Membrane fusion requires Rab-family GTPases and SNARE proteins. SNAREs constitute four families, termed R, Qa, Qb, and Qc (Fasshauer et al., 1998). Each of them has an N-domain, an α-helical SNARE domain of 50–60 aminoacyl residues with heptad-repeat apolar residues, and often a C-terminal membrane anchor. Each SNARE α-helical turn is termed a ‘layer.’ The central ‘0-layer’ of each fully-assembled SNARE complex has inwardly-oriented arginyl (for R-SNAREs) or glutaminyl (for Qa, Qb, and Qc SNAREs) residues, forming a polar center to the otherwise hydrophobic core of the 4-helical SNARE bundle (Sutton et al., 1998). The SNARE domain layers are numbered from the 0-layer, in the positive direction toward the SNARE C-termini and in the negative direction toward the N-domains. Prior to 4-SNARE assembly, individual SNARE domains are random coil (Fasshauer et al., 1997; Hazzard et al., 1999). Sec1/Munc18 (SM) family proteins catalyze the N- to C-directional assembly of SNAREs anchored to each tethered membrane (Fiebig et al., 1999; Sørensen et al., 2006; Baker et al., 2015; Orr et al., 2017; Jiao et al., 2018). Each SNARE domain transitions from random coil to α-helix as the heptad-repeat apolar amino acyl residues become sequestered into the interior of the coiled coils (Fasshauer et al., 1997; Sutton et al., 1998). This hydrophobic collapse relies on the exclusion of water and is the driving force for SNARE assembly (Sørensen et al., 2006). Completion of SNARE zippering can release up to 40 kBT per SNARE complex (Gao et al., 2012; Min et al., 2013; Zhang, 2017) to overcome the 40–90 kBT hydration barrier for membrane stalk formation, the dominant energy barrier for fusion (Aeffner et al., 2012). Upon fusion, the trans-SNARE complex becomes a cis-complex, anchored to the fused membrane bilayer. Sec17 (αSNAP) and SNAREs are receptors for the Sec18 (NSF) AAA ATPase (Clary et al., 1990; Winter et al., 2009; Zick et al., 2015). Sec18 uses the energy from ATP binding and hydrolysis to disassemble SNAREs for further fusion cycles (Söllner et al., 1993; Ungermann et al., 1998; Zhao et al., 2015) and to disassemble dead-end SNARE complexes (Xu et al., 2010; Lai et al., 2017; Choi et al., 2018; Song and Wickner, 2019; Jun and Wickner, 2019).

The molecular interactions between Sec18/NSF, Sec17/αSNAP, and neuronal SNAREs were illuminated by determination of their structures when assembled without membrane anchors into the NSF/αSNAP/SNARE complex, also referred to as the 20S particle (Zhao et al., 2015; White et al., 2018). The heart of these structures is the 4-helical bundle of the R, Qa, Qb, and Qc SNARE domains. Between two and four Sec17/αSNAP form a right-handed assembly surrounding the left-handed superhelical coiled coils of the SNARE complex. In this structure, the N-terminal apolar loop of each αSNAP is poised to enter a lipid bilayer adjacent to the SNARE transmembrane (TM) domains.

Yeast vacuole fusion, a model of non-neuronal fusion, has been studied in vivo (Wada et al., 1992), in vitro with the isolated organelle (Wickner, 2010), and in a reconstituted proteoliposome-based reaction with purified components (Mima et al., 2008; Stroupe et al., 2009; Zick and Wickner, 2016). Each protein implicated by the in vivo genetics is required for the reconstitution: the Rab Ypt7, the R-SNARE Nyv1, and Q-SNAREs Vam3, Vti1, and Vam7 (hereafter referred to as R, Qa, Qb, and Qc), and a large hexameric protein termed HOPS (homotypic fusion and vacuole protein sorting) with multiple direct affinities. Two HOPS subunits bind Ypt7, anchored on each membrane (Brett et al., 2008), to mediate tethering (Hickey and Wickner, 2010). A third HOPS subunit is Vps33, the vacuolar Sec1/Munc18 (SM) protein, with direct capacity to bind R and Qa SNARE domains, in parallel and in register (Baker et al., 2015). HOPS also has direct affinity for the Qb and Qc SNAREs (Stroupe et al., 2006; Song et al., 2020) and for vacuolar lipids (Orr et al., 2015). Some functions of HOPS correspond to fusion factors in other systems; neuronal Munc13 cooperates with Munc18 in SNARE assembly (Richmond et al., 2001; Ma et al., 2011; Lai et al., 2017). Munc18 corresponds to the HOPS subunit Vps33, but the protein that mediates the Munc13 function for vacuole fusion is unclear. The association of HOPS with Ypt7 and vacuolar lipids allosterically activates HOPS to catalyze SNARE assembly (Torng et al., 2020). When the SNAREs are initially in 4-SNARE complexes on two apposed membranes, fusion requires Sec17, Sec18, and ATP to disassemble these cis-SNARE complexes and liberate the SNAREs for assembly into trans-complexes (Mayer et al., 1996; Nichols et al., 1997; Zick et al., 2015).

Though early studies have suggested that disassembly of cis-SNARE complexes might be the sole function of Sec17 and Sec18 (Mayer et al., 1996), recent findings have broadened our understanding of their roles. While SNAREs are a core fusion machine (Weber et al., 1998), a complete fusion machine (Mima et al., 2008; Zick and Wickner, 2016) also requires the Rab, its tethering effector, an SM-family protein, and the NSF/Sec18 and αSNAP/Sec17 SNARE chaperones. In this more complete context, Sec17 and Sec18 also contribute to fusion per se: (1) trans-SNARE complexes, which form in a Ypt7-dependent manner between vacuoles, bear Sec17 in comparable abundance to the SNAREs (Xu et al., 2010). (2) Fusion between proteoliposomes has been reconstituted with purified components. When tethering is by non-specific agents, fusion is inhibited by Sec17, Sec18, and ATP (Mima et al., 2008; Song et al., 2017). However, Sec17, Sec18, and ATP stimulate fusion with HOPS (Mima et al., 2008; Zick et al., 2015; Song et al., 2017). (3) A pioneering study by Schwartz and Merz, 2009, showed that the Qc3Δ deletion of several heptads at the C-terminus of the Qc SNARE blocks the fusion of isolated yeast vacuoles, but this block is overcome by the addition of Sec17. Qc3Δ, ending at the SNARE domain layer +3 and thus lacking layers +4 to +8, blocks vacuole fusion in vivo as well, and overexpression of Sec17 partially restores cellular vacuole morphology (Schwartz et al., 2017). Reconstituted in vitro fusion with limiting Sec17 concentrations, where Sec17 will not restore fusion, also requires Sec18 (Schwartz et al., 2017). It has been unclear whether the Sec17 bypass of this deletion of the C-terminal Qc region is particular to just this one SNARE or is general for any Q-SNARE, and whether Sec17 simply contributes its SNARE-binding energy to the energy of 3-SNARE zippering or whether it drives fusion by other means. (4) Fusion reactions with initially separate SNAREs can require Sec17, and this fusion is stimulated by Sec18 without ATP hydrolysis (Zick et al., 2015; Song et al., 2017). Sec17 alone stimulates the fusion of reconstituted proteoliposomes with wild-type SNAREs, and the degree of stimulation is a function of the lipid headgroup and fatty acyl composition (Zick et al., 2015). An intermediate in fusion accumulates during HOPS-dependent fusion without Sec17, allowing a sudden burst of fusion upon Sec17 addition (Zick et al., 2015). Single-molecule pulling studies have also revealed that αSNAP stabilizes SNARE complexes (Ma et al., 2016, but see Ryu et al., 2015). However, the mechanism by which HOPS-dependent fusion is stimulated by Sec17/Sec18 has been unclear.

Complete SNARE zippering is considered essential for SNARE-dependent fusion (Sørensen et al., 2006). We now report that Sec17 has a second mode of promoting fusion, which can compensate for incomplete zippering. Fusion with SNAREs and HOPS is completely arrested when several heptad repeats in the C-terminal region of the SNARE core complex are removed from any one of the three Q-SNAREs. With any such C-terminally truncated Q-SNARE domain, or even with C-terminal truncations to both Qb and Qc, blocked fusion is restored by Sec17 and Sec18 without ATP hydrolysis. Association between the C-terminal heptads of the R and the single remaining full-length Qa-SNARE, each anchored to one of the docked membranes, would not yield the same assembly energy as with wild-type SNAREs (Sutton et al., 1998) and would contribute far less force toward the bilayer rearrangements of fusion. The N-terminal apolar loop of Sec17 is particularly important for this function of Sec17, and it may stabilize SNARE bundles or trigger fusion by insertion into lipid bilayers. Zippering-driven fusion is also arrested with full-length SNARE domains when the apolar, inward-facing residues of the Qa SNARE layers +4 to +8 are replaced by Ala, Ser, or Gly, but in each case fusion is restored by Sec17, Sec18, and non-hydrolyzable ATPγS. Strikingly, even fusion that is blocked by the concurrent replacement of apolar residues from the +4 to +8 layers of Qa and the deletion of the +4 to +8 layers of both Qb and Qc, removing all capacity for hydrophobic collapse between the +4 and +8 layers of the R and Q SNAREs, is fully restored by Sec17 and Sec18. We propose that Sec17 either creates a favorable folding environment for the assembly of the remaining full-length SNARE domains or directly promotes bilayer remodeling through insertion of the apolar loops of several SNARE-bound Sec17s or acts by a combination of these two mechanisms.

Vacuole SNAREs (Figure 1A) have an N-domain and a SNARE domain. Several of them have a TM anchor, but Qc lacks a hydrophobic membrane anchor, and instead associates with membranes through its affinities for the other SNAREs, for HOPS (Stroupe et al., 2006), and for phosphatidylinositol 3-phosphate through its N-terminal PX domain (Cheever et al., 2001). SNARE domain layers are numbered from the central 0-layer, as shown. Fusion requires that R- and at least one Q-SNARE be anchored to docked membranes (Song and Wickner, 2017). When they are, soluble forms of the other Q-SNAREs without membrane anchors, termed sQ (Figure 1A), will support Ypt7/HOPS-dependent fusion. Vacuolar SNAREs with C-terminal truncations, corresponding to partial zippering, form stable complexes (Figure 1—figure supplement 1), supporting their use in fusion studies. Based on the single-particle cryo-EM (Electron Microscope) structure of the homologous human neuronal NSF/αSNAP/SNARE complex (Zhao et al., 2015), we modeled the associations of Sec17 (αSNAP) (Figure 1B) and Sec18 (NSF) with vacuolar SNAREs, viewed in profile or in an end-on view from the membrane (Figure 1C). In this model, we assume that four Sec17 monomers (Figure 1B) assemble together, surrounding the 4-SNARE coiled coil (Figure 1C). In contrast, only two αSNAP molecules have been observed in EM structures of the NSF/αSNAP/V7-SNARE complex (Zhao et al., 2015) and the NSF/αSNAP/SNARE complex that included the linker between the two SNAP-25 SNARE domains (White et al., 2018). The presence of the SNAP-25 linker in these two complexes may interfere with the binding of the other two αSNAP molecules. Since the vacuolar SNARE complex does not contain a linker between SNARE domains, it is reasonable to postulate that four Sec17 molecules bind to the vacuolar SNARE complex, but the precise number of Sec17 molecules is yet to be determined for the Sec18/Sec17/vacuolar SNARE complex. Rapid fusion needs Sec17 and Sec18 in addition to HOPS and SNAREs (Song et al., 2017). To understand how they work together, we exploited direct assays of SNARE associations to show that incompletely zippered SNAREs can associate stably and that HOPS allows Sec17 to promote the completion of zippering and SNARE complex stability. We then examined their functional relationships to show that SNARE-bound Sec17 and Sec18 can promote rapid fusion when energy from zippering is greatly reduced or lost.

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Sec17 alters SNARE complex conformation

The capacity of vacuolar SNAREs to form stable partially zippered structures raised the question of whether these SNAREs can zipper efficiently, especially when anchored to membranes or associated with other fusion proteins such as HOPS. To study the kinetics of SNARE interactions, we employed an ensemble fluorescence resonance energy transfer (FRET) assay of vacuolar SNARE associations (Torng et al., 2020). The Qc-SNARE was prepared with a unique cysteinyl residue in any of three positions (Figure 2A), either the native Cys208, which is upstream (U) of the SNARE domain or, after substitution of serine for this cysteine, with Met250Cys at the N-terminal end of the SNARE domain or with Ala316Cys at the C-terminal end of the SNARE domain. Each was derivatized with Oregon Green 488. Fusion proteins were also prepared with a unique cysteine either immediately N-terminal, or C-terminal, to the Qb SNARE domain, and each was derivatized with Alexa Fluor 568. As a model for exploring the effects of HOPS and Sec17 on SNARE dynamics, these fluorescent proteins were co-incubated with proteoliposomes bearing Ypt7, R, and Qa. cis-SNARE complex assembly can occur spontaneously on these proteoliposomes, but assembly is stimulated by HOPS, allowing direct comparison of HOPS-dependent and HOPS-independent SNARE assembly (Torng et al., 2020). The average FRET efficiency in these studies is modest because they include, in bulk reactions, all the fluorescent Qb and Qc, many of which do not enter SNARE complexes. SNARE complex assembly with HOPS gave a high average FRET efficiency when fluorophores were at the N-terminus of the Qb SNARE domain and at or near the N-terminal end of the Qc SNARE domain (Figure 2B, red and yellow curves). There was a lower FRET efficiency when the fluorophores were at opposite ends of the Qc and Qb SNARE domains (Figure 2B; blue, green, and indigo). When both fluorophores were at the C-terminal ends of the Qb and Qc SNARE domains, a low signal was seen (Figure 2B, purple), similar to the average FRET efficiency when fluorophores were at opposite ends of the SNARE domains, thus suggesting incomplete zippering. After 1 hr, Sec17 was added to each reaction. Strikingly, Sec17 only enhanced the average FRET efficiency between C-terminal fluorophores, rapidly rising to the level seen when the fluorophores were together at the N-terminii of the SNARE domains (Figure 2B, purple; Figure 2—figure supplement 1), indicating a Sec17-induced change at the C-terminal end of the SNARE complex. Sec17/αSNAP may promote the zippering of isolated SNARE domains (Ma et al., 2016) but is now seen to act in the context of membranes and HOPS.

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Generality of Sec17 and Sec18 bypass of arrested zippering

Sec17 can restore fusion when Qc has truncations at the C-terminal end of its SNARE domain (Schwartz and Merz, 2009), stimulated by Sec18 (Schwartz et al., 2017). We asked whether Sec17 can restore fusion with similar deletions of residues after the +3 layer in the other Q-SNAREs. Proteoliposomes bearing Ypt7 and R-SNARE were assayed for fusion with proteoliposomes bearing this Rab and any two anchored Q-SNAREs. Proteoliposome mixtures were incubated with HOPS, and the soluble form of the remaining Q-SNARE was deprived of its membrane anchor and a C-terminal portion of its SNARE domain (Figure 3; A, Qc3Δ; B, sQb3Δ; C, sQa3Δ). As reported by Schwartz et al., 2017, there was no fusion with Qc3Δ unless 600 nM Sec17, 300 nM Sec18, and ATP or ATPγS were present (Figure 3A); these concentrations are in the physiological concentration ranges of Sec17 (150–1100 nM) and Sec18 (250–760 nM) (Ho et al., 2018). While ATP and its non-hydrolyzable analog ATPγS support comparable fusion with wild-type SNAREs (Song et al., 2017), hydrolyzable ATP inhibits fusion through Sec17/Sec18-mediated disassembly of SNARE complexes when defective SNAREs such as Qc3Δ are present, a proofreading function. The same pattern was seen for fusion with sQb3Δ (Figure 3B) and sQa3Δ (Figure 3C). The unique spatial disposition of the Sec17/αSNAP molecules with respect to each SNARE (Figure 1C and Zhao et al., 2015) makes it unlikely that Sec17 could somehow fill each of the gaps left by each of these deletions to shield apolar residues within the SNARE bundle and thereby continue to drive zippering, or that Sec17 binding could induce the remaining R and two Q +4 to +8 layers to somehow rotate to form a hydrophobic two- or three-layered core.

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Fusion assays were also performed with Ypt7/R proteoliposomes and each of the three Ypt7/single-anchored Q-SNARE proteoliposomes in the presence of HOPS and the other two soluble Q-SNAREs (Figure 4). With membrane-anchored Qa and with sQb and Qc having complete SNARE domains, there was HOPS-dependent fusion without further addition (Figure 4A, black line), though Sec17 and Sec18 with AMP-PNP, ATPγS, or ATP did stimulate (compare black curves, A–D). Deletion of the +4 to +8 layers from either soluble Qb or Qc abolished fusion (Figure 4A), which was restored by Sec17 and Sec18 with either AMP-PNP, ATPγS, or ATP (B–D, red and blue curves). There was no fusion when both soluble Q-SNAREs had truncated SNARE domains (Figure 4A, orange), but, strikingly, the fusion was partially restored by Sec17 and Sec18 with ATP (Figure 4D, orange) and more fully restored when the adenine nucleotide was resistant to hydrolysis (Figure 4, B and C, orange). With two Q-SNAREs lacking the C-terminal portion of their SNARE domains, the apolar amino acyl residues of the remaining two SNAREs would not be as effectively shielded from water if they continued zippering together. Fusion could not be restored by Sec17 and Sec18 if either soluble SNARE was omitted instead of truncated (green and purple). This fusion with sQb3Δ and Qc3Δ occurs in stages, initially sensitive to antibody to either the HOPS SM subunit Vps33 or Sec18, then acquiring resistance to Vps33 antibody while remaining sensitive to the Sec18 ligand (Figure 4—figure supplement 2). When only the Qb-SNARE was membrane anchored, there was little fusion without Sec17 and Sec18 (Figure 4E, black curve). When the SNARE domain of sQa or Qc had been truncated, fusion was strictly dependent on non-hydrolyzable ATP analogs, and little fusion was seen with dual SNARE domain truncation. Similar patterns were seen with anchored Qc (Figure 4, I–L). We term the fusion induced by Sec17 and Sec18 in the presence of 3Δ SNARE domain truncations ‘zippering bypass fusion’.

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To explore the capacity of Sec17 and Sec18 to compensate for partial SNARE zippering, we assayed the fusion of Ypt7/R and Ypt7/Qa proteoliposomes with sQb3Δ, Qc3Δ, and HOPS, using various concentrations of wild-type or mutant Sec17, and with or without Sec18 and ATPγS (Figure 5). Without Sec17, fusion is not supported by Sec18 (A and B, blue curves). Limited fusion is possible with 1 or 2 μM wild-type Sec17 alone (A, black and red), but Sec18 allows faster fusion and with less Sec17 (A and B; tan). Fusion requires the apolar loop near the N-terminus of Sec17, as the F21S,M22S mutation (FSMS) blocks fusion entirely (C and D). The K159E,K163E mutation (KEKE) diminishes Sec17:SNARE association (Marz et al., 2003). The KEKE mutation prevents fusion without Sec18 (E), but a slow and limited fusion with KEKE-Sec17 is restored by Sec18 (F). The C-terminal L291A,L292A mutation of Sec17 (LALA), which interferes with its cooperation with Sec18 for SNARE complex disassembly (Barnard et al., 1997; Schwartz and Merz, 2009; Zick et al., 2015), diminishes zippering bypass fusion (A vs. G, red curves), and a limited fusion is restored through the addition of Sec18 (H). These data suggest that Sec17 action directly requires its apolar loop domain, since the loss of this apolar region is not bypassed by Sec18. Sec18 may stimulate fusion by modulating the conformation of Sec17 associations with trans-SNARE complexes, but Sec18 is not simply promoting Sec17 binding, since it is still needed for fusion when Sec17 is joined to an integral N-terminal membrane anchor (Figure 5—figure supplement 2). Basic residues in the +3 to +8 layers near the C-terminus of the R and Qa SNARE domains are near acidic residues on the interior of the Sec17 assembly (Figure 5I). To determine whether Sec17 might rely on these ionic interactions to support fusion, we converted these acidic residues of Sec17 to alanine or serine, creating the mutant Sec17-E34S,E35S,D38S,E73A,D74A,E75A (termed Sec17 6 Acidic to Neutral or Sec17-6AtoN), but this mutant Sec17 still supports fusion (Figure 5, J and K). Interestingly, mutation of acidic residues of αSNAP near the C-terminal end of the neuronal SNARE complex also only had a modest effect on disassembly activity of NSF/αSNAP (Zhao et al., 2015).

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Fusion despite triply-crippled SNARE zippering

Since SNARE zippering is driven by the sequestration of apolar residues into the interior of the 4-SNARE bundle, we examined the effect of converting the apolar residues of the Qa SNARE domain +4 to +8 layers to Ala, Ser, or Gly. Fusion between Ypt7/R and Ypt7/Qa proteoliposomes in the presence of HOPS, sQb, and Qc, but without Sec17 or Sec18 (Figure 6A, black curve), was diminished by replacing each of the +4 to +8 layer apolar residues of Qa with Ala (Figure 6A, green curve) and was abolished when they were replaced by Ser (red curve) or by Gly (blue curve). The persistence of some fusion with the Ala substitutions may reflect that two of the residues were already Ala, that Ala has the greatest propensity among the amino acids to form α-helices, Gly the least, and Ser is in-between (Pace and Scholtz, 1998), and that alanine itself is the least hydrophilic of these three amino acids. When these same incubations were performed with Sec17, Sec18, and ATPγS, rapid and comparable fusion was seen in each case (Figure 6B). When hydrolyzable ATP was used instead of ATPγS, there was little effect on the fusion kinetics with wild-type SNARE domain sequences (Figure 6, B vs. C, black curves). In contrast, hydrolyzable ATP led to fusion inhibition when SNARE packing stability was reduced by substitution of apolar residues by Ala, Ser, or Gly (Figure 6C). Though the apolar residues are not required for fusion aided by Sec17 and Sec18 (Figure 6B), they apparently stabilize the SNAREs against ATP-driven proofreading disassembly (Figure 6C). To triply weaken the completion of zippering, the same proteoliposomes with wild-type Qa or the Qa with +4 to +8 layers having small side-chain residues instead of apolar residues were incubated with HOPS, sQb3Δ, and Qc3Δ, either without Sec17 or Sec18 (Figure 6D) or with Sec17, Sec18, and ATPγS (Figure 6E) or ATP (Figure 6F). Fusion was optimally supported by Sec17, Sec18, and ATPγS (Figure 6E). The independence of this fusion from energy derived by zippering is underscored by the similar fusion rates in all incubations with Sec17, Sec18, and ATPγS, whether with apolar or polar Qa +4 to +8 residues or with full-length or +4to +8-truncated sQb and Qc (Figure 6, B vs. E). With the Qb and Qc SNARE domains truncated, and the Qa lacking apolar inward-facing amino acyl side chains, little or no energy could be gained from the completion of R and mutant-Qa zippering. Thus, Sec17 acts in three ways: triggering a zippering-dependent SNARE conformational change in the presence of HOPS and full-length SNARE domains (Figure 2), acting with Sec18 to promote fusion independent of energy from zippering (Figure 6), and supporting the disassembly of post-fusion cis-SNARE complexes by Sec18.

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The catalytic roles of fusion proteins have been gleaned from functional reconstitution studies. These studies initially showed that the zippering of concentrated SNAREs can drive slow fusion (Weber et al., 1998; Fukuda et al., 2000). As proteoliposome SNARE levels were lowered toward physiological levels, reconstituted vacuolar and neuronal fusion reactions required additional factors (Zick and Wickner, 2016; Stepien and Rizo, 2021). In addition to SNAREs, reconstituted neuronal fusion requires Munc18, Munc13, calcium, NSF, and αSNAP (Ma et al., 2013; Lai et al., 2017) while reconstituted vacuole fusion needs HOPS (Stroupe et al., 2006), the Rab Ypt7 (Stroupe et al., 2009; Ohya et al., 2009), and specific lipid head-group composition and fatty acyl fluidity (Zick and Wickner, 2016). HOPS and other SM proteins catalyze SNARE assembly (Baker et al., 2015; Orr et al., 2017; Jiao et al., 2018), regulated by an activated Rab (Zick and Wickner, 2016; Torng et al., 2020), and may confer resistance to Sec17/αSNAP- and Sec18/NSF-mediated trans-SNARE disassembly (Xu et al., 2010; Jun and Wickner, 2019). Sec17/αSNAP and Sec18/NSF stimulate fusion with HOPS and wild-type SNAREs (Mima et al., 2008; Song et al., 2017).

Fusion can be supported by either complete 4-SNARE zippering without Sec17 or Sec18, or by Sec17 and Sec18 association with only partially zippered SNAREs, but the most rapid fusion requires both (Song et al., 2017). While Sec17 and Sec18 are known to bypass the fusion blockade by Qc C-terminal truncation alone (Schwartz and Merz, 2009; Schwartz et al., 2017), the generality of this bypass with respect to any one Q-SNARE or even two Q-SNAREs (Figures 3 and 4) shows that it is not specific to Qc alone. Moreover, when zippering with full-length SNARE domains is weakened by the substitution of small amino acyl residues for large apolar residues in the +4 to +8 layers of Qa, Sec17 and Sec18 will also restore fusion (Figure 6A,B). Strikingly, Sec17 and Sec18 drive fusion despite a triple blockade to complete zippering, namely the absence of two C-terminal Q-SNARE domains and the lack of apolarity in the third (Figure 6E). In the NSF/αSNAP/SNARE complex, the 4-SNAREs wrap around each other in a left-handed super helix, and the Sec17s wrap around them in a right-handed fashion, yet they form a specific structure (Zhao et al., 2015; White et al., 2018). It seems unlikely that residues from one or more Sec17 could substitute for the missing residues when two heptads are removed from the C-termini of one or even two Q-SNAREs and the bulky apolar residues are removed from the third.

From the earliest reconstitutions of HOPS-dependent fusion (Mima et al., 2008) and in subsequent studies (e.g., Zick et al., 2015), Sec17 and Sec18 gave strong stimulation. In contrast, Sec17 and Sec18 inhibit SNARE-only fusion, or fusion with non-physiological tethers (Mima et al., 2008; Zick and Wickner, 2014; Schwartz et al., 2017; Song and Wickner, 2019). HOPS not only binds each SNARE, but also has direct affinity for Sec17 (Figure 2—figure supplement 2). In model studies with cis-SNARE complexes, HOPS is necessary for Sec17 to enhance zippering per se (Figure 2C). HOPS:Sec17 binding may underlie their synergy for fusion, but further studies are needed to test this idea.

Earlier studies and our current work suggest a working model of vacuole membrane fusion, encompassing findings here and elsewhere (Figure 7). HOPS exploits the affinity of its Vps39 and Vps41 subunits for the Rab Ypt7 (Brett et al., 2008) on each fusion partner membrane (Figure 7A) to mediate (Step 1) tethering (Hickey and Wickner, 2010). Tethered membranes (Figure 7B) are a prerequisite for SNARE assembly in an active conformation, likely a common N to C SNARE domain orientation (Song and Wickner, 2019). HOPS has direct affinity for each of the four vacuolar SNAREs (Song et al., 2020) and is allosterically activated by vacuolar lipids and Ypt7:GTP (Torng et al., 2020) as a catalyst of SNARE assembly (Step 2). SNAREs begin to zipper (Figure 7C) from the N- toward the C-terminal end of their SNARE domain. When any one Q-SNARE is omitted, fusion intermediates assemble, which undergo very rapid fusion when the missing Q-SNARE is supplied (Song et al., 2020), suggesting that fusion without Sec17/Sec18 is rate-limited by the completion of SNARE zippering and/or the spontaneous release of bulky HOPS. Sec17 has direct affinity for SNAREs (Söllner et al., 1993), HOPS (Figure 2—figure supplement 2), lipids (Clary et al., 1990; Zick et al., 2015), and Sec18 (Söllner et al., 1993). Sec17 displaces HOPS from the SNAREs (Figure 7, Step 3), as shown by earlier studies: (1) vacuolar SNAREs are found in complex with Sec17 or HOPS, but not with both, and Sec17 can displace HOPS from SNAREs (Collins et al., 2005); (2) when vacuolar HOPS-dependent reconstituted fusion is arrested by Qc3Δ, HOPS is bound to the incompletely zippered SNARE complex until it is displaced by Sec17 (Schwartz et al., 2017); and (3) trans-SNARE complexes, which assemble between isolated vacuoles, are largely associated with Sec17 (Xu et al., 2010). The Sec17 association with partially zippered trans-SNARE complex (Figure 7D) promotes a conformational change (Figure 2) leading to complete zippering. The Sec17: 4-SNARE complex will bind Sec18 (Step 4) through its affinities for both SNAREs (Zick et al., 2015) and for Sec17. Sec18 regulates, in some unknown fashion, the Sec17/αSNAP assembly into an oligomeric structure surrounding the SNARE complex intermediate (Zhao et al., 2015), a trans-anchored Sec18/Sec17/SNARE pre-fusion complex (Figure 7E). Some ATP hydrolysis-dependent disassembly can occur, which may represent proofreading of incorrect and unstable trans-SNARE complexes (Choi et al., 2018). Sec17 oligomerization may also be stabilized or guided by the insertion of its N-terminal loop into the membranes. While SNAREs can slowly complete zippering and support fusion without Sec17 or Sec18 (Mima et al., 2008; Song et al., 2017), and slow fusion can occur with Sec17 and Sec18 when sQb and Qc lack the C-terminal portion of their zippering domain (Figure 4D), optimal fusion requires four complete SNARE domains, Sec17/αSNAP, and Sec18/NSF. The energy for fusion (Step 5) derives from multiple sources: the completion of SNARE zippering, the binding energies which create the Sec17 structure surrounding the SNAREs, and the energy of bilayer distortion through Sec17 apolar loop insertion. Cis-SNARE complexes (Figure 7F) formed by fusion (Söllner et al., 1993; Mayer et al., 1996) are disassembled by Sec17, Sec18, and ATP (Step 6), freeing SNAREs from each other (Figure 7A) for later assembly in trans. Each of these components – the four SNAREs, the Rab Ypt7, HOPS, Sec17, and Sec18 – is part of this ordered pathway of associations and disassembly, constituting a holoenzyme for vacuole membrane fusion.

Figure 7

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Our current studies reveal a general capacity of Sec17/αSNAP and Sec18/NSF to support fusion, even when little or no energy would be derived from completion of zippering. Sec17 provides a cage-like environment (Chakraborty et al., 2010), albeit with side fenestrations (Schwartz et al., 2017), which may favor SNARE zippering or, where zippering is blocked, allow the remaining SNARE domains to attain positions and conformations that approximate zippering. The apolar N-domains of the four Sec17s are clearly essential for fusion (Figure 5 and Figure 5—figure supplement 2), whether through positioning the Sec17s, facilitating their assembly, or directly inserting as a ‘membrane wedge’ and thereby contributing to bilayer disruption at the fusion site. The continued need for this apolar loop when Sec17 is integrally membrane-anchored (Figure 5—figure supplement 2) shows that it does not simply contribute to Sec17 membrane association. Elements of ‘Sec17 cage’ and ‘membrane wedge’ action are not mutually exclusive. Further work will be needed to determine the relative energies of Sec17 and Sec18 binding to the assembling 4-SNARE trans-complex, each of the four Sec17s inserting its apolar loops into the membrane, Sec17 forming lateral associations with other Sec17 molecules in the cage around the SNAREs, and complete SNARE zippering, as each of these helps to achieve the bilayer distortions of fusion.

Our current studies further our understanding of how Sec17(αSNAP), Sec18 (NSF), and SNAREs cooperate to promote membrane fusion. Sec17 has multiple functions: (a) It supports Sec18 association with SNARE complexes for their ATP-driven disassembly for subsequent rounds of fusion (Ungermann et al., 1998; Choi et al., 2018). Sec17/αSNAP and Sec18/NSF are also part of a quality control system that, for vacuoles, includes HOPS (Starai et al., 2008) and, in the neuronal system, includes Munc18 and Munc13 (Ma et al., 2013; Lai et al., 2017). These disassembly reactions require ATP hydrolysis and can be interrupted by the Sec17 LALA mutation. (b) Sec17 promotes a conformational change that leads to complete SNARE zippering. While neuronal SNAREs, which are properly assembled (i.e., involving the Munc18/Munc13 pathway), will fully zipper with a high energy yield, this may not be true for all non-neuronal SNAREs. (c) Sec17 promotes fusion even when SNARE zippering is incomplete. Sec17 uses the partially zippered SNAREs as a platform to bind to the fusion site and its apolar N-domain loop to trigger fusion. Sec18 also has multiple functions: (a) Sec18/NSF drives ATP-driven SNARE disassembly (Zhao et al., 2015), blocked by the Sec17 LALA mutation. (b) Sec18 supports Sec17 for direct promotion of fusion, built on a platform of partially zippered SNAREs. Note that the loading of SNARE N-terminal residues into the pore of the D1 ring of NSF does not require hydrolysis (White et al., 2018), so initial assembly of the NSF/αSNAP/SNARE complex occurs in the absence of hydrolysis. This does not need ATP hydrolysis and is insensitive to the LALA mutation. Thirdly, SNAREs also have multiple functions: (a) They can completely zipper, thereby stressing the bilayer and triggering fusion (Weber et al., 1998; Sutton et al., 1998). As previously reported (Mima et al., 2008; Song et al., 2017), HOPS alone, without Sec17 or Sec18, will support slow SNARE-dependent fusion. (b) SNAREs support the assembly of a microdomain in which multiple fusion proteins and lipids become highly enriched (Fratti et al., 2004). (c) As shown here, SNAREs form an essential platform for Sec17 and Sec18 to contribute to fusion independently of completion of zippering. With physiological levels of wild-type vacuolar SNAREs, rapid fusion requires Rab-activated HOPS, Sec17, Sec18, and ATP.

While intracellular fusion reactions share many requirements, such as for SNAREs and SM protein, there are major differences as well. Synaptic vesicle fusion and other calcium-dependent secretion systems require calcium, synaptotagmin, Munc13, and complexin, none of which have their obvious counterparts in calcium-independent systems. Vacuole and endosome fusion have their SM protein as an integral subunit of the tethering complex, which is not seen in other organelles. The similarities and differences in fusion pathways at each organelle will be clarified as each is more thoroughly studied.

PI3P was from Echelon (Salt Lake City, Utah), ergosterol from Sigma (St. Louis, MO), fluorescent lipids from Thermo Fisher (Waltham, MA), and other lipids were from Avanti (Alabaster, AL). Biobeads SM2 were from BioRad, Cy5-Streptavidin from SeraCare (Milford, MA), biotinylated phycoerythrin from Invitrogen (Eugene, OR), and underivatized streptavidin from Thermo Fisher. Spectrapor six dialysis tubing (7.5 mm diameter, 25 kDa cutoff) was from Spectrum Labs (Las Vegas, NV). Octyl-b-D-glucopyranoside was purchased from Anatrace (Maumee, OH).

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

1. 1. Zhao M
   2. Wu S
   3. Cheng Y
   4. Brunger AT

   (2015) RCSB Protein Data Bank

   ID 3J96. Structure of 20S supercomplex determined by single particle cryoelectron microscopy (State I).

   https://www.rcsb.org/structure/3j96
2. 1. Rice LM
   2. Brunger AT

   (1999) RCSB Protein Data Bank

   ID 1QQE. CRYSTAL STRUCTURE OF THE VESICULAR TRANSPORT PROTEIN SEC17.

   https://www.rcsb.org/structure/1QQE

1. 1. Aeffner S
   2. Reusch T
   3. Weinhausen B
   4. Salditt T

   (2012) Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

   PNAS 109:E1609–E1618.

   https://doi.org/10.1073/pnas.1119442109

   • PubMed
   • Google Scholar
2. 1. Baker RW
   2. Jeffrey PD
   3. Zick M
   4. Phillips BP
   5. Wickner WT
   6. Hughson FM

   (2015) A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly

   Science 349:1111–1114.

   https://doi.org/10.1126/science.aac7906

   • PubMed
   • Google Scholar
3. 1. Barnard RJ
   2. Morgan A
   3. Burgoyne RD

   (1997) Stimulation of NSF ATPase activity by alpha-SNAP is required for SNARE complex disassembly and exocytosis

   Journal of Cell Biology 139:875–883.

   https://doi.org/10.1083/jcb.139.4.875

   • PubMed
   • Google Scholar
4. 1. Brett CL
   2. Plemel RL
   3. Lobingier BT
   4. Lobinger BT
   5. Vignali M
   6. Fields S
   7. Merz AJ

   (2008) Efficient termination of vacuolar rab GTPase signaling requires coordinated action by a GAP and a protein kinase

   Journal of Cell Biology 182:1141–1151.

   https://doi.org/10.1083/jcb.200801001

   • PubMed
   • Google Scholar
5. 1. Chakraborty K
   2. Chatila M
   3. Sinha J
   4. Shi Q
   5. Poschner BC
   6. Sikor M
   7. Jiang G
   8. Lamb DC
   9. Hartl FU
   10. Hayer-Hartl M

   (2010) Chaperonin-catalyzed rescue of kinetically trapped states in protein folding

   Cell 142:112–122.

   https://doi.org/10.1016/j.cell.2010.05.027

   • PubMed
   • Google Scholar
6. 1. Cheever ML
   2. Sato TK
   3. de Beer T
   4. Kutateladze TG
   5. Emr SD
   6. Overduin M

   (2001) Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes

   Nature Cell Biology 3:613–618.

   https://doi.org/10.1038/35083000

   • PubMed
   • Google Scholar
7. 1. Choi UB
   2. Zhao M
   3. White KI
   4. Pfuetzner RA
   5. Esquivies L
   6. Zhou Q
   7. Brunger AT

   (2018) NSF-mediated disassembly of on- and off-pathway SNARE complexes and inhibition by complexin

   eLife 7:e36497.

   https://doi.org/10.7554/eLife.36497

   • PubMed
   • Google Scholar
8. 1. Clary DO
   2. Griff IC
   3. Rothman JE

   (1990) SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast

   Cell 61:709–721.

   https://doi.org/10.1016/0092-8674(90)90482-T

   • Google Scholar
9. 1. Collins KM
   2. Thorngren NL
   3. Fratti RA
   4. Wickner WT

   (2005) Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion

   The EMBO Journal 24:1775–1786.

   https://doi.org/10.1038/sj.emboj.7600658

   • PubMed
   • Google Scholar
10. 1. Fasshauer D
    2. Bruns D
    3. Shen B
    4. Jahn R
    5. Brünger AT

    (1997) A structural change occurs upon binding of syntaxin to SNAP-25

    Journal of Biological Chemistry 272:4582–4590.

    https://doi.org/10.1074/jbc.272.7.4582

    • PubMed
    • Google Scholar
11. 1. Fasshauer D
    2. Sutton RB
    3. Brunger AT
    4. Jahn R

    (1998) Conserved structural features of the synaptic fusion complex: snare proteins reclassified as Q- and R-SNAREs

    PNAS 95:15781–15786.

    https://doi.org/10.1073/pnas.95.26.15781

    • PubMed
    • Google Scholar
12. 1. Fiebig KM
    2. Rice LM
    3. Pollock E
    4. Brunger AT

    (1999) Folding intermediates of SNARE complex assembly

    Nature Structural Biology 6:117–123.

    https://doi.org/10.1038/5803

    • PubMed
    • Google Scholar
13. 1. Fratti RA
    2. Jun Y
    3. Merz AJ
    4. Margolis N
    5. Wickner W

    (2004) Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

    Journal of Cell Biology 167:1087–1098.

    https://doi.org/10.1083/jcb.200409068

    • PubMed
    • Google Scholar
14. 1. Fukuda R
    2. McNew JA
    3. Weber T
    4. Parlati F
    5. Engel T
    6. Nickel W
    7. Rothman JE
    8. Söllner TH

    (2000) Functional architecture of an intracellular membrane t-SNARE

    Nature 407:198–202.

    https://doi.org/10.1038/35025084

    • PubMed
    • Google Scholar
15. 1. Gao Y
    2. Zorman S
    3. Gundersen G
    4. Xi Z
    5. Ma L
    6. Sirinakis G
    7. Rothman JE
    8. Zhang Y

    (2012) Single reconstituted neuronal SNARE complexes zipper in three distinct stages

    Science 337:1340–1343.

    https://doi.org/10.1126/science.1224492

    • PubMed
    • Google Scholar
16. 1. Haas A
    2. Wickner W

    (1996) Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF)

    The EMBO Journal 15:3296–3305.

    https://doi.org/10.1002/j.1460-2075.1996.tb00694.x

    • PubMed
    • Google Scholar
17. 1. Hazzard J
    2. Südhof TC
    3. Rizo J

    (1999) NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin

    Journal of Biomolecular NMR 14:203–207.

    https://doi.org/10.1023/a:1008382027065

    • PubMed
    • Google Scholar
18. 1. Hickey CM
    2. Wickner W

    (2010) HOPS initiates vacuole docking by tethering membranes before trans-SNARE complex assembly

    Molecular Biology of the Cell 21:2297–2305.

    https://doi.org/10.1091/mbc.e10-01-0044

    • PubMed
    • Google Scholar
19. 1. Ho B
    2. Baryshnikova A
    3. Brown GW

    (2018) Unification of protein abundance datasets yields a quantitative Saccharomyces cerevisiae proteome

    Cell Systems 6:192–205.

    https://doi.org/10.1016/j.cels.2017.12.004

    • PubMed
    • Google Scholar
20. 1. Izawa R
    2. Onoue T
    3. Furukawa N
    4. Mima J

    (2012) Distinct contributions of vacuolar qabc- and R-SNARE proteins to membrane fusion specificity

    Journal of Biological Chemistry 287:3445–3453.

    https://doi.org/10.1074/jbc.M111.307439

    • PubMed
    • Google Scholar
21. 1. Jiao J
    2. He M
    3. Port SA
    4. Baker RW
    5. Xu Y
    6. Qu H
    7. Xiong Y
    8. Wang Y
    9. Jin H
    10. Eisemann TJ
    11. Hughson FM
    12. Zhang Y

    (2018) Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

    eLife 7:e41771.

    https://doi.org/10.7554/eLife.41771

    • PubMed
    • Google Scholar
22. 1. Jun Y
    2. Wickner W

    (2019) Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

    PNAS 116:23573–23581.

    https://doi.org/10.1073/pnas.1913985116

    • PubMed
    • Google Scholar
23. 1. Lai Y
    2. Choi UB
    3. Leitz J
    4. Rhee HJ
    5. Lee C
    6. Altas B
    7. Zhao M
    8. Pfuetzner RA
    9. Wang AL
    10. Brose N
    11. Rhee J
    12. Brunger AT

    (2017) Molecular mechanisms of synaptic vesicle priming by Munc13 and Munc18

    Neuron 95:591–607.

    https://doi.org/10.1016/j.neuron.2017.07.004

    • PubMed
    • Google Scholar
24. 1. Ma C
    2. Li W
    3. Xu Y
    4. Rizo J

    (2011) Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex

    Nature Structural & Molecular Biology 18:542–549.

    https://doi.org/10.1038/nsmb.2047

    • PubMed
    • Google Scholar
25. 1. Ma C
    2. Su L
    3. Seven AB
    4. Xu Y
    5. Rizo J

    (2013) Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release

    Science 339:421–425.

    https://doi.org/10.1126/science.1230473

    • Google Scholar
26. 1. Ma L
    2. Kang Y
    3. Jiao J
    4. Rebane AA
    5. Cha HK
    6. Xi Z
    7. Qu H
    8. Zhang Y

    (2016) α-SNAP enhances SNARE zippering by stabilizing the SNARE Four-Helix bundle

    Cell Reports 15:531–539.

    https://doi.org/10.1016/j.celrep.2016.03.050

    • PubMed
    • Google Scholar
27. 1. Marz KE
    2. Lauer JM
    3. Hanson PI

    (2003) Defining the SNARE complex binding surface of alpha-SNAP: implications for SNARE complex disassembly

    The Journal of Biological Chemistry 278:27000–27008.

    https://doi.org/10.1074/jbc.M302003200

    • PubMed
    • Google Scholar
28. 1. Mayer A
    2. Wickner W
    3. Haas A

    (1996) Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles

    Cell 85:83–94.

    https://doi.org/10.1016/S0092-8674(00)81084-3

    • PubMed
    • Google Scholar
29. 1. Mima J
    2. Hickey CM
    3. Xu H
    4. Jun Y
    5. Wickner W

    (2008) Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones

    The EMBO Journal 27:2031–2042.

    https://doi.org/10.1038/emboj.2008.139

    • PubMed
    • Google Scholar
30. 1. Min D
    2. Kim K
    3. Hyeon C
    4. Hoon Cho Y
    5. Shin Y-K
    6. Yoon T-Y

    (2013) Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism

    Nature Communications 4:ncomms2692.

    https://doi.org/10.1038/ncomms2692

    • Google Scholar
31. 1. Nichols BJ
    2. Ungermann C
    3. Pelham HR
    4. Wickner WT
    5. Haas A

    (1997) Homotypic vacuolar fusion mediated by t- and v-SNAREs

    Nature 387:199–202.

    https://doi.org/10.1038/387199a0

    • PubMed
    • Google Scholar
32. 1. Ohya T
    2. Miaczynska M
    3. Coskun U
    4. Lommer B
    5. Runge A
    6. Drechsel D
    7. Kalaidzidis Y
    8. Zerial M

    (2009) Reconstitution of rab- and SNARE-dependent membrane fusion by synthetic endosomes

    Nature 459:1091–1097.

    https://doi.org/10.1038/nature08107

    • PubMed
    • Google Scholar
33. 1. Orr A
    2. Wickner W
    3. Rusin SF
    4. Kettenbach AN
    5. Zick M

    (2015) Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and rab:gtp for membrane binding and to catalyze tethering and fusion

    Molecular Biology of the Cell 26:305–315.

    https://doi.org/10.1091/mbc.E14-08-1298

    • PubMed
    • Google Scholar
34. 1. Orr A
    2. Song H
    3. Rusin SF
    4. Kettenbach AN
    5. Wickner W

    (2017) HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex

    Molecular Biology of the Cell 28:975–983.

    https://doi.org/10.1091/mbc.e16-10-0743

    • PubMed
    • Google Scholar
35. 1. Pace CN
    2. Scholtz JM

    (1998) A Helix propensity scale based on experimental studies of peptides and proteins

    Biophysical Journal 75:422–427.

    https://doi.org/10.1016/S0006-3495(98)77529-0

    • PubMed
    • Google Scholar
36. 1. Rice LM
    2. Brunger AT

    (1999) Crystal structure of the vesicular transport protein Sec17

    Molecular Cell 4:85–95.

    https://doi.org/10.1016/S1097-2765(00)80190-2

    • Google Scholar
37. 1. Richmond JE
    2. Weimer RM
    3. Jorgensen EM

    (2001) An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming

    Nature 412:338–341.

    https://doi.org/10.1038/35085583

    • PubMed
    • Google Scholar
38. 1. Ryu JK
    2. Min D
    3. Rah SH
    4. Kim SJ
    5. Park Y
    6. Kim H
    7. Hyeon C
    8. Kim HM
    9. Jahn R
    10. Yoon TY

    (2015) Spring-loaded unraveling of a single SNARE complex by NSF in one round of ATP turnover

    Science 347:1485–1489.

    https://doi.org/10.1126/science.aaa5267

    • PubMed
    • Google Scholar
39. 1. Schwartz ML
    2. Nickerson DP
    3. Lobingier BT
    4. Plemel RL
    5. Duan M
    6. Angers CG
    7. Zick M
    8. Merz AJ

    (2017) Sec17 (α-SNAP) and an SM-tethering complex regulate the outcome of SNARE zippering in vitro and in vivo

    eLife 6:e27396.

    https://doi.org/10.7554/eLife.27396

    • PubMed
    • Google Scholar
40. 1. Schwartz ML
    2. Merz AJ

    (2009) Capture and release of partially zipped trans-SNARE complexes on intact organelles

    Journal of Cell Biology 185:535–549.

    https://doi.org/10.1083/jcb.200811082

    • PubMed
    • Google Scholar
41. 1. Seals DF
    2. Eitzen G
    3. Margolis N
    4. Wickner WT
    5. Price A

    (2000) A ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

    PNAS 97:9402–9407.

    https://doi.org/10.1073/pnas.97.17.9402

    • PubMed
    • Google Scholar
42. 1. Sheffield P
    2. Garrard S
    3. Derewenda Z

    (1999) Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors

    Protein Expression and Purification 15:34–39.

    https://doi.org/10.1006/prep.1998.1003

    • PubMed
    • Google Scholar
43. 1. Söllner T
    2. Bennett MK
    3. Whiteheart SW
    4. Scheller RH
    5. Rothman JE

    (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion

    Cell 75:409–418.

    https://doi.org/10.1016/0092-8674(93)90376-2

    • PubMed
    • Google Scholar
44. 1. Song H
    2. Orr A
    3. Duan M
    4. Merz AJ
    5. Wickner W

    (2017) Sec17/Sec18 act twice, enhancing membrane fusion and then disassembling cis-SNARE complexes

    eLife 6:e26646.

    https://doi.org/10.7554/eLife.26646

    • PubMed
    • Google Scholar
45. 1. Song H
    2. Orr AS
    3. Lee M
    4. Harner ME
    5. Wickner WT

    (2020) HOPS recognizes each SNARE, assembling ternary trans-complexes for rapid fusion upon engagement with the 4th SNARE

    eLife 9:e53559.

    https://doi.org/10.7554/eLife.53559

    • PubMed
    • Google Scholar
46. 1. Song H
    2. Wickner W

    (2017) A short region upstream of the yeast vacuolar Qa-SNARE heptad-repeats promotes membrane fusion through enhanced SNARE complex assembly

    Molecular Biology of the Cell 28:2282–2289.

    https://doi.org/10.1091/mbc.e17-04-0218

    • PubMed
    • Google Scholar
47. 1. Song H
    2. Wickner W

    (2019) Tethering guides fusion-competent trans-SNARE assembly

    PNAS 116:13952–13957.

    https://doi.org/10.1073/pnas.1907640116

    • PubMed
    • Google Scholar
48. 1. Sørensen JB
    2. Wiederhold K
    3. Müller EM
    4. Milosevic I
    5. Nagy G
    6. de Groot BL
    7. Grubmüller H
    8. Fasshauer D

    (2006) Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles

    The EMBO Journal 25:955–966.

    https://doi.org/10.1038/sj.emboj.7601003

    • PubMed
    • Google Scholar
49. 1. Starai VJ
    2. Hickey CM
    3. Wickner W

    (2008) HOPS proofreads the trans-SNARE complex for yeast vacuole fusion

    Molecular Biology of the Cell 19:2500–2508.

    https://doi.org/10.1091/mbc.e08-01-0077

    • PubMed
    • Google Scholar
50. 1. Stepien KP
    2. Rizo J

    (2021) Synaptotagmin-1-, Munc18-1-, and Munc13-1-dependent liposome fusion with a few neuronal SNAREs

    PNAS 118:e2019314118.

    https://doi.org/10.1073/pnas.2019314118

    • PubMed
    • Google Scholar
51. 1. Stroupe C
    2. Collins KM
    3. Fratti RA
    4. Wickner W

    (2006) Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p

    The EMBO Journal 25:1579–1589.

    https://doi.org/10.1038/sj.emboj.7601051

    • PubMed
    • Google Scholar
52. 1. Stroupe C
    2. Hickey CM
    3. Mima J
    4. Burfeind AS
    5. Wickner W

    (2009) Minimal membrane docking requirements revealed by reconstitution of rab GTPase-dependent membrane fusion from purified components

    PNAS 106:17626–17633.

    https://doi.org/10.1073/pnas.0903801106

    • PubMed
    • Google Scholar
53. 1. Sutton RB
    2. Fasshauer D
    3. Jahn R
    4. Brunger AT

    (1998) Crystal structure of a SNARE complex involved in Synaptic exocytosis at 2.4 A resolution

    Nature 395:347–353.

    https://doi.org/10.1038/26412

    • PubMed
    • Google Scholar
54. 1. Torng T
    2. Song H
    3. Wickner W

    (2020) Asymmetric rab activation of vacuolar HOPS to catalyze SNARE complex assembly

    Molecular Biology of the Cell 31:1060–1068.

    https://doi.org/10.1091/mbc.E20-01-0019

    • PubMed
    • Google Scholar
55. 1. Ungermann C
    2. Nichols BJ
    3. Pelham HR
    4. Wickner W

    (1998) A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion

    Journal of Cell Biology 140:61–69.

    https://doi.org/10.1083/jcb.140.1.61

    • PubMed
    • Google Scholar
56. 1. Wada Y
    2. Ohsumi Y
    3. Anraku Y

    (1992) Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. I. isolation and characterization of two classes of vam mutants

    Journal of Biological Chemistry 267:18665–18670.

    https://doi.org/10.1016/S0021-9258(19)37012-7

    • PubMed
    • Google Scholar
57. 1. Webb B
    2. Sali A

    (2016) Comparative protein structure modeling using MODELLER

    Current Protocols in Bioinformatics 54:139–148.

    https://doi.org/10.1002/cpbi.3

    • Google Scholar
58. 1. Weber T
    2. Zemelman BV
    3. McNew JA
    4. Westermann B
    5. Gmachl M
    6. Parlati F
    7. Söllner TH
    8. Rothman JE

    (1998) SNAREpins: minimal machinery for membrane fusion

    Cell 92:759–772.

    https://doi.org/10.1016/S0092-8674(00)81404-X

    • PubMed
    • Google Scholar
59. 1. White KI
    2. Zhao M
    3. Choi UB
    4. Pfuetzner RA
    5. Brunger AT

    (2018) Structural principles of SNARE complex recognition by the AAA+ protein NSF

    eLife 7:e38888.

    https://doi.org/10.7554/eLife.38888

    • PubMed
    • Google Scholar
60. 1. Wickner W

    (2010) Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a rab, all dancing in a ring on yeast vacuoles

    Annual Review of Cell and Developmental Biology 26:115–136.

    https://doi.org/10.1146/annurev-cellbio-100109-104131

    • Google Scholar
61. 1. Winter U
    2. Chen X
    3. Fasshauer D

    (2009) A conserved membrane attachment site in alpha-SNAP facilitates N-ethylmaleimide-sensitive factor (NSF)-driven SNARE complex disassembly

    Journal of Biological Chemistry 284:31817–31826.

    https://doi.org/10.1074/jbc.M109.045286

    • PubMed
    • Google Scholar
62. 1. Xu H
    2. Jun Y
    3. Thompson J
    4. Yates J
    5. Wickner W

    (2010) HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion

    The EMBO Journal 29:1948–1960.

    https://doi.org/10.1038/emboj.2010.97

    • PubMed
    • Google Scholar
63. 1. Xu Z
    2. Wickner W

    (1996) Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae

    Journal of Cell Biology 132:787–794.

    https://doi.org/10.1083/jcb.132.5.787

    • PubMed
    • Google Scholar
64. 1. Zhang Y

    (2017) Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers

    Protein Science 26:1252–1265.

    https://doi.org/10.1002/pro.3116

    • PubMed
    • Google Scholar
65. 1. Zhao M
    2. Wu S
    3. Zhou Q
    4. Vivona S
    5. Cipriano DJ
    6. Cheng Y
    7. Brunger AT

    (2015) Mechanistic insights into the recycling machine of the SNARE complex

    Nature 518:61–67.

    https://doi.org/10.1038/nature14148

    • Google Scholar
66. 1. Zick M
    2. Orr A
    3. Schwartz ML
    4. Merz AJ
    5. Wickner WT

    (2015) Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

    PNAS 112:E2290–E2297.

    https://doi.org/10.1073/pnas.1506409112

    • PubMed
    • Google Scholar
67. 1. Zick M
    2. Wickner W

    (2013) The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes

    Molecular Biology of the Cell 24:3746–3753.

    https://doi.org/10.1091/mbc.e13-07-0419

    • PubMed
    • Google Scholar
68. 1. Zick M
    2. Wickner WT

    (2014) A distinct tethering step is vital for vacuole membrane fusion

    eLife 3:e03251.

    https://doi.org/10.7554/eLife.03251

    • PubMed
    • Google Scholar
69. 1. Zick M
    2. Wickner W

    (2016) Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric rab(GTP) requirement

    Molecular Biology of the Cell 27:2590–2597.

    https://doi.org/10.1091/mbc.e16-04-0230

    • PubMed
    • Google Scholar
70. 1. Zucchi PC
    2. Zick M

    (2011) Membrane fusion catalyzed by a rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis

    Molecular Biology of the Cell 22:4635–4646.

    https://doi.org/10.1091/mbc.e11-08-0680

    • PubMed
    • Google Scholar

Author details

1. Hongki Song

   Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, Hanover, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   Additional information

   HS and TLT are co-first authors of this work

   "This ORCID iD identifies the author of this article:" 0000-0002-3761-5434

2. Thomas L Torng

   Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, Hanover, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   Additional information

   HS and TLT are co-first authors of this work

   "This ORCID iD identifies the author of this article:" 0000-0003-2295-2777

3. Amy S Orr

   Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, Hanover, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0266-0390

4. Axel T Brunger

   Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology Stanford University, Stanford, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Visualization, Project administration, Writing - review and editing

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-5121-2036

5. William T Wickner

   Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth, Hanover, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   William.T.Wickner@dartmouth.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8431-0468

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