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Fusion of intracellular transport vesicles requires soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18-family (SM) proteins. Membrane-bridging SNARE complexes are critical for fusion, but their spontaneous assembly is inefficient and may require SM proteins in vivo. We report x-ray structures of Vps33, the SM subunit of the yeast homotypic fusion and vacuole protein–sorting (HOPS) complex, bound to two individual SNAREs. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent complex assembly. Vps33 and potentially other SM proteins could thus act as templates for generating partially zipped SNARE assembly intermediates. HOPS was essential to mediate SNARE complex assembly at physiological SNARE concentrations. Thus, Vps33 appears to catalyze SNARE complex assembly through specific SNARE motif recognition.

Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex. Cells of higher organisms contain compartments called organelles and structures called vesicles that transfer molecules and proteins between these organelles. Each organelle and each vesicle is enclosed within a membrane, and these membranes must fuse together to allow these transfers to take place. A certain group of proteins, called SNAREs, have a central role in these fusion events. Since membrane fusion is difficult to observe directly, many researchers have used a method called ‘fluorescent lipid dequenching’ to study it indirectly. In this approach, one fraction of vesicles is labeled with two fluorescent molecules, with one of these molecules quenching the fluorescence of the other. However, when a labeled vesicle fuses with an unlabeled vesicle, the surface concentrations of the fluorescent molecules are diluted. This reduces the amount of quenching and the resulting increase in fluorescence can be measured. Experiments utilizing this technique had suggested that SNARE proteins are sufficient for fusion to take place, and that no other protein complexes need to be present. However, when a different assay method called ‘lumenal compartment mixing’ was used, little fusion was seen when the only proteins present were the SNAREs. The lumenal compartment mixing approach relies on measuring the degree of mixing between the contents of two vesicles. To address these conflicting results, Zick and Wickner used both methods to study fusion in a yeast-based system. The lumenal compartment mixing approach, which is the more reliable method, revealed that rapid and efficient membrane fusion in fact requires another protein complex, called HOPS, to hold the two membrane vesicles together. Zick and Wickner found that the HOPS complex does not enable fusion by just increasing the amount of interactions between the SNARE proteins. Rather, it seems to facilitate the formation of a particular quality of SNARE interactions. Future work is needed to work out how the SNARE complexes become ‘fusion-competent’, and to explore the mechanism that allows the HOPS complex to assist in the formation of fusion-competent SNARE complexes.

Yeast vacuole fusion requires R-SNARE, Q-SNAREs, and HOPS. A HOPS SM-family subunit binds the R- and Qa-SNAREs. We now report that HOPS binds each of the four SNAREs. HOPS catalyzes fusion when the Q-SNAREs are not pre-assembled, ushering them into a functional complex. Co-incubation of HOPS, proteoliposomes bearing R-SNARE, and proteoliposomes with any two Q-SNAREs yields a rapid-fusion complex with 3 SNAREs in a trans-assembly. The missing Q-SNARE then induces sudden fusion. HOPS can ‘template’ SNARE complex assembly through SM recognition of R- and Qa-SNAREs. Though the Qa-SNARE is essential for spontaneous SNARE assembly, HOPS also assembles a rapid-fusion complex between R- and QbQc-SNARE proteoliposomes in the absence of Qa-SNARE, awaiting Qa for fusion. HOPS-dependent fusion is saturable at low concentrations of each Q-SNARE, showing binding site functionality. HOPS thus tethers membranes and recognizes each SNARE, assembling R+Qa or R+QbQc rapid fusion intermediates.

Sec17 [soluble N -ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis -SNARE complexes, liberating SNAREs for subsequent assembly of trans -complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans -SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans -SNARE complex but triggered sudden fusion of trans -SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17 ᴸ²⁹¹ᴬ,ᴸ²⁹²ᴬ did not stimulate Sec18 to disassemble cis -SNARE complex but triggered the fusion of trans -SNARE paired membranes. Sec17 F²¹S,ᴹ²²S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans -SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17 F²¹S,ᴹ²²S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans -SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion. Significance Intracellular membrane trafficking relies on SNARE proteins from apposed membranes to form trans -complexes. Sec18 ( N -ethylmaleimide–sensitive factor; NSF) and its cochaperone Sec17 (soluble NSF attachment protein; α-SNAP) disassemble cis -SNARE complexes, liberating SNAREs for trans -complex assembly. We now describe an additional function of Sec17, its ability to trigger the fusion of trans -SNARE paired membranes. We propose a model in which Sec17 oligomerizes on trans -SNARE complexes, inserting apolar loops into the adjacent membranes. This precisely localized membrane interaction may disturb the lipid bilayer, lowering the energy barrier that prevents the two membranes from merging, and thereby facilitate fusion.

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

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. All cells are enclosed with a membrane that is made of phospholipid molecules, and many of the structures found inside cells—such as the vacuoles in plant and fungal cells—are also enclosed with a phospholipid membrane. To form a membrane, the phospholipid molecules—which have a phosphate head and two fatty acid tails—arrange themselves in two layers, with the fatty acid tails pointing into the membrane, and the phosphate heads pointing outwards. This structure is known as a phospholipid bilayer. Vacuoles are filled with water that contains various proteins and molecules in solution, and adjust their volume to keep the concentrations of substances in the cell in balance. To do this, the vacuoles fuse with each other. This fusion process requires dramatic spatial rearrangements of the phospholipid molecules. The SNARE family of proteins plays a key role in membrane fusion. As the two membranes come together, SNARE proteins located on each membrane form a complex known as a trans-SNARE complex. This docks the vacuole in place beside another vacuole while the phospholipid molecules in the two membranes rearrange. However, much less is known about the phospholipid molecules that are involved in the fusion process. Now, Zick et al. have shown that three types of phospholipid molecules must be present for membrane fusion to be completed. These have in common that their phosphate ‘headgroups’ are small and they do not tend to form bilayers. The vacuoles can dock beside each other if these small headgroup phospholipid molecules are not present, but the bilayer lipids in the vacuole membranes cannot rearrange themselves in the absence of these particular lipids. The importance of these nonbilayer lipid molecules had not previously been established, as the majority of experiments investigating membrane fusion used concentrations of SNARE proteins that were much higher than those found physiologically. At such high concentrations, fusion can go ahead without the nonbilayer lipid molecules being present.

SNARE‐dependent membrane fusion requires the disassembly of cis ‐SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans ‐SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans ‐SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans ‐SNARE complex, like cis ‐SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans ‐, but not in the cis ‐, configuration. This selective HOPS preservation of trans ‐SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X‐100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans ‐SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic.

Yeast vacuole fusion requires 4 SNAREs, 2 SNARE chaperone systems (Sec17p/Sec18p/ATP and the HOPS complex), and 2 phosphoinositides, phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. By reconstituting proteoliposomal fusion with purified components, we now show that phosphoinositides have 4 distinct roles: PI(3)P is recognized by the PX domain of the SNARE Vam7p; PI(3)P enhances the capacity of membrane-bound SNAREs to drive fusion in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ can activate SNARE chaperones for the recruitment of Vam7p into fusion-competent SNARE complexes; and either PI(3)P or PI(4,5)P₂ strikingly promotes synergistic SNARE complex remodeling by Sec17p/Sec18p/ATP and HOPS. This ternary synergy of phosphoinositides and 2 SNARE chaperone systems is required for rapid fusion.

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.