes between VSyt1- and T-liposomes against disassembly by NSF-αSNAP. Similar to (B), VSyt1- and T-liposomes were incubated with Syb49-93 (but for 24 hr at 4°C) to preform trans-SNARE complexes, and then they were incubated for five minutes with different combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and Ca2+. Fluorescence emission spectra were acquired before and after addition of NSF-αSNAP and the ratio r between the donor fluorescence intensities at 518 nm measured after and before NSF-αSNAP addition was calculated. Representative examples of the spectra acquired under different conditions are shown in Figure 5—figure supplement 4. In (B,C), ‘No additions’ indicates experiments where none of these factors were included before addition of NSF-αSNAP. Control experiments with no additions and replacing ATP with ATPγS or replacing Mg2+ with EDTA were also performed. All experiments were performed in triplicate. Values indicate means ±standard deviations. A few examples of statistical significance are indicated to illustrate which differences among the r values obtained under different conditions are meaningful. Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Holm-Sidak test (*p<0.05; ***p<0.001).

https://doi.org/10.7554/eLife.38880.013

The fluorescence spectra obtained after incubation of the preformed trans-SNARE complexes with different combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, synaptotagmin-1 C2AB and complexin-1 before addition of NSF-αSNAP were very similar for all samples, indicating that the amount of trans-SNARE complexes was not affected by the incubations. However, substantial differences were observed in the donor emission intensities in the spectra obtained after addition of NSF-αSNAP (Figure 5—figure supplement 1), indicating different extents of SNARE complex disassembly. To derive a quantitative idea of how much the different combinations of proteins protect against disassembly, we calculated the ratio r between the donor fluorescence intensity after adding NSF-αSNAP and that before addition of NSF-αSNAP. This ratio was 1.30 for control experiments with no additions before disassembly with NSF-αSNAP (Figure 5B). This value was variable in experiments performed with different liposome preparations and depended on the extent of trans-SNARE complex assembly achieved, but the relative changes in r values obtained in the presence of different factors were comparable for the different preparations.

The r values measured showed that Ca2+-free synaptotagmin-1 C2AB provided no protection but Ca2+-bound C2AB prevented disassembly considerably. Complexin-1 afforded similar protection as Ca2+-bound C2AB. In experiments with Munc13-1 C1C2BMUNC2C alone in the absence or presence of Ca2+, r was slightly larger than that observed in the control with no additions, which can be attributed to the slight increase in donor fluorescence caused by Munc13-1 C1C2BMUNC2C on the V*+T control (Figure 5—figure supplement 2C) and shows that there is no protection against disassembly under these conditions. The r value observed with Munc18-1 alone was slightly smaller than 1.3; although the difference with respect to the controls with no additions was not statistically significant, there was a significant difference between the (smaller) r value observed in experiments with Munc18-1 and Munc13-1 C1C2BMUNC2C in the absence of Ca2+ and the control with no additions. A dramatic decrease in r was observed when Ca2+ was included with Munc18-1 and Munc13-1 C1C2BMUNC2C. Adding complexin-1 or of C2AB together with Munc18-1 and Munc13-1 C1C2BMUNC2C, with or without Ca2+, also decreased the corresponding r values, and the smallest r was observed in experiments that included all these components (Figure 5B), showing almost complete protection under these conditions (Figure 5A). Overall, these results support the notion that Munc18-1 and Munc13-1 C1C2BMUNC2C can protect trans-SNARE complexes against disassembly to a moderate extent in the absence of Ca2+ and that such protection is increased by Ca2+, in correlation with the results of the kinetic assays (Figure 3). In addition, these data also indicate that complexin-1 also protects against disassembly and that Ca2+-free synaptotagmin-1 C2AB alone does not prevent disassembly but can help to protect against disassembly.

To investigate how protection of trans-SNARE complexes is influenced by membrane-anchored synaptotagmin-1, we performed additional experiments where we preformed trans-SNARE complexes between VSyt1- and T-liposomes in the presence of Syb49-93, and we incubated the resulting samples with different combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and Ca2+ before adding NSF-αSNAP to test for disassembly. Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and Ca2+ did not significantly alter the fluorescence spectra acquired before addition of NSF-αSNAP, but markedly affected the spectra obtained after such addition (black and red curves, respectively, in the different panels of Figure 5—figure supplement 4). The ratio r between the donor fluorescence emission intensities observed after and before addition of NSF-αSNAP without other proteins was 1.46 and, surprisingly, addition of Ca2+ did not lead to protection against disassembly (Figure 5C), which contrasts with the protection provided by Ca2+-bound synaptotagmin-1 C2AB in experiments with V-liposomes (Figure 5B) and suggests that the latter result might arise from excessive accumulation of C2AB molecules at the membrane-membrane interface (Araç et al., 2006). Munc18-1 alone again appeared to have a tendency to prevent disassembly, compared to the control with no additions, but the difference was not statistically significant, and Munc13-1 C1C2BMUNC2C alone provided no protection. Together, Munc18-1 and Munc13-1 C1C2BMUNC2C did provide moderate protection in the absence of Ca2+ and strong protection in its presence. Interestingly, complexin-1 alone afforded robust protection against disassembly (Figure 5C) that appeared to be stronger than that observed with V- and T-liposomes (Figure 5B), suggesting that membrane-anchored synaptotagmin-1 can cooperate with complexin-1 in protecting trans-SNARE complexes against disassembly. Maximal protection of the trans-SNARE complexes between VSyt1- and T-liposomes against disassembly by NSF-αSNAP was again observed when all components (Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and Ca2+) were included (Figure 5C).

The levels of protection afforded by different proteins in these experiments need to be examined with caution, as they are expected to depend on their concentrations. This is exemplified by the lower protection provided by complexin-1 as we decreased its concentration from 2 μM (used in our standard assays, Figure 5B,C) to 0.2 μM (Figure 5—figure supplement 5). Note also that we kept the concentration of Munc13-1 C1C2BMUNC2C at 0.3 μM because higher concentrations sometimes led to sample precipitation, but in vivo the local concentrations of Munc13-1 at the active zone may be highly increased due to binding to RIMs (see discussion). Nevertheless, the overall results presented in Figures 35 provide strong evidence that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to protect trans-SNARE complexes against disassembly by NSF-αSNAP.

Disassembly of cis-SNARE complexes

To further investigate the functional interplay between NSF, αSNAP, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 in the SNARE complex assembly-disassembly cycle, we performed kinetic assays where we analyzed the assembly and disassembly of cis-SNARE complexes mixing V-liposomes containing Alexa488-synaptobrevin with SNAP-25m and a soluble fragment spanning the cytoplasmic region of syntaxin-1 (residues 2–253) labeled with TMR at residue 186. Cis-SNARE complex assembly was efficient in the presence of Syb49-93 but was abolished if NSF-αSNAP were included from the beginning (Figure 6A, dark and light gray curves, respectively). Munc18-1 plus Munc13-1 C1C2BMUNC2C or complexin-1 plus synaptotagmin-1 C2AB, or the four proteins together, were unable to support cis-SNARE complex formation in the presence of NSF-αSNAP even upon addition of Ca2+ (Figure 6A). These results are in stark contrast to the efficient formation of trans-SNARE complexes observed in the presence of NSF-αSNAP when Munc18-1 and Munc13-1 C1C2BMUNC2C were included (Figure 3A).

Figure 6 with 1 supplement see all
Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 C2AB do not decrease the overall amount of cis-SNARE complex disassembly caused by NSF-αSNAP.

(A) Kinetic assays monitoring changes in the donor fluorescence emission intensity due to cis-SNARE complex formation upon mixing V-liposomes containing Alexa488-synaptobrevin with an excess of TMR-labeled syntaxin-1 (2–253) and SNAP-25m in the presence of NSF-αSNAP with no additions (ctrl) (light gray trace) or with different combinations of Munc18-1 (M18), Munc13-1 C1C2BMUNC2C (M13), complexin-1 (Cpx) and synaptotagmin-1 C2AB as indicated. Ca2+ was added at 550 s. For comparison purposes, the dark gray trace shows a cis-SNARE complex assembly reaction performed in the presence of Syb49-93 and absence of NSF-αSNAP. (B) Kinetic assays of cis-SNARE complex assembly analogous to those of (A), but performed in the absence of NSF-αSNAP and the presence of Syb49-93 alone (black trace) or together with complexin-1 (Cpx) (red trace) or synaptotagmin-1 C2AB plus Ca2+ (blue trace). NSF-αSNAP were added when the reactions reached a plateau (black arrow) to monitor cis-SNARE complex disassembly. (C) Kinetic assays analogous to those in (B), but in the presence of Munc18-1 (M18) and Munc13-1 C1C2BMUNC2C (M13) without (red trace) or with (dark gray trace) Syb49-93. Ca2+ was added after 950 s. (D) Kinetic assays where cis-SNARE complex formation was initially catalyzed by Syb49-93 and, after reaching a plateau, Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1, synaptotagmin-1 C2AB and Ca2+ were added (red arrow); after five minutes, NSF-αSNAP were added to test for disassembly (red trace). The black trace shows a control experiment where the four proteins were not included before adding NSF-αSNAP. In the experiments shown in (C–D), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of (A–D), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents.

https://doi.org/10.7554/eLife.38880.019

In experiments performed initially without NSF-αSNAP, cis-SNARE complex assembly was strongly stimulated by complexin-1 or by Ca2+-bound synaptotagmin-1 C2AB, but most of the donor fluorescence was recovered upon addition of NSF-αSNAP at the end of the reaction due to disassembly of the cis-SNARE complexes (Figure 6B). Munc18-1 and the Munc13-1 C1C2BMUNC2C fragment were unable to support cis-SNARE complex assembly even after addition of Ca2+ (Figure 6C, red curve), and they partially inhibited cis-SNARE complex assembly catalyzed by Syb49-93, without protecting against disassembly upon addition of NSF-αSNAP (Figure 6C, gray curve). We also performed additional experiments where we preformed cis-SNARE complexes in the presence of Syb49-93 and tested whether incubation of these complexes with Munc18-1, Munc13-1, synaptotagmin-1 C2AB, complexin-1 and Ca2+ for five minutes protected against disassembly by NSF-αSNAP, but disassembly was as efficient as a control experiment where the four proteins were not added (Figure 6D). We note that these results are not necessarily inconsistent with the finding that complexin-1 slows down the kinetics of cis-SNARE complex disassembly by NSF-αSNAP (Choi et al., 2018; Winter et al., 2009), as we did not attempt to measure the kinetics of disassembly in our experiments (see Materials and methods). Nevertheless, to test for potential effects on the overall extent of disassembly arising from different relative concentrations of complexin-1 versus αSNAP, or perhaps from the mutation in SNAP-25m, we performed additional experiments where we incubated pre-formed cis-SNARE complexes with different concentrations of complexin-1 or we replaced SNAP-25m with WT SNAP-25. We observed comparable, nearly complete levels of disassembly in all of these experiments (Figure 6—figure supplement 1). Overall, the contrast of the results obtained with cis-SNARE complexes with those observed with trans-SNARE complexes provides a dramatic demonstration of how the apposition of two membranes tilts the balance in favor of SNARE complex assembly, whereas disassembly dominates on a single membrane.

Discussion

Extensive research has yielded a wealth of information on the mechanism of neurotransmitter release, including the notions that assembly of the trans-SNARE complex four-helix bundle between the synaptic vesicle and plasma membranes is crucial for membrane fusion, that NSF-αSNAP disassemble cis-SNARE complexes after fusion to recycle the SNAREs, and that priming of synaptic vesicles to a readily releasable state involves formation of partially assembled trans-SNARE complexes, which is organized by Munc18-1 and Munc13-1 in an NSF-αSNAP-resistant manner. However, the nature of the primed state of synaptic vesicles remained enigmatic and recent reports indicating that NSF-SNAPs also disassemble trans-SNARE complexes (Yavuz et al., 2018) and can de-prime synaptic vesicles (He et al., 2017) raised the question of how trans-SNARE complexes are protected to prevent vesicle de-priming. More generally, it was unclear how the functions of Munc18-1 and Munc13-1, as well as those of other proteins that have been implicated in vesicle priming such as synaptotagmin-1 and complexin-1, are related to roles in promoting trans-SNARE complex assembly and/or in preventing their disassembly by NSF-αSNAP. The results presented here now show that Munc18-1 and Munc13-1 are crucial to form trans-SNARE complexes in the presence of NSF-αSNAP, as expected, and help to maintain trans-SNARE complexes assembled. Complexin-1 does not appear to play a role in NSF-αSNAP-resistant trans-SNARE complex assembly, but protects against disassembly, while synaptotagmin-1 may play a role in both assembly and protection. These results raise the possibility that Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 form macromolecular assemblies with trans-SNARE complexes that constitute the core of the primed state of synaptic vesicles.

Our FRET data showing that trans-SNARE complexes can be disassembled by NSF-αSNAP agree with recent results obtained by Yavuz et al. (2018) using a similar approach, and with earlier studies of yeast vacuolar fusion showing that Sec18-Sec17 disassemble trans-SNARE complexes (Xu et al., 2010). However, our FRET assays and those of Yavuz et al. (2018) also show that a substantial fraction of trans-SNARE complexes is resistant to disassembly by NSF-αSNAP, which might explain the finding that NSF-αSNAP inhibited lipid mixing between synaptobrevin- and syntaxin-1-SNAP-25 liposomes if added from the beginning but not if added after the liposomes were pre-incubated at low temperature (Weber et al., 2000). NSF-αSNAP resistant, tightly docked liposomes were attributed to the formation of large, flat interfaces between the liposomes (Yavuz et al., 2018). Our cryo-EM images also revealed extended interfaces between liposomes but the interfaces were generally smaller (Figure 2—figure supplement 3), perhaps because we used a much lower synaptobrevin-to-lipid ratio. It is unclear whether such extended interfaces are physiologically relevant, as inclusion of other key components of the release machinery favors the formation of point contacts between liposomes over extended interfaces (Gipson et al., 2017). These observations emphasize the difficulty of reconstituting with a few components the steps that lead to synaptic vesicle fusion, particularly the formation of the primed state, because of the metastable, transient nature of this state and because off-pathway, kinetically trapped states can be formed in the absence of some components that are important for vesicle priming (e.g. RIM and CAPS in our assays, see (Rizo and Südhof, 2012). We speculate that the population of trans-SNARE complexes that can be disassembled by NSF-αSNAP in our assays is more closely related to the partially assembled trans-SNARE complexes present in primed synaptic vesicles. This proposal is supported by electrophysiological studies showing that readily-releasable vesicles can be de-primed and that de-priming is prevented by N-ethylmaleimide, an agent that inactivates NSF (He et al., 2017). Although N-ethylmaleimide could potentially alter other proteins in vivo, the correlation with the finding that trans-SNARE complexes can be disassembled by NSF-αSNAP in vitro strongly supports the notion that de-priming is mediated by NSF. Since NSF-SNAPs can also disassemble syntaxin-1-SNAP-25 heterodimers (Hayashi et al., 1995), there is little doubt that the cytoplasm provides an environment that favors SNARE complex disassembly in general, and hence that trans-SNARE complexes need to be protected to maintain vesicles primed.

The decreases in the RRP of primed vesicles observed in mice lacking Munc18-1, Munc13-1, complexins or synaptotagmin-1/7 (Bacaj et al., 2015; Chang et al., 2018; Rosenmund et al., 2002; Verhage et al., 2000; Xue et al., 2010; Yang et al., 2010) could arise because they mediate vesicle priming and/or because they protect against de-priming. With the underlying hypothesis that trans-SNARE complex assembly in our in vitro assays recapitulates at least to some extent the process of vesicle priming, we used different types of assays to dissect the contributions of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 to assembling trans-SNARE complexes in the presence of NSF-αSNAP and to protecting these complexes against disassembly once they are formed. Our assays that included NSF-αSNAP from the beginning clearly show that Munc18-1 and Munc13-1 C1C2BMUNC2C are essential to assemble trans-SNARE complexes in the presence of NSF-αSNAP (Figure 3A,B,E), as expected from the results of our previous liposome fusion assays (Liu et al., 2016; Ma et al., 2013). The progressive formation of trans-SNARE complexes observed in these assays suggests that Munc18-1 and Munc13-1 C1C2BMUNC2C prevent their disassembly, in addition to mediating assembly, but we could not rule out that the assembled trans-SNARE complexes are NSF-αSNAP resistant and Munc18-1 and/or Munc13-1 C1C2BMUNC2C become dispensable after assembly, particularly in the absence of Ca2+. The experiments where we added EGTA after allowing efficient Ca2+-dependent assembly show that at least a population of the trans-SNARE complexes formed could be disassembled by NSF-αSNAP, but a substantial amount of complexes remained assembled even after addition of EGTA (Figure 3C,F). These data suggest that Munc18-1 and Munc13-1 C1C2BMUNC2C do protect trans-SNARE complexes against disassembly by NSF-αSNAP to some extent, and that Ca2+ enhances the protective activity. This conclusion was further supported by experiments where we preformed trans-SNARE complexes in the absence of NSF-αSNAP and monitored disassembled by NSF-αSNAP in the presence of Munc18-1 and Munc13-1 C1C2BMUNC2C (Figure 5B,C).

An alternative interpretation of these results is that Munc18-1 and Munc13-1 C1C2BMUNC2C do not prevent disassembly but instead mediate fast re-assembly of trans-SNARE complexes after they are disassembled by NSF-αSNAP. Although we cannot completely rule out this possibility, multiple arguments support the notion that that Munc18-1 and Munc13-1 C1C2BMUNC2C directly or indirectly hinder the disassembly reaction. First, this mechanism makes more sense from an energetic point of view, as it does not involve futile cycles of disassembly and re-assembly. Second, in kinetic assays where trans-SNARE complexes were assembled in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C and Ca2+, and NSF-αSNAP were added at the end (Figure 4B), we observed similar results if NSF-αSNAP were added together with an excess of the synaptobrevin SNARE motif (Figure 4—figure supplement 1). If continued disassembly and re-assembly of SNARE complexes occurred under these conditions, the gradual incorporation of the soluble synaptobrevin fragment into SNARE complexes would be expected to decrease the observed FRET, but no such decrease was observed. Third, the finding that both Munc18-1 and Munc18-2 can mediate priming but only Munc18-1 prevents de-priming by NSF in neurons (He et al., 2017) suggests that both isoforms can mediate trans-SNARE complex assembly but only Munc18-1 prevents disassembly. Fourth, αSNAP was reported to strongly inhibit liposome lipid mixing by binding to trans-SNARE complexes (Park et al., 2014). Hence, fusion might be arrested after Munc18-1 and Munc13-1 C1C2BMUNC2C organize trans-SNARE complex assembly unless they block αSNAP binding. And fifth, Munc18-1 and Munc13-1 exhibit weak interactions with SNARE complexes in solution that are strengthened by membranes (Dulubova et al., 2007; Guan et al., 2008; Ma et al., 2011; Shen et al., 2007; Weninger et al., 2008), and could compete with binding of αSNAP to the SNARE four-helix bundle. Indeed, αSNAP covers much of the surface of the SNARE four-helix bundle in the cryo-EM structure of the 20S complex formed by NSF, αSNAP and the SNAREs (Zhao et al., 2015) (Figure 7A), and therefore almost any protein that interacts with the SNARE four-helix bundle might compete with αSNAP for binding. Note also that Munc13-1 C1C2BMUNC2C was recently proposed to bridge the vesicle and plasma membranes (Liu et al., 2016), which might provide an additional mechanism to protect trans-SNARE complexes against disassembly by NSF-αSNAP by imposing steric constraints that hinder formation of the 20S complex. Moreover, Ca2+-binding to the Munc13-1 C2B domain is expected to change the orientation of Munc13-1 C1C2BMUNC2C with respect to the plasma membrane, bringing the two membranes into closer proximity (Xu et al., 2017) and potentially increasing the steric constraints that impair 20S complex assembly. This model can explain why Ca2+ increases the ability of Munc13-1 C1C2BMUNC2C (together with Munc18-1) to protect trans-SNARE complexes against disassembly by NSF-αSNAP (Figure 5B,C).

Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly.

(A,B) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) (Zhao et al., 2015) assembled on a cis-SNARE complex on one membrane (A) or on a trans-SNARE complex between two membranes (B). Synaptobrevin is in red, syntaxin-1 in yellow, SNAP-25 in green, NSF in gray and the four molecules of αSNAP in cyan, orange, blue and pink. The positions of the αSNAP N-terminal hydrophobic loops (N-loops) are indicated. The orientation of the 20S complex in (A) was chosen to favor simultaneous interactions of the N-loops of the four αSNAP molecules with the membrane. In (B), the orientation of the 20S complex is arbitrary and is meant to illustrate the difficulty of simultaneous interactions of the N-loops from the four αSNAP molecules with membranes in the trans configuration. Note that, at the same time, the apposition of both membranes may enhance the affinity of Munc18-1, Munc13-1, synaptotagmin-1 and complexin-1 for SNARE complexes in the trans configuration due to simultaneous interactions with the membranes that are not possible or less favorable in the cis configuration, while the SNARE four-helix bundle is likely to be only partially assembled, which may weaken binding to αSNAP.

https://doi.org/10.7554/eLife.38880.021

Overall, the crucial nature of Munc18-1 and Munc13-1 C1C2BMUNC2C for trans-SNARE complex assembly provides a clear explanation for the complete abrogation of synaptic vesicle priming observed in mice lacking Munc18-1 or Munc13-1/2 (Varoqueaux et al., 2002; Verhage et al., 2000), while the finding that Munc18-1 and Munc13-1 C1C2BMUNC2C maintain trans-SNARE complexes assembled in the presence of NSF-αSNAP can explain the key importance of Munc18-1 and Munc13-1 to prevent de-priming of the RRP (He et al., 2017). These correlations support the physiological relevance of our in vitro results. It is also worth noting that the strong Ca2+-dependent stimulation of the ability of Munc18-1 and Munc13-1 C2B domain to mediate NSF-αSNAP-resistant trans-SNARE complex assembly (Figure 3A,E) and to maintain trans-SNARE complexes assembled (Figure 5B,C) likely underlies at least in part the importance of Ca2+ binding to the Munc13 C2B domain for facilitating release during repetitive stimulation, when there is a strong demand to rapidly refill the RRP to prevent its depletion (Shin et al., 2010).

Synaptotagmin-1 and complexin-1 are not essential to form trans-SNARE complexes in the presence of NSF-αSNAP, but they do enhance the amount of trans-SNARE complexes formed (Figure 3). These findings correlate with the observation that deletion of synaptotagmin-1 or complexins leads to decreases in the RRP of vesicles (Bacaj et al., 2015; Chang et al., 2018; Xue et al., 2010; Yang et al., 2010), but not as dramatic as those observed in Munc18-1 KO and Munc13-1/2 DKO neurons. Complexin-1 appeared to inhibit in the initial states of Ca2+-independent trans-SNARE complex assembly between VSyt1- and T-liposomes in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C and NSF-αSNAP, but increased the overall efficiency of assembly at longer time scales (Figure 3E, Figure 3—figure supplement 1). Moreover, complexin-1 clearly protected against disassembly by NSF-αSNAP in analogous experiments where EGTA was added after efficient Ca2+-dependent assembly (Figure 3D,F), and in experiments where NSF-αSNAP were added after trans-SNARE complexes were pre-formed (Figure 5B,C). These results suggest that complexin-1 does not assist in synaptic vesicle priming but protects the RRP against de-priming. Conversely, membrane-anchored synaptotagmin-1 accelerated Ca2+-independent trans-SNARE complex assembly (Figure 3A,E), suggesting a role in priming, but did not protect by itself against disassembly of pre-formed trans-SNARE complexes (Figure 5C). Moreover, Ca2+-free synaptotagmin-1 C2AB did not protect pre-formed trans-SNARE complexes against disassembly and the protection provided by Ca2+-bound C2AB may not be meaningful (see above) (Figure 5B). However, C2AB appeared to enhance the protection against disassembly provided by complexin-1 and by Munc18-1 plus Munc13-1 in the absence of Ca2+ (Figure 5B), and membrane-anchored synaptotagmin-1 also seemed to enhance the protection of pre-formed trans-SNARE complexes afforded by complexin-1 (Figure 5C), as well as the levels of trans-SNARE complexes formed in the presence of NSF-αSNAP, Munc18-1 and Munc13-1 C1C2BMUNC2C (Figure 3A,C,E,F). These results suggest that synaptotagmin-1 cooperates with Munc18-1-Munc13-1 in priming and may also help in preventing de-priming.

Mechanistically, it is not surprising that complexin-1 can hinder formation of the 20S complex, preventing disassembly of trans-SNARE complexes, as it binds to SNARE complexes with nanomolar affinity (Pabst et al., 2002) through a binding mode (Chen et al., 2002) that is incompatible with formation of the 20S complex. The various binding modes that have been observed between synaptotagmin-1 and the SNARE complex (Brewer et al., 2015; Zhou et al., 2015; Zhou et al., 2017) are also incompatible with αSNAP binding. Although the affinities of these interactions are in the micromolar range, binding could be enhanced by the localization of synaptotagmin-1 on the vesicle membrane and by cooperativity with complexin-1 binding (Zhou et al., 2017) as well as with interactions of synaptotagmin-1 with one or two membranes (Bai et al., 2004; Brewer et al., 2015). It is also plausible that trans-SNARE complexes bound to complexin-1 and synaptotagmin-1 form macromolecular assemblies with Munc18-1 and Munc13-1, and that all underlying interactions cooperate with each other. In such an assembly, Munc18-1 and/or Munc13-1 may not interact directly with the SNAREs, but the cooperativity of the interactions and the resulting geometry might block access of αSNAP and NSF to the SNARE four-helix bundle and thus prevent its disassembly.

It is interesting to note the dramatic effects that the membrane topology has on SNARE complex assembly and on protection against disassembly: Munc18-1 and Munc13-1 C1C2BMUNC2C mediate efficient formation of trans-SNARE complexes but not of cis-SNARE complexes in the presence of NSF-αSNAP (Figures 3A and 6A), and pre-formed trans-SNARE complexes remain assembled in the presence of NSF-αSNAP when Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 are included (Figure 5B,C), unlike cis-SNARE complexes (Figure 6D). These differences must arise from distinct balances among the interactions of these proteins with the SNAREs and the membranes. In the cis configuration, up to four αSNAP molecules interact with much of the surface of the SNARE four-helix bundle (Zhao et al., 2015) and at the same time a hydrophobic N-terminal loop from all αSNAP molecules, which is known to strongly stimulate disassembly of membrane-anchored cis-SNARE complexes (Winter et al., 2009), can bind simultaneously to the membrane, likely with high cooperativity (Figure 7A). Interactions of αSNAP with the SNAREs and the membranes are also important for disassembly of trans-SNARE complexes by NSF-αSNAP (Figure 2D,E), but the geometry of the system (Figure 7B) is expected to hinder simultaneous binding of all αSNAP molecules to membranes, and incomplete assembly of the SNARE four-helix bundle may also limit the extent of αSNAP-SNARE interactions (perhaps enhancing other interactions). Hence, NSF-αSNAP are expected to be less active in disassembling trans- than cis-SNARE complexes. Conversely, the trans-configuration favors simultaneous binding of Munc13-1 C1C2BMUNC2C to the apposed membranes, which is likely key for its activity in promoting trans-SNARE complex assembly (Liu et al., 2016) and is impossible in the cis-configuration. The protection of trans- but not cis-SNARE complexes by complexin-1 under the conditions of our experiments may arise simply because complexin-1 binds tighter to the former than NSF-αSNAP, while the opposite is true for the latter. This model also explains that complexin-1 does hinder the speed of disassembly of cis-SNARE complexes (Choi et al., 2018; Winter et al., 2009), which we did not analyze in our experiments.

As mentioned above, the levels of protection of trans-SNARE complex against disassembly by NSF-αSNAP that we observed (Figure 5B,C) are also expected to depend on the experimental conditions, including protein concentrations (e.g. Figure 5—figure supplement 5), and hence need be examined with caution. Moreover, other proteins that were not included in this study may also influence the protection of trans-SNARE complexes directly and/or by enhancing the local concentrations of protecting factors. For instance, RIMs are intrinsic components of pre-synaptic active zones that bind to Munc13-1 (Betz et al., 2001; Dulubova et al., 2005), an interaction that is important for optimal vesicle priming (Camacho et al., 2017) and can dramatically increase the local concentrations of Munc13-1 at release sites. Thus, more systematic studies of how the components of the release machinery protect trans-SNARE complexes against disassembly in vitro and against de-priming of the RRP in neurons will be required to better understand the nature of the primed state of synaptic vesicles. Based on the available data, we propose that the core of this primed state is formed by a macromolecular assembly that includes trans-SNARE complexes, Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1.

Materials and methods

Recombinant proteins

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The following constructs were used for protein expression in E. coli BL21 (DE3) cells: Full-length rat syntaxin-1A, rat syntaxin 2–253, full-length rat SNAP-25A (C84S, C85S, C90S, C92S), full-length rat synaptobrevin, rat synaptobrevin 49–93, rat synaptotagmin-1 57–421 (C74S, C75A, C77S, C79I, C82L, C277S) (a kind gift from Thomas Sollner), rat synaptotagmin-1 C2AB (131–421 C277A), full-length rat complexin, full-length Chinese hamster NSF (a kind gift from Minglei Zhao), full-length Bos Taurus αSNAP, full length rat Munc18-1, and a rat Munc13-1 fragment spanning the C1C2BMUNC2C regions (529–1725 Δ1408–1452). Expression and purification of the corresponding proteins were performed as previously reported (Chen et al., 2006; Chen et al., 2002; Dulubova et al., 1999; Liang et al., 2013; Liu et al., 2017; Ma et al., 2011; Ma et al., 2013; Xu et al., 2013; Zhao et al., 2015) with the modifications described below. His6-full-length syntaxin-1A was induced with 0.4 mM IPTG and expressed overnight at 25°C. Purification was done using Ni-NTA resin (Thermo Fisher) in 20 mM Tris pH 7.4, 500 mM NaCl, 8 mM imidazole, 2% Triton X-100, and 6M urea followed by elution in 20 mM Tris pH 7.4, 500 mM NaCl, 400 mM imidazole, and 0.1% DPC. The His6 tag was removed using thrombin cleavage, followed by size exclusion chromatography on a Superdex 200 column (GE 10/300) in 20 mM Tris pH 7.4, 125 mM NaCl, 1 mM TCEP, 0.2% DPC (Liang et al., 2013). GST-syntaxin-1A 2–253 was induced with 0.4 mM IPTG and expressed overnight at 25°C. Purification was done using glutathione sepharose resin (GE) followed by thrombin cleavage of the GST-tag and anion exchange chromatography on a HiTrap Q column (GE) in 25 mM Tris pH 7.4, 1 mM TCEP using a linear gradient from 0 mM to 1000 mM NaCl. GST-Syb49-93 was induced with 0.4 mM IPTG and expressed overnight at 23°C. Purification was done using glutathione sepharose resin (GE) followed by cleavage of the GST-tag and size exclusion chromatography on a Superdex 75 column (GE 16/60) in 20 mM Tris pH 7.4, 125 mM NaCl. His6-full-length complexin-1 was induced with 0.5 mM IPTG and expressed for 4 hr at 37°C. Purification was done using Ni-NTA resin followed by TEV cleavage of the His6-tag and size exclusion chromatography on a Superdex 75 column (GE 16/60) in 20 mM Tris pH 7.4, 125 mM NaCl, 1 mM TCEP. His6-full-length NSF was induced with 0.4 mM IPTG and expressed overnight at 20°C. Purification was done in 5 steps (Zhao et al., 2015): i) Ni-NTA affinity chromatography; ii) size exclusion chromatography of hexameric NSF on a Superdex S200 column (GE 16/60) in 50 mM Tris pH 8.0, 100 mM NaCl, 1 mM ATP, 1 mM EDTA, 1 mM DTT, and 10% glycerol; iii) TEV cleavage of the His6-tag and monomerization with apyrase during 36 hr dialysis with nucleotide-free buffer; iv) three rounds of size exclusion chromatography to separate monomeric and hexameric NSF (re-injecting the latter) on a Superdex S200 column (GE 16/60) in 50 mM NaPi pH 8.0, 100 mM NaCl, 0.5 mM TCEP; and v) reassembly of the NSF monomers and size exclusion chromatography of reassembled hexameric NSF on a Superdex S200 column (GE 16/60) in 50 mM Tris pH 8.0, 100 mM NaCl, 1 mM ATP, 1 mM EDTA, 1 mM DTT, and 10% glycerol. For experiments requiring the use of a non-hydrolyzable analog of ATP, reassembly of monomeric NSF was done in the presence of ATPγS followed by size exclusion chromatography of the hexamer in a similar buffer as before substituting ATPγS for ATP. His6-Munc13-1 C1C2BMUNC2C (529–1725 Δ1408–1452) was induced with 0.5 mM IPTG and expressed overnight at 16°C. Purification was done using Ni-NTA resin (Thermo Fisher) followed by thrombin cleavage of the His6-tag and anion exchange chromatography on a HiTrap Q column (GE) in 20 mM Tris pH 8.0, 10% glycerol, 1 mM TCEP using a linear gradient from 0 to 500 mM NaCl.

Mutant proteins

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All mutations were performed using QuickChange site-directed mutagenesis (Stratagene). These include the S186C mutation in full length syntaxin-1A (C145A, C271A, C272A) and in syntaxin-1A 2–253 (C145A), the M71D,L78D mutation in full-length SNAP-25A (C84S, C85S, C90S, C92S), the L26C mutation in full-length synaptobrevin (C103A), and the F27S,F28S and K122E,K163E mutations in αSNAP. For synaptobrevin L26C, the construct was cloned into a pet28A vector with an N-terminal His6 tag for soluble expression. All mutant proteins were purified as the wild type proteins.

Labeling proteins with Alexa Fluor 488 and tetramethylrhodamine

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Single cysteine mutants were labeled with Alexa Fluor 488 (Alexa488, for full length synaptobrevin L26C) or with tetramethylrhodamine (TMR, for full-length syntaxin-1A S186C and syntaxin-1A 2–253 S186C) using maleimide reactions (Thermo Fisher). Full length synaptobrevin L26C was first buffered exchanged to 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM TCEP, 1% octyl β-glucopyranoside (β-OG) using a PD Miditrap G25 column to provide buffer conditions that allow the reactive thiol group to be sufficiently nucleophilic so that they exclusively react with the dye. Buffered exchanged proteins at a concentration of 75 µM were incubated with a 20-fold excess of dye for 2 hr at room temperature. Unreacted dye was separated from the labeled protein through cation exchange chromatography on a HiTrap SP column (GE) in 25 mM NaAc pH 5.5, 1 mM TCEP, 1% β-OG using a linear gradient from 0 to 1000 mM NaCl. Full length Syntaxin S186C and Syntaxin 2–253 S186C were tagged with tetramethylrhodamine (Thermo Fisher) using a similar protocol. After labeling full length syntaxin-1A S186C, unreacted dye was separated from the labeled protein though anion exchange chromatography on a HiTrap Q column (GE) in 20 mM Tris pH 7.4, 1 mM TCEP, 0.1% DPC using a linear gradient from 0 to 1000 mM NaCl. After labeling syntaxin-1A 2–253 L26C, unreacted dye was separated from the labeled protein using multiple PD Miditrap G25 columns. The concentration of fluorescently tagged proteins was determined using UV-vis absorbance and a Bradford assay.

Simultaneous lipid mixing and content mixing assays

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Assays that simultaneously monitor lipid and content mixing (Figure 1B,C) were performed as described in detail in Liu et al. (2017) except for a few modifications. Briefly, V-liposomes with full length synaptobrevin contained 39% POPC, 19% DOPS, 19% POPE, 20% cholesterol, 1.5% NBD-PE, and 1.5% Marina Blue DHPE. T-liposomes with full-length syntaxin-1A and full-length SNAP25 (WT or M71D,L78D mutant) contained 38% POPC, 18% DOPS, 20% POPE, 20% cholesterol, 2% PIP2, and 2% DAG. Dried lipid mixtures were re-suspended in 25 mM HEPES pH 7.4, 150 KCl, 1 mM TCEP, 10% glycerol, 2% β-OG. Purified SNARE proteins and fluorescently labeled content mixing molecules were added to the lipid mixtures to make the syntaxin-1:SNAP25:lipid ratio 1:5:800 and Phycoerythrin-Biotin (4 µM) for T-liposomes, and the synaptobrevin:lipid ratio 1:500 and Cy5-Streptavidin (8 µM) for V-liposomes. The mixtures were incubated at room temperature and dialyzed against the reaction buffer (25 mM HEPES pH 7.4, 150 mM KCl, 1 mM TCEP, 10% glycerol) with 2 g/L Amberlite XAD-2 beads (Sigma) 3 times at 4°C. Proteoliposomes were purified by floatation on a three-layer histodenz gradient (35%, 25%, and 0%) and harvested from the topmost interface. To simultaneously measure lipid mixing from de-quenching of Marina Blue lipids and content mixing from the development of FRET between Phycoerythrin-Biotin trapped in T-liposomes and Cy5-streptavidin trapped in V-liposomes, T-liposomes (0.25 mM lipid) were mixed with V-liposomes (0.125 mM lipid) in a total volume of 200 µL. Acceptor T-liposomes were first incubated with 0.8 µM NSF, 2 µM αSNAP, 2.5 mM MgCl2, 2 mM ATP, 0.1 mM EGTA, and 1 µM Munc18-1 at 37°C for 25 min. They were then mixed with donor V-liposomes, 0.5 µM Munc13-1 C1C2BMUNC2C, and 1 µM excess SNAP25. All experiments were performed at 30°C and 0.6 mM Ca2+ was added at 300 s. The fluorescence signal from Marina Blue (excitation at 370 nm, emission at 465 nm) and Cy5 (excitation at 565 nm, emission at 670 nm) were recorded to monitor lipid and content mixing, respectively. At the end of the reaction, 1% β-OG was added to solubilize the liposomes and the lipid mixing data were normalized to the maximum fluorescence signal. Most experiments were performed in the presence of 5 µM streptavidin, and control experiments without streptavidin were performed to measure the maximum Cy5 fluorescence after detergent addition for normalization of the content mixing data. Analogous procedures were followed for the lipid and content mixing assays of Figure 3—figure supplement 2, except that we used Vsyt1-liposomes instead of V-liposomes. The V-Syt1 liposomes contained 41% POPC, 6.8% DOPS, 29.2% POPE, 20% cholesterol, 1.5% NBD-PE, and 1.5% Marina, as well as full-length synaptobrevin and synaptotagmin-1 (57–421) at 1:10,000 and 1:1000 ratios with the lipids, respectively. Fusion reactions were performed with a 1:4 ratio of VSyt1- to T-liposomes as the trans-SNARE complex assembly assays of Figure 3E.

FRET assays to monitor trans-SNARE complex assembly and disassembly

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Reconstituted liposomes were made similarly to those used for the lipid and content mixing assay. V-liposomes with full-length synaptobrevin L26C-Alexa488 contained 42% POPC, 19% DOPS, 19% POPE, and 20% cholesterol. VSyt1-liposomes with Synaptotagmin 57–421 and full length synaptobrevin L26C-Alexa488 contained 43% POPC, 6.8% DOPS, 30.2% POPE, and 20% cholesterol. T-liposomes with full-length syntaxin-1A S186C-TMR and full length SNAP25-A M71D, L78D mutant contained 38% POPC, 18% DOPS, 20% POPE, 20% cholesterol, 2% PIP2, and 2% DAG (note that for selected experiments WT SNAP-25 was used instead of the mutant). Dried lipid mixtures were re-suspended in 25 mM HEPES pH 7.4, 150 KCl, 1 mM TCEP, 2% β-OG. Purified SNARE proteins were added to the lipid mixtures to make the syntaxin-1:SNAP25:lipid ratio 1:5:800 for T-liposomes, the synaptobrevin:lipid ratio 1:10,000 for V-liposomes, and the synaptotagmin-1:synaptobrevin:lipid ratio 1:0.1:1000 for VSyt1-liposomes. The mixtures were incubated at room temperature and dialyzed against the reaction buffer (25 mM HEPES pH 7.4, 150 mM KCl, 1 mM TCEP) with 2 g/L Amberlite XAD-2 beads (Sigma) 3 times at 4°C. All FRET experiments were performed at 37°C on a PTI Quantamaster 400 spectrofluorometer (T-format) equipped with a rapid Peltier temperature controlled four-position sample holder. All slits were set to 1.25 mm. A GG495 longpass filter (Edmund optics) was used to filter scattered light. SNARE complex formation was measured by the development of FRET between Alexa488-Synaptobrevin on V- or VSyt1-liposomes and TMR-syntaxin-1 on T-liposomes or TMR-syntaxin-1 (2–253). Typically, four parallel reactions were monitored simultaneously, which allowed performance of multiple experiments under various conditions (each in triplicate) with the same fresh liposome preparations in the same day.

For kinetic traces, the fluorescence signal at 518 nm (excitation at 468 nm) was recorded to monitor the Alexa488 donor fluorescence intensity. The signal of donor V-or VSyt1-liposomes (0.0625 mM lipid) in reaction buffer containing 0.1 mM EGTA, 2.5 mM MgCl2, 2 mM ATP and various additions was first recorded for 180 s to check for signal stability and then either acceptor T-liposomes (0.25 mM lipid) or soluble acceptor TMR-syntaxin-1 2–253 (300 nM) were added. The initial additions included the following in different combinations as specified in the Figures and their legends: 10 µM Syb49-93, 2 µM complexin-1, 1 µM synaptotagmin-1 C2AB, 0.6 mM Ca2+, 1 µM Munc18-1, and 0.3 μM Munc13-1 C1C2BMUNC2C. For experiments where disassembly was tested after recording an assembly reaction (Figures 4 and 6B–D), 2 µM αSNAP and 0.4 µM NSF were added at the indicated time points. Because adding these reagents to four reactions running in parallel took more than one minute and disassembly reactions are relatively fast at this time scale, we did not attempt to monitor the kinetics of disassembly and focused on the overall amount of disassembly. For homogeneity among the reactions, we let a short amount of time pass before starting to monitor the donor fluorescence intensity again, which thus was re-initiated 2–5 min after the addition of NSF-αSNAP. For experiments designed to test the assembly of cis- or trans-SNARE complexes in the presence of NSF-αSNAP (Figures 3 and 6A), 0.1 mM EGTA, 2.5 mM MgCl2, 2 mM ATP, 2 µM αSNAP and 0.4 µM NSF were mixed with the V- or VSyt1-liposomes from the start, together with the corresponding additional proteins. For some experiments, Ca2+ (0.6 mM) was added at the time points indicated in the Figures. In the experiments of Figures 3C,D,F and 240 μM Ca2+ was added at 2 min and 500 μM EGTA at the indicated time points. Data points were collected (1 s acquisition) every 20 s for 30 min for reactions where saturation was reached, and for longer times for slower reactions. Only a small amount of photobleaching of the donor was observed under these conditions in control experiments with donor alone.

Pre-formed trans-SNARE complexes were made by incubating V-liposomes, T-liposomes, SNAP25m, Synaptobrevin 49–93, and 0.1 mM EGTA together for 5 hr at 37°C. For experiments with WT SNAP-25 or samples with VSyt1 liposomes, these reagents were mixed together for 24 hr at 4°C. Various combinations of the proteins listed above, as well as 2.5 mM MgCl2 and 2 mM ATP, were added to the pre-formed trans-SNARE complex and incubated for 5 min at 37°C. An emission scan was then collected (excitation 468 nm, emission from 490 nm to 700 nm) to detect how much trans-SNARE complex was formed. Two µM αSNAP and 0.4 µM NSF were then added to each reaction and allowed to incubate for 5 min at 37°C to disassemble the trans-SNARE complex. A second wavelength scan was then collected to determine how much of the complex was disassembled. All experiments were repeated at least 3 times with a single preparation and the results were verified in multiple experiments with different preparations. For some control experiments, ATPγS (2 mM) was used instead of ATP, or Mg2+ was replaced with EDTA (1 mM).

Cryo-electron microscopy

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Samples of pre-formed trans-SNARE complexes between V- and T-liposomes were prepared by incubating them with Syb49-93 for five hours at 37°C as for the assays used to measure protection against disassembly by NSF-αSNAP. Cryo-EM grids were prepared by applying 3 μL of the sample solution to a negatively glow discharged Lacey carbon copper grid (200-mesh; Electron Microscopy Sciences) and blotted for 4.0 s under 100% humidity at 4°C before plunge-freezing in liquid ethane using a Mark IV Vitrobot (FEI). Micrographs were acquired on a Talos Arctica microsope (FEI) operated at 200 kV with a K2 Summit direct electron detector (Gatan). A nominal magnification of 11,000 was used for imaging, and 20 dose-fractionation frames were recorded over a 10 s exposure at a dose rate of 2.1 electrons/ Å2/s for each micrograph. Motion correction was performed using the MotionCorr2 program (Zheng et al., 2017).

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Decision letter

  1. Axel T Brunger
    Reviewing Editor; Stanford University, United States
  2. Randy Schekman
    Senior Editor; Howard Hughes Medical Institute, University of California, Berkeley, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Multiple factors synergistically protect trans-SNARE complexes against disassembly by NSF and aSNAP" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Axel Brunger as the Reviewing Editor and Randy Schekman as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yongli Zhang (Reviewer #2).

Summary:

This study addresses the effects of NSF, αSNAP, synaptotagmin, CA, Munc 18, Munc 13, and complexin on the assembly, disassembly, and stability of trans-SNARE complexes.

Overall, this study contains a mixture of results that were already known (such as the ability of NSF to disassemble trans SNARE complexes, Yavuz et al.., 2018, or the effects of various factors on trans SNARE complex assembly, Ma et al., 2013), along with new results, in particular those shown in Figures 3 and 6. This mixture of some old and some new results makes the paper somewhat difficult to read and reduces its impact.

In addition, the reviewers and reviewing editor identified a number of serious concerns as outlined below. In the current form, the manuscript is not therefore not acceptable. However, we are open to consider a re-written manuscript that focusses on new and interesting findings and address the concerns raised below.

Major comments:

1) One major conclusion is that NSF/SNAP can disassemble trans-SNARE complexes. As the authors acknowledge, however, the novelty of this conclusion is unfortunately undermined by a recent report from the Jahn lab (Yavuz, 2018). Another major conclusion is that Munc18 and Munc13 together support trans-SNARE complex assembly in the presence of NSF/SNAP. This is important, and consistent with the results presented in this manuscript, but it was already well established by previous work, especially from the Rizo lab (especially Ma et al., 2013). Please re-write the paper, focusing on new insights, and remove material that is similar to previously published work (or relegate it to a supplement).

2) Throughout the work, the authors used mutant SNAP-25 with two mutations in its C-terminal region, which attenuates SNARE zippering and stabilizes the partially zippered SNARE conformation for trans-SNAREs. The authors should address the impact of such mutations on the major conclusions derived for wild-type SNAREs. A clarification on this issue is important, which affects comparison of this work with previous work (especially, Weber et al., 2000).

3) t-SNARE (3Q) proteoliposomes tend to form a dead-end conformation of 2 syntaxins (2Qa) associated with 1 SNAP-25 (QbQc); to prevent this, the authors start with a SNARE domain fragment Syb49-93 which can enter the complex to form [Sb49-93]:Syntaxin:SNAP25. The synaptobrevin coming in trans purportedly displaces the Syb49-93 and gives the authentic parallel trans-SNARE complex. For clarity, the authors really must expand their presentation of this in Figure 2A. The cartoon in Figure 2A does not present where and how Syb49-93 binds in the initial v-SNARE (3Q) complex, and they add a second synthetic SNARE fragment as well, Syb29-93, to prevent trans-SNARE complex reassembly. If as claimed (Figure 2B) NSF and SNAP are disassembling trans-SNARE complex, why would the reassembly of that complex be a problem? Wouldn't it just be again disassembled by NSF/SNAP? Presumably they found that Syb29-93 was need to keep the bulk of the system in a NSF/SNAP-induced disassembled structure at steady-state. In sum, the proteoliposomes the authors are using have 2 SNARE-domain mutations, introducing charged residues, to block the completion of zippering, multiple mutations to eliminate the cysteines, two mutations to introduce not just Cys residues at the desired positions but then stoichiometric derivatization of these Cys residues with fluorophores, and there are two (2) extra SNARE domain peptides introduced as well. Moreover, starting the system with syntaxin in complex with Munc18-1 and then using Munc13, the fundamental insight of their Science paper of 2013 (Ma et al.), should get around the need for Syb49-93 and Syb29-93.

4) Figure 2B: The fluorescence emission spectrum of the v-SNARE liposomes is clear, just one peak at about 525nm (black), and there is a big reduction in this peak due to quenching when the t-SNARE proteoliposomes are added (red) and a new FRET peak appears at around 580 nm. Addition of NSF + αSNAP relieves most of the quenching at 525nm without removing most of the presumptive FRET peak at 580. Presumably, this is due to direct excitation of the acceptor dye, as pointed out by the authors in a parenthetical note in the fourth paragraph of the subsection “Munc18-1, Munc13-1, complexin-1 and the synaptotagmin-1 C2 domains stabilize trans-SNARE complexes”. Please measure this effect for by collecting an emission spectrum of the same sample preparation with only acceptor dyes, but no donor dyes, using the same excitation wavelength that was used in Figure 2. Of course, all the data need to be viewed in the context of this direct donor excitation, and ideally, all the data need to be corrected for it, although this is difficult with bulk fluorescence experiments. Another complication in interpretation of such bulk fluorescence experiments is that differences could be due to affecting the acceptor dye by molecular interactions rather than trans SNARE complex disassembly per se. For example, might the green curve in Figure 2B be due to an altered distance and conformation change in the 2 fluorophores when α-SNAP and NSF bind rather than to true disassembly? Thus, controls are essential (e.g., Mg2+ vs EDTA, and no ATP, ATP, or ATPγS). Comparison of ATP vs. ATPγS should be done throughout.

5) Related to the above concern, in Figure 3 the major effect of synaptotagmin, Munc18, Mun13, and complexin seems to be in suppressing the donor:acceptor ratio before the NSF/SNAP is added. In this figure, trans-SNARE complex spontaneous assembly was first allowed, then synaptotagmin, Munc18, Mun13, and/or complexin added, then NSF/SNAP. It is possible that these might substantially modify the environment of the fluorophores and affect FRET as much as they actually affect trans-SNARE disassembly. As mentioned above, controls need to be carried out to roll out such effects on the fluorophores.

6) The paper would benefit tremendously if the authors could complement their fluorescence study with another assay of SNARE association, such as pull-down experiments. Also, it would strengthen the study to know which combinations of factors would give fusion (they use an assay of protected fluorescent protein mixing in Figure 1) if they used wild-type or less permanently arrested SNAREs. In Figure 2E, there is a striking change in donor fluorescence, which I presume represents trans-SNARE complex assembly, which depends on Munc18, Munc13, and calcium. With unblocked SNAREs, does this lead to fusion?

7) A key question is how NSF/α-SNAP distinguish trans- and cis-SNARE complexes. The authors argue that α-SNAP cannot bind to two membranes well as in trans-SNARE complex, which prevents NSF/α-SNAP from disassembling trans-SNARE complexes. However, membrane binding by α-SNAP is not required for SNARE disassembly, as NSF/α-SNAP efficiently disassemble cytoplasmic SNARE complexes. Thus, it is possible that NSF/alpha-SNAP recognize certain SNARE conformational differences between trans- and cis-SNAREs. For example, as is previously proposed (Minglei Zhao et al., Nature, 2015), trans-SNAREs may be partially zippered. The authors are encouraged to reveal more insights into the key question by more experiments if possible.

8) The authors claim, in the title as well as the text, that Munc18, Munc13, synaptotagmin, and complexin act synergistically to protect trans-SNARE complexes from NSF/SNAP. However, this is not obvious based on the data presented in Figure 3C/D, where greater-than-additive effects are not observed at least for most combinations. Complexin and synaptotagmin on their own, at the concentration used (but see the next point #9), have only modest activity, which might simply reflect the ability of any binding partner to get in the way of NSF/SNAP. In the absence of calcium, there is a bit of additional protection afforded by complexin (but not by synaptotagmin) when Munc18 and Munc13 are already present.

9) The claim that complexin does not prevent disassembly of cis SNARE complexes (Figure 4) is at odds with other results in the literature. Previously, at a SNAP:complexin ratio of ~ 18:1, complexin had no effect (Pabst et al., 2000), whereas at a ratio of ~ 3:1, complexin had a substantial effect on NSF-mediated disassembly of the soluble SNARE complex (Winter et al., 2009). More recently, concentration-dependent complexin inhibition of NSF-mediated disassembly of the cis SNARE complex was observed (Choi et al., 2018). Please perform the experiments shown in Figures 3, 4, 6 at different complexin concentrations, i.e., different complexin:SNAP molar ratios.

10) Figures 3 and 6 show protection of disassembly of trans SNARE complexes by a variety of factors. A key difference between experiments in these two figures relates to synaptotagmin. The C2AB fragment is used in Figure 6, whereas full-length synaptotagmin is used in Figure 6. Why? Please redo all experiments in Figure 3 with membrane anchored synaptotagmin (as in Figure 6).

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Multiple factors protect neuronal trans-SNARE complexes against disassembly by NSF and aSNAP" for further consideration at eLife. Your revised article has been favorably evaluated by Randy Schekman as the Senior Editor, Axel Brunger as the Reviewing Editor, and three reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.

Major comments:

1) The data make a convincing case that complexin can bind trans-SNARE complexes to block NSF/SNAP-driven disassembly. This conclusion, moreover, seems entirely plausible based on previous findings, including high-resolution structures. The role(s) of Munc18/13 on inhibiting trans SNARE complex disassembly is more problematic. It was known that, working together, they powerfully promote trans-SNARE complex assembly. The simplest model, call it Model I, is that Munc18/13 assembly activity simply opposes NSF/SNAP disassembly activity. Model I does not require that Munc18/13 interact with trans-SNARE complexes. By contrast the author's model, call it Model II, is that Munc18/13 remains associated with assembled trans-SNARE complexes. In so doing, Munc18/13 blocks NSF/SNAP from binding, and therefore disassembling, trans-SNARE complexes.

Moreover, NSF/SNAP plays a dual role: not only does it catalyze SNARE disassembly, but it also promotes Munc18/13-mediated SNARE assembly by rescuing SNAREs that are kinetically trapped in off-pathway, dead end products. No one, of course, knows this better than the authors, who indeed point out this effect in Figure 4B. Nevertheless, the dual role of NSF/SNAP further complicates the task of unambiguously distinguishing models I and II by using the author's assays.

The revised manuscript does not conclusively rule out Model I since every manipulation that strengthens the postulated 'protective' activity of Munc18/13, including the addition of Ca2+, also increases its assembly activity. Even the authors, upon occasion, seem to adopt the language of Model I. For example, "[T]he contrast of the results obtained with cis-SNARE complexes with those observed with trans-SNARE complexes provides a dramatic demonstration of how the apposition of two membranes tilts the balance in favor of SNARE complex assembly, whereas disassembly dominates on a single membrane".

What would seem to be needed for distinguishing Model I from Model II is some manipulation or mutation that separates the proposed Munc18/13's protective function from its assembly function. For example, this could be accomplished by determining if Munc18 and Munc13 are both associated with trans SNARE complexes prior to NSF/aSNAP-mediated disassembly. However, we realize that such a direct observation may not be possible with the author's assays. At the minimum, we request a more balanced discussion of these points, presenting both models I and II as being consistent with the data presented in this paper and other available data, as well as adjusting the title, Abstract and other relevant parts of the text.

2) An additional model to explain the data is as follows (call it Model III): Munc18/13 catalyze the proper assembly of the SNARE complex, and thereby, supercomplexes with complexin and synaptotagmin. Once such properly assembled supercomplexes are situated between membranes, it might be difficult for SNAP/NSF to bind to the trans-SNARE complex even if Munc13/Munc18 are not associated with such supercomplexes anymore. The absence of Munc18/Munc13 may lead to a mixture of properly and improperly assembled trans-SNARE complexes where the latter are more readily disassembled by NSF/SNAP. Please discuss this possible model as well.

3) The authors performed additional experiments to test inhibition of cis-SNARE complexes by complexin (Figure 6, Figure 6—figure supplement 1, Results section "Disassembly of cis-SNARE complexes”; Discussion section, seventh paragraph), and state: "It is plausible that the differences observed with the results of Winter et al.. 2009, and Choi et al., 2018, arose because syntaxin-1 did not include the N-terminal region containing the Habc domain in both of these studies, and in the latter NSF- αSNAP might have been less active because of the absence of membranes." However, contrary to what is stated here, the Winter et al. experiments did include a membrane. The reviewers are not convinced that there is any discrepancy between the data presented in the present work and the previous work by Winter et al., and Choi et al. since the experiment shown in Figure 6—figure supplement 1 does not have the necessary time resolution to discern the effect of complexin on the kinetics of NSF/aSNAP-mediated SNARE complex disassembly observed by Winter et al. and Choi et al.

Moreover, the drawing in Figure 6—figure supplement 1 is somewhat misleading since upon injection of NSF/aSNAP, at least a minute (or more) passes before the fluorescence intensity measurements continue. Thus, this break period should be clearly marked in the figure and explained in the figure caption or the Materials and methods. The presentation and discussion of the results in the subsection “Disassembly of cis-SNARE complexes” and the Discussion, should be adjusted to present a unified view of the action of complexin on slowing the kinetics of NSF/aSNAP-mediated for both cis and trans SNARE complexes (i.e., complexin slows the kinetics in both cases to different degrees).

https://doi.org/10.7554/eLife.38880.024

Author response

Summary:

This study addresses the effects of NSF, αSNAP, synaptotagmin, CA, Munc 18, Munc 13, and complexin on the assembly, disassembly, and stability of trans-SNARE complexes.

Overall, this study contains a mixture of results that were already known (such as the ability of NSF to disassemble trans SNARE complexes, Yavuz et al., 2018, or the effects of various factors on trans SNARE complex assembly, Ma et al., 2013), along with new results, in particular those shown in Figures 3 and 6. This mixture of some old and some new results makes the paper somewhat difficult to read and reduces its impact.

In addition, the reviewers and reviewing editor identified a number of serious concerns as outlined below. In the current form, the manuscript is not therefore not acceptable. However, we are open to consider a re-written manuscript that focusses on new and interesting findings and address the concerns raised below.

We fully understand these concerns and appreciate the opportunity to address them in the revised manuscript. There are a few points that we would like to emphasize about the complexity of the system and the difficulty of investigating trans-SNARE complexes between two membranes before we provide answers to the specific concerns.

SNAREs are highly abundant in the vesicle and plasma membranes, but assembly of many trans-SNARE complexes may lead to uncontrolled membrane fusion and perhaps to fusion pathways that are not physiologically relevant. Cryo-EM images of liposome fusion reactions with SNAREs have shown extended membrane interfaces [e.g. Hernandez et al. Science 336, 1581 (2012)] that we believe are unlikely to occur in the pathway to synaptic vesicle fusion. Indeed, inclusion of other proteins affects the morphology of the membrane interfaces [e.g. Bharat et al. EMBO Rep. 15, 308 (2014)] and favors ‘point contact’ interfaces that are more likely to be physiologically relevant [Gipson et al., 2017]. We believe that Munc13-1 and other components of the release machinery limit the number of trans-SNARE complexes that are formed before priming, but it is difficult to faithfully recapitulate the primed state of synaptic vesicles with a limit set of proteins.

The interpretation of the data is further complicated by the finding that some trans-SNARE complexes can be disassembled by NSF-αSNAP while others are NSF-αSNAP resistant, as described in the recent Yavuz et al. 2018., which attributes such resistance to formation of the extended liposome interfaces mentioned above. In our experiments we used a very low synaptobrevin-to-lipid ratio (1:10,000) in part to maximize the percent of synaptobrevin molecules that engage in trans-SNARE complex formation, thus optimizing the decrease in donor fluorescence, and in part to avoid accumulation of too many trans-SNARE complexes between each membrane-membrane interface. Even with this design, we still observe that 50% of trans-SNARE complexes are resistant to NSF-αSNAP, and cryo-EM images of liposomes with pre-formed trans-SNARE complexes still show some extended interfaces between liposomes (new Figure 2—figure supplement 3), albeit smaller than those observed by Yavuz et al., 2018. In the paper we suggest that the trans-SNARE complexes that can be disassembled by NSF-αSNAP may be more representative of those present in primed synaptic vesicles (Discussion, second paragraph), which is a speculation but correlates with the finding that synaptic vesicles can be de-primed and that de-priming is NEM-sensitive [He et al., 2017].

The observation of NSF-αSNAP-resistant trans-SNARE complexes provides a plausible explanation for the results of Weber et al., 2000, which showed that lipid mixing between V- and T-liposomes could occur in the presence of NSF-αSNAP only if the liposomes were pre-incubated (we now suggest this explanation in the second paragraph of the Discussion). We have tried to use liposome fusion assays to investigate this issue but so far have not obtained sufficiently conclusive results. We believe that resolving this issue is beyond the scope of this paper. In the revised manuscript we do include new fusion assays (Figure 3—figure supplement 2) that illustrate that trans-SNARE complex assembly does not necessarily lead to membrane fusion (see points 1 and 6 below).

We would also like to point out that we attempted to perform pulldown assays, as suggested by the reviewers, and also co-sedimentation assays between heavy V-liposomes containing sucrose and T-liposomes, to obtain data that could complement the FRET results, but it soon became clear that both approaches cannot yield meaningful results because Munc13-1 and synaptotagmin-1 can bridge membranes even if there are no trans-SNARE complexes. We hope that the reviewers will agree that the FRET approach that we used is the most appropriate for direct measurement of transSNARE complex assembly and disassembly, and that, with the controls that were requested and that extensive additional data that we acquired to address the reviewer concerns, this is a strong story that provides key insights into how central components of the release machinery protect trans-SNARE complexes against disassembly by NSFαSNAP.

Major comments:

1) One major conclusion is that NSF/SNAP can disassemble trans-SNARE complexes. As the authors acknowledge, however, the novelty of this conclusion is unfortunately undermined by a recent report from the Jahn lab (Yavuz, 2018). Another major conclusion is that Munc18 and Munc13 together support trans-SNARE complex assembly in the presence of NSF/SNAP. This is important, and consistent with the results presented in this manuscript, but it was already well established by previous work, especially from the Rizo lab (especially Ma et al., 2013). Please re-write the paper, focusing on new insights, and remove material that is similar to previously published work (or relegate it to a supplement).

In the revised manuscript we do not present the disassembly of trans-SNARE complexes by NSF-αSNAP as a conclusion of the paper and we have increased the emphasis on the newest aspect of our results, namely the analysis of protection of trans-SNARE complexes against disassembly by NSF-αSNAP. However, we would like to clarify that, while it is generally assumed that trans-SNARE complex assembly is required for lipid and content mixing, and in the Ma et al., 2013 paper we did propose that Munc18-1 and Munc13-1 orchestrate SNARE complex assembly in an NSF-αSNAP resistant manner, we did not really measure trans-SNARE complexes in that paper. The contrast between trans-SNARE complex assembly and fusion is now illustrated by comparing the transSNARE complex assembly assays between VSyt1- and T-liposomes of Figure 3E (red curve), where Ca2+-independent assembly is observed, and the fusion assays shown in the new Figure 3—figure supplement 2, where there is no fusion before addition of Ca2+. Moreover, the analysis of protection against disassembly is tightly linked to the analysis of trans-SNARE complex assembly in the presence of NSF and αSNAP because the former may depend on the latter. Hence, we cannot really separate the assembly data from the disassembly results.

2) Throughout the work, the authors used mutant SNAP-25 with two mutations in its C-terminal region, which attenuates SNARE zippering and stabilizes the partially zippered SNARE conformation for trans-SNAREs. The authors should address the impact of such mutations on the major conclusions derived for wild-type SNAREs. A clarification on this issue is important, which affects comparison of this work with previous work (especially, Weber et al., 2000).

We have performed some experiments with WT SNAP-25 instead of SNAP-25m (Figure 2—figure supplement 4B), verifying that the SNAP-25 mutation does not impair trans-SNARE complex disassembly by NSF-αSNAP. Moreover, we now also show that the mutation in SNAP-25m does not interfere with disassembly of cis-SNARE complexes (new Figure 6—figure supplement 1B, C).

3) t-SNARE (3Q) proteoliposomes tend to form a dead-end conformation of 2 syntaxins (2Qa) associated with 1 SNAP-25 (QbQc); to prevent this, the authors start with a SNARE domain fragment Syb49-93 which can enter the complex to form [Sb49-93]:Syntaxin:SNAP25. The synaptobrevin coming in trans purportedly displaces the Syb49-93 and gives the authentic parallel trans-SNARE complex. For clarity, the authors really must expand their presentation of this in Figure 2A. The cartoon in Figure 2A does not present where and how Syb49-93 binds in the initial v-SNARE (3Q) complex, and they add a second synthetic SNARE fragment as well, Syb29-93, to prevent trans-SNARE complex reassembly. If as claimed (Figure 2B) NSF and SNAP are disassembling trans-SNARE complex, why would the reassembly of that complex be a problem? Wouldn't it just be again disassembled by NSF/SNAP? Presumably they found that Syb29-93 was need to keep the bulk of the system in a NSF/SNAP-induced disassembled structure at steady-state. In sum, the proteoliposomes the authors are using have 2 SNARE-domain mutations, introducing charged residues, to block the completion of zippering, multiple mutations to eliminate the cysteines, two mutations to introduce not just Cys residues at the desired positions but then stoichiometric derivatization of these Cys residues with fluorophores, and there are two (2) extra SNARE domain peptides introduced as well. Moreover, starting the system with syntaxin in complex with Munc18-1 and then using Munc13, the fundamental insight of their Science paper of 2013 (Ma et al.), should get around the need for Syb49-93 and Syb29-93.

We have improved Figure 2A to help visualizing the effects of Syb49-93. In our original experiments we used Syb29-93 to prevent re-assembly of SNARE complexes, similar to the approach used in Winter et al., 2009. However, we have performed additional experiments and found that Syb29-93 does not affect the results when we add NSFαSNAP to disassemble trans-SNARE complexes, most likely because re-assembly is much slower than disassembly. We have repeated all the protection assays without Syb29-93 and obtained analogous results to those that we described originally. All the data presented in the revised manuscript were obtained without Syb29-93.

We understand the concern about so many other manipulations, but many of them are common in the field and are based on solid grounds. Mutations of native cysteines have been widely used in studies where cysteines were introduced in non-native positions of SNARE proteins to attach fluorescent probes. The positions where we attached probes on synaptobrevin and syntaxin-1 were chosen carefully to minimize the possibility that they might hinder binding of the various factors used in our experiments, based on the available structural information. The protection against disassembly provided by these factors suggests that the probes indeed did not prevent binding.

We did perform experiments where NSF-αSNAP were added from the beginning and trans-SNARE complex assembly was monitored in the presence of different factors, which confirmed that Munc13-1 and Munc13-1 C1C2BMUNC2C are essential for transSNARE complex assembly in the presence of NSF-αSNAP (Figures 3A, B, E). This is the ideal set up to guide the system through the Munc18-1-Munc13-1-dependent pathway of trans-SNARE complex assembly, and these experiments do suggest that Munc18-1 and Munc13-1 C1C2BMUNC2C must somehow protect against disassembly, particularly in the presence of Ca2+. However, it was less clear whether Munc18-1 and Munc13-1 C1C2BMUNC2C protect against disassembly in the absence of Ca2+, as Ca2+-independent assembly was inefficient. We have performed additional experiments where EGTA was added after efficient Ca2+-dependent assembly of trans-SNARE complexes to test whether they could be disassembled by the NSF-αSNAP present in the reaction, and found that there is some but limited disassembly (Figures 3C, D). These results support the notion that Munc18-1 and Munc13-1 C1C2BMUNC2C protect against disassembly in the absence of Ca2+ and that such protection is enhanced by Ca2+. Using this approach, we further show that complexin-1 strongly protects against disassembly (Figures 3D, F).

While these experiments were very informative, we also wanted to test whether Munc18-1, Munc13-1 C1C2BMUNC2C, synaptotagmin-1 and complexin-1 individually or in different combinations can protect trans-SNARE complexes against disassembly. For this purpose, we use the approach of pre-forming trans-SNARE complexes in the absence of any of these proteins and then testing whether incubation of the pre-formed complexes with the proteins prevents disassembly by NSF-αSNAP. Including the Syb4993 peptide in these experiments was critical to accelerate trans-SNARE complex assembly and reach optimal assembly in a reasonable amount of time, as we show now in Figure 2—figure supplement 2. Yavuz et al., 2018 showed that this peptide is released upon trans-SNARE complex assembly; hence, Syb49-93 should not interfere with the disassembly reaction.

4) Figure 2B: The fluorescence emission spectrum of the v-SNARE liposomes is clear, just one peak at about 525nm (black), and there is a big reduction in this peak due to quenching when the t-SNARE proteoliposomes are added (red) and a new FRET peak appears at around 580 nm. Addition of NSF + αSNAP relieves most of the quenching at 525nm without removing most of the presumptive FRET peak at 580. Presumably, this is due to direct excitation of the acceptor dye, as pointed out by the authors in a parenthetical note in the fourth paragraph of the subsection “Munc18-1, Munc13-1, complexin-1 and the synaptotagmin-1 C2 domains stabilize trans-SNARE complexes”. Please measure this effect for by collecting an emission spectrum of the same sample preparation with only acceptor dyes, but no donor dyes, using the same excitation wavelength that was used in Figure 2. Of course, all the data need to be viewed in the context of this direct donor excitation, and ideally, all the data need to be corrected for it, although this is difficult with bulk fluorescence experiments.

We acquired fluorescence emission spectra for separate samples of donor and acceptor (Figure 2—figure supplement 4A). There is indeed some excitation of the acceptor even using the donor excitation wavelength, and the emission intensity is considerable because the acceptor is used in large excess over the donor. We now use the addition of the two separate spectra as the control for ‘no assembly’ (Figure 2B, Figure 2—figure supplements 4B, C). As we explain in point 5, our assessment of the ability of different factors to protect against disassembly now relies on measurements of donor fluorescence at 518 nm, where its emission is highest. The fluorescence emission spectra that we acquired for the acceptor alone showed that the emission of the acceptor at 518 nm is negligible, although there is a small signal intensity at this wavelength that arises from scattering (Figure 2—figure supplement 4A). We did not attempt to correct for this contribution to the intensity at 518 nm because we did not calculate FRET efficiencies and the small contribution from scattering is analogous under the various conditions where we measured donor fluorescence intensities.

Another complication in interpretation of such bulk fluorescence experiments is that differences could be due to affecting the acceptor dye by molecular interactions rather than trans SNARE complex disassembly per se. For example, might the green curve in Figure 2B be due to an altered distance and conformation change in the 2 fluorophores when α-SNAP and NSF bind rather than to true disassembly? Thus, controls are essential (e.g., Mg2+ vs EDTA, and no ATP, ATP, or ATPγS). Comparison of ATP vs. ATPγS should be done throughout.

We address the issue of altered distance of conformation in point 5. In the revised manuscript we now present controls for trans-SNARE complex disassembly where Mg2+ was replaced by EDTA or ATP was replaced by ATPγS (Figures 2C, 5B, C, Figure 5—figure supplements 1, 4).

5) Related to the above concern, in Figure 3 the major effect of synaptotagmin, Munc18, Mun13, and complexin seems to be in suppressing the donor:acceptor ratio before the NSF/SNAP is added. In this figure, trans-SNARE complex spontaneous assembly was first allowed, then synaptotagmin, Munc18, Mun13, and/or complexin added, then NSF/SNAP. It is possible that these might substantially modify the environment of the fluorophores and affect FRET as much as they actually affect trans-SNARE disassembly. As mentioned above, controls need to be carried out to roll out such effects on the fluorophores.

This is a very important point. First we would like to clarify that Figure 5A of the revised manuscript (which corresponds to Figure 3B in the original paper) shows two spectra, one acquired after incubating pre-formed trans-SNARE complexes with Munc18-1, Munc13-1 C1C2BMUNC2C, synaptotagmin-1 C2AB, complexin-1 and Ca2+ and another acquired on the same sample after adding NSF-αSNAP. Analogous spectra were acquired with different combinations of these proteins to dissect their abilities to protect against trans-SNARE complex disassembly. Representative spectra for all the different conditions are now shown in Figure 5—figure supplement 1. As we now mention in the manuscript (subsection “Multiple factors stabilize trans-SNARE complexes against disassembly by NSF-αSNAP”, sixth paragraph), all the spectra acquired after incubating trans-SNARE complexes with different combinations of Munc18-1, Munc13-1 C1C2BMUNC2C, synaptotagmin-1 C2AB, complexin-1 and Ca2+ were very similar, showing that these proteins do not substantially affect the fluorophores in trans-SNARE complexes. However, there were clear differences among the spectra obtained after NSF-αSNAP addition, some of which could arise from proteins affecting the fluorophores rather than disassembly per se. To assess the potential effects of the various proteins on the fluorescence emission spectra of the donor and acceptor in samples as similar as possible to those used to examine protection against disassembly, we prepared control samples with V-liposomes containing Alexa488-labeled synaptobrevin (V*) and T-liposomes containing no fluorescence acceptor (T), as well as controls samples where the V-liposomes did not have a fluorescence donor (V) and T-liposomes with TMR-labeled syntaxin-1 (T*). The spectra obtained with different additions showed that the different proteins did not affect the donor fluorescence substantially (Figure 5—figure supplement 2), but NSF-αSNAP did affect the acceptor fluorescence considerably and this effect was altered under some conditions, particularly by the inclusion of Munc18-1 (Figure 5—figure supplement 3).

As it would be difficult to account for these different effects on the acceptor fluorescence, to obtain a quantitative measure of protection against disassembly we now rely only on donor fluorescence intensities, and calculated the ratio of these intensities after and before NSF-αSNAP addition (Figures 5B, C). Note that in the original manuscript we calculated the ratio between donor and acceptor intensities, which in retrospective was clearly wrong. We are very thankful to the reviewers for helping us to correct this mistake. The overall results that we obtained in terms of the protection afforded by different proteins are similar to those described in the original manuscript, but there is an important distinction in that Munc18-1 alone appeared previously to provide robust protection against disassembly but with the new data such protection appears to be rather limited or none. Nevertheless, Munc18-1 still cooperates with Munc13-1 in protecting against disassembly, particularly in the presence of Ca2+, and optimal protection is observed when all four proteins are included, as concluded in the original manuscript.

6) The paper would benefit tremendously if the authors could complement their fluorescence study with another assay of SNARE association, such as pull-down experiments. Also, it would strengthen the study to know which combinations of factors would give fusion (they use an assay of protected fluorescent protein mixing in Figure 1) if they used wild-type or less permanently arrested SNAREs. In Figure 2E, there is a striking change in donor fluorescence, which I presume represents trans-SNARE complex assembly, which depends on Munc18, Munc13, and calcium. With unblocked SNAREs, does this lead to fusion?

We agree that it would be desirable to use other methods to confirm the results from the FRET assays. However, as we explain in our response to the Summary, pulldown assays did not provide useful information because Munc13-1 and synaptotagmin-1 can bridge membranes regardless of the presence of trans-SNARE complexes.

With regard to the change in fluorescence in Figure 2E (now Figure 4B), this Ca2+dependent change does correlate with the Ca2+-dependence of liposome fusion in our reconstitution assays (Ma et al., 2013; Liu et al., 2016), although more closely related experiments are those where NSF-αSNAP were included from the beginning (red traces in what are now Figure 3A, for experiments with V-liposomes and Figure 3E for experiments with VSyt1-liposomes). To examine whether there is fusion under conditions that are more similar to those used for the trans-SNARE complex assembly assays, we performed fusion assays using VSyt1- and T-liposomes with the same protein-to-lipid ratios as in the assembly assays, but using WT SNAP-25 instead of the SNAP-25 mutant to allow fusion to occur. The results show that there is no fusion before Ca2+ addition and efficient but slow fusion upon Ca2+ addition (Figure 3—figure supplement 2); in contrast, trans-SNARE complex assembly does occur in the absence of Ca2+ and is very fast upon addition of Ca2+ (Figure 3E). These results show that transSNARE complex assembly does not necessarily lead to membrane fusion and that the FRET assay that we have developed provides a much better tool to analyze protection of trans-SNARE complexes against disassembly by NSF-αSNAP than liposome fusion assays.

7) A key question is how NSF/α-SNAP distinguish trans- and cis-SNARE complexes. The authors argue that α-SNAP cannot bind to two membranes well as in trans-SNARE complex, which prevents NSF/α-SNAP from disassembling trans-SNARE complexes. However, membrane binding by α-SNAP is not required for SNARE disassembly, as NSF/α-SNAP efficiently disassemble cytoplasmic SNARE complexes. Thus, it is possible that NSF/alpha-SNAP recognize certain SNARE conformational differences between trans- and cis-SNAREs. For example, as is previously proposed (Minglei Zhao et al., Nature, 2015), trans-SNAREs may be partially zippered. The authors are encouraged to reveal more insights into the key question by more experiments if possible.

This is an important issue but we believe that obtaining definitive data to address this question is outside the scope of this study. However, we did perform trans-SNARE complex disassembly assays with αSNAP bearing mutations that disrupt binding of its N-terminal loop to membranes (FS mutation), thus decreasing the effective activity of NSF-αSNAP (Winter et al., 2009) or that disrupt SNARE complex binding (KE mutation) (Zhao et al., 2015). Both mutations impaired the disassembly reaction (new Figures 2D, E), showing that binding of αSNAP to the membranes and to the SNAREs is important for disassembly.

8) The authors claim, in the title as well as the text, that Munc18, Munc13, synaptotagmin, and complexin act synergistically to protect trans-SNARE complexes from NSF/SNAP. However, this is not obvious based on the data presented in Figure 3C/D, where greater-than-additive effects are not observed at least for most combinations. Complexin and synaptotagmin on their own, at the concentration used (but see the next point #9), have only modest activity, which might simply reflect the ability of any binding partner to get in the way of NSF/SNAP. In the absence of calcium, there is a bit of additional protection afforded by complexin (but not by synaptotagmin) when Munc18 and Munc13 are already present.

We fully agree with this criticism. We had misunderstood the meaning of the term synergy and we do not use it in the revised manuscript.

9) The claim that complexin does not prevent disassembly of cis SNARE complexes (Figure 4) is at odds with other results in the literature. Previously, at a SNAP:complexin ratio of ~ 18:1, complexin had no effect (Pabst et al., 2000), whereas at a ratio of ~ 3:1, complexin had a substantial effect on NSF-mediated disassembly of the soluble SNARE complex (Winter et al., 2009). More recently, concentration-dependent complexin inhibition of NSF-mediated disassembly of the cis SNARE complex was observed (Choi et al., 2018). Please perform the experiments shown in Figures 3, 4, 6 at different complexin concentrations, i.e., different complexin:SNAP molar ratios.

We measured the dependence of cis-SNARE complex disassembly by NSF-αSNAP on complexin-1 concentration and αSNAP/complexin-1 ratio (Figure 6—figure supplement 1A, C). We did not see protection under any of these conditions. In the revised manuscript we suggest that differences with respect to the results of these previous papers may arise because our experiments used full-length syntaxin-1 whereas the previous results were obtained with syntaxin-1 lacking the N-terminal region containing the Habc domain; some of the previous results may also have been influenced by the lack of membranes, which renders NSF-αSNAP much less active (subsection “Disassembly of cis-SNARE complexes”, last paragraph).

We also measured the dependence of trans-SNARE complex disassembly on complexin1 concentration, which is now presented in Figure 5—figure supplement 5.

10) Figures 3 and 6 show protection of disassembly of trans SNARE complexes by a variety of factors. A key difference between experiments in these two figures relates to synaptotagmin. The C2AB fragment is used in Figure 6, whereas full-length synaptotagmin is used in Figure 6. Why? Please redo all experiments in Figure 3 with membrane anchored synaptotagmin (as in Figure 6).

We performed the experiments of Figure 3 of the old manuscript with the synaptotagmin-1 C2AB fragment so that we could measure the effects of including the fragment or not in the presence or absence of the other proteins. We have repeated these experiments without including Syb29-93 with NSF-αSNAP (now Figure 5B) and in addition performed protection experiments for trans-SNARE complexes formed between liposomes containing synaptobrevin and synaptotagmin-1 (VSyt1-liposomes) and T-liposomes (Figure 5C).

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Major comments:

1) The data make a convincing case that complexin can bind trans-SNARE complexes to block NSF/SNAP-driven disassembly. This conclusion, moreover, seems entirely plausible based on previous findings, including high-resolution structures. The role(s) of Munc18/13 on inhibiting trans SNARE complex disassembly is more problematic. It was known that, working together, they powerfully promote trans-SNARE complex assembly. The simplest model, call it Model I, is that Munc18/13 assembly activity simply opposes NSF/SNAP disassembly activity. Model I does not require that Munc18/13 interact with trans-SNARE complexes. By contrast the author's model, call it Model II, is that Munc18/13 remains associated with assembled trans-SNARE complexes. In so doing, Munc18/13 blocks NSF/SNAP from binding, and therefore disassembling, trans-SNARE complexes.

Moreover, NSF/SNAP plays a dual role: not only does it catalyze SNARE disassembly, but it also promotes Munc18/13-mediated SNARE assembly by rescuing SNAREs that are kinetically trapped in off-pathway, dead end products. No one, of course, knows this better than the authors, who indeed point out this effect in Figure 4B. Nevertheless, the dual role of NSF/SNAP further complicates the task of unambiguously distinguishing models I and II by using the author's assays.

The revised manuscript does not conclusively rule out Model I since every manipulation that strengthens the postulated 'protective' activity of Munc18/13, including the addition of Ca2+, also increases its assembly activity. Even the authors, upon occasion, seem to adopt the language of Model I. For example, "[T]he contrast of the results obtained with cis-SNARE complexes with those observed with trans-SNARE complexes provides a dramatic demonstration of how the apposition of two membranes tilts the balance in favor of SNARE complex assembly, whereas disassembly dominates on a single membrane".

What would seem to be needed for distinguishing Model I from Model II is some manipulation or mutation that separates the proposed Munc18/13's protective function from its assembly function. For example, this could be accomplished by determining if Munc18 and Munc13 are both associated with trans SNARE complexes prior to NSF/aSNAP-mediated disassembly. However, we realize that such a direct observation may not be possible with the author's assays. At the minimum, we request a more balanced discussion of these points, presenting both models I and II as being consistent with the data presented in this paper and other available data, as well as adjusting the title, Abstract and other relevant parts of the text.

We understand the overall concern about distinguishing between models I and II; this was one of the reasons why in the experiments described in our original manuscript we had used the synaptobrevin SNARE motif (Syb29-93) during our disassembly assays, as the large excess of this soluble fragment would be expected to favor its incorporation into newly formed SNARE complexes, preventing the re-assembly of trans-SNARE complexes. Following the instructions from the first round of review, we removed these data in the revised manuscript and repeated all the experiments without Syb29-93. We believe it is not worth putting all the data that we acquired with Syb29-93 back into the paper, but in the new revised manuscript we did include one experiment where transSNARE complexes were assembled in the presence of Munc18-1, Mun13-1 C1C2BMUNC2C and Ca2+, and NSF-αSNAP were added at the end together with an excess of Syb29-93 (new Figure 4—figure supplement 1). The finding that Syb29-93 does not affect the results (compare with Figure 4B) provides strong evidence that disassembly-reassembly of SNARE complex is not occurring substantially under the conditions of these experiments, since we would have observed a gradual decrease in FRET as Syb29-93 becomes incorporated into the complexes.

There are multiple other arguments that support model II against model I, as described in the paragraph that we have included in the Discussion (fourth paragraph) and is copied below. One of these arguments is that αSNAP was reported to strongly inhibit lipid mixing between liposomes by binding to trans-SNARE complexes (Park et al., 2014). Hence, fusion might be arrested if Munc18-1 and Munc13-1 do not prevent αSNAP binding. We have an extensive study of αSNAP function that supports these conclusions and we hope to submit for publication soon. We cannot cite these unpublished results in the paper because this goes against journal policy, but we do cite the data from Park et al. as one of the arguments favoring model II.

We do realize that none of the arguments that we provide is ‘full-proof’ and hence there is a small but reasonable possibility that Model I might be correct. It is too cumbersome to change the wording of the entire Results and Discussion sections to always accommodate the two models, and it might be confusing to the readers. To avoid being too conclusive and to make sure that readers understand the existence of the two possibilities, we have changed the title, the Abstract and some other key parts of the manuscript to make our wording consistent with both models. Moreover, in the new revised manuscript we have included the following warning:

‘Note however that we cannot completely rule out the possibility that, instead of physically preventing disassembly, Munc18-1 and Munc13-1 C1C2BMUNC2C mediate fast re-assembly of trans-SNARE complexes after they are disassembled. For simplicity, below we use terms like ‘prevent’ or ‘protect against disassembly’ to reflect the observation that a particular factor(s) increases the amount of assembled trans-SNARE complexes observed in the presence of NSF-αSNAP, but it is important to keep in mind both possible interpretations (see discussion).’

We still propose model II in last sentence of the Abstract for the reasons explained above and in the paragraph below, but we do not claim that this model has been demonstrated.

We also note that we have re-written some parts of the Discussion to accommodate the paragraph that discusses the merits of model II over model, which reads as follows:

‘An alternative interpretation of these results is that Munc18-1 and Munc13-1 C1C2BMUNC2C do not prevent disassembly but instead mediate fast re-assembly of trans-SNARE complexes after they are disassembled by NSF-αSNAP. […] This model can explain why Ca2+ increases the ability of Munc13-1 C1C2BMUNC2C (together with Munc18-1) to protect trans-SNARE complexes against disassembly by NSF-αSNAP (Figure 5B, C).’

2) An additional model to explain the data is as follows (call it Model III): Munc18/13 catalyze the proper assembly of the SNARE complex, and thereby, supercomplexes with complexin and synaptotagmin. Once such properly assembled supercomplexes are situated between membranes, it might be difficult for SNAP/NSF to bind to the trans-SNARE complex even if Munc13/Munc18 are not associated with such supercomplexes anymore. The absence of Munc18/Munc13 may lead to a mixture of properly and improperly assembled trans-SNARE complexes where the latter are more readily disassembled by NSF/SNAP. Please discuss this possible model as well.

We are not sure what the reviewers mean by properly and improperly assembled transSNARE complexes. There is evidence for formation of antiparallel trans-SNARE complexes, but those would not be detected by our FRET assays because our FRET probes right before the N-termini of the synaptobrevin and syntaxin-1 SNARE motifs. Since the Discussion is already very long and we are not sure of the relevance of these ideas to the data that we present, we prefer not discussing them in the paper. We hope that this is acceptable.

3) The authors performed additional experiments to test inhibition of cis-SNARE complexes by complexin (Figure 6, Figure 6—figure supplement 1, Results section "Disassembly of cis-SNARE complexes”; Discussion section, seventh paragraph), and state: "It is plausible that the differences observed with the results of Winter et al.. 2009, and Choi et al., 2018, arose because syntaxin-1 did not include the N-terminal region containing the Habc domain in both of these studies, and in the latter NSF- αSNAP might have been less active because of the absence of membranes." However, contrary to what is stated here, the Winter et al. experiments did include a membrane. The reviewers are not convinced that there is any discrepancy between the data presented in the present work and the previous work by Winter et al., and Choi et al. since the experiment shown in Figure 6—figure supplement 1 does not have the necessary time resolution to discern the effect of complexin on the kinetics of NSF/aSNAP-mediated SNARE complex disassembly observed by Winter et al. and Choi et al.

Moreover, the drawing in Figure 6—figure supplement 1 is somewhat misleading since upon injection of NSF/aSNAP, at least a minute (or more) passes before the fluorescence intensity measurements continue. Thus, this break period should be clearly marked in the figure and explained in the figure caption or the Materials and methods. The presentation and discussion of the results in the subsection “Disassembly of cis-SNARE complexes” and the Discussion, should be adjusted to present a unified view of the action of complexin on slowing the kinetics of NSF/aSNAP-mediated for both cis and trans SNARE complexes (i.e., complexin slows the kinetics in both cases to different degrees).

We apologize for not describing some of the details of these experiments and not properly illustrating in the figures the delay occurring between addition of NSF-αSNAP and the moment we re-started monitoring the donor fluorescence. In the new revised manuscript we have modified Figures 4 and 6, as well as Figure 6—figure supplement 1, to illustrate this delay. We have also indicated the existence of this delay in the figure legends, and we give more details in the Materials and methods section (subsection “FRET assays to monitor trans-SNARE complex assembly and disassembly”, second paragraph). We also made it clear in the text that we did not make any attempt to monitor the kinetics of disassembly (subsection “Multiple factors stabilize trans-SNARE complexes against disassembly by NSF-αSNAP”, first paragraph), and that our data are not inconsistent with those of Choi et al., 2018 and Winter et al., 2009 (subsection “Disassembly of cis-SNARE complexes”, last paragraph and Discussion, eighth paragraph).

https://doi.org/10.7554/eLife.38880.025

Article and author information

Author details

  1. Eric A Prinslow

    1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
    3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  2. Karolina P Stepien

    1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
    3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Formal analysis, Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Yun-Zu Pan

    1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
    3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  4. Junjie Xu

    1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
    3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  5. Josep Rizo

    1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
    3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration
    For correspondence
    Jose.Rizo-Rey@UTSouthwestern.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1773-8311

Funding

National Institute of Neurological Disorders and Stroke (R35 NS097333)

  • Josep Rizo

Welch Foundation (I-1304)

  • Josep Rizo

National Institute of General Medical Sciences (T32 GM008297)

  • Eric A Prinslow

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

Acknowledgements

We thank Minglei Zhao for providing the plasmid to express NSF, and Bradley Quade for providing purified proteins. We also thank the reviewers of this paper for their constructive criticisms, which have helped to considerably improve its quality. Cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy (Cryo-EM) Facility that is funded in part by the CPRIT Core Facility Support Award RP170644. We thank Daniel Stoddard for training and maintenance of the UTSW Cryo-EM Facility. Eric Prinslow was supported by NIH Training Grant T32 GM008297. This work was supported by grant I-1304 from the Welch Foundation (to JR) and by NIH Research Project Award R35 NS097333 (to JR).

Senior Editor

  1. Randy Schekman, Howard Hughes Medical Institute, University of California, Berkeley, United States

Reviewing Editor

  1. Axel T Brunger, Stanford University, United States

Publication history

  1. Received: June 3, 2018
  2. Accepted: January 17, 2019
  3. Accepted Manuscript published: January 18, 2019 (version 1)
  4. Version of Record published: January 30, 2019 (version 2)

Copyright

© 2019, Prinslow et al.

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

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