PI3P regulates multiple stages of membrane fusion.

The conserved catalysts of intracellular membrane fusion are Rab-family GTPases, effector complexes that bind Rabs for membrane tethering, SNARE proteins of the R, Qa, Qb, and Qc families, and SNARE chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. Yeast vacuole fusion is regulated by phosphatidylinositol-3-phosphate (PI3P). PI3P binds directly to the vacuolar Qc-SNARE and to HOPS, the vacuolar tethering/SM complex. We now report several distinct functions of PI3P in fusion. PI3P binds the N-terminal PX domain of the Qc-SNARE to enhance its engagement for fusion. Even when Qc has been preassembled with the Qa- and Qb-SNAREs, PI3P still promotes trans-SNARE assembly and fusion between these 3Q proteoliposomes and those with R-SNARE, whether with the natural HOPS tether or with a synthetic tether. With HOPS, efficient trans-SNARE complex formation needs PI3P on the 3Q-SNARE proteoliposomes, in cis to the Qc. PI3P is also needed for HOPS to confer resistance to Sec17/Sec18. With a synthetic tether, fusion is supported by PI3P on either fusion partner membrane, but this fusion is blocked by Sec17/Sec18. PI3P thus supports multiple stages of fusion: the engagement of the Qc-SNARE, trans-SNARE complex formation with preassembled Q-SNAREs, HOPS protection of SNARE complexes from Sec17/Sec18, and fusion per se after tethering and Q-SNARE assembly.

Sec18 supports membrane fusion by promoting Sec17 membrane association.

Membrane fusion is driven by Sec17, Sec18, and SNARE zippering. Sec17 bound to SNAREs promotes fusion through its membrane-proximal N-terminal apolar loop domain. At its membrane-distal end, Sec17 serves as a high-affinity receptor for Sec18. At that distance from the fusion site, it has been unclear how Sec18 can aid Sec17 to promote fusion. We now report that Sec18, with ATPγS, lowers the Km of Sec17 for fusion. A C-terminal and membrane-distal Sec17 mutation, L291A,L292A, diminishes Sec17 affinity for Sec18. High levels of wild-type Sec17 or Sec17-L291AL292A show equivalent fusion without Sec18, but Sec18 causes far less fusion enhancement with low levels of Sec17-L291AL292A than with wild-type Sec17. Another mutant, Sec17-F21SM22S, has reduced N-loop apolarity. Only very high levels of this mutant protein support fusion, but Sec18 still lowers the apparent fusion Km for Sec17-F21SM22S. Thus Sec18 stimulates fusion through Sec17 and acts at the well-described interface between Sec18 and Sec17. ATP acts as a ligand to activate Sec18 for Sec17-dependent fusion, but ATP hydrolysis is not required. Even without SNAREs, Sec18 and Sec17 exhibit interdependent stable association with lipids, with several Sec17 bound for each Sec18 hexamer, explaining how Sec18 stabilization of surface-concentrated clusters of Sec17 lowers the Sec17 Km for assembly with SNAREs. Each of the associations, between SNARE complex, Sec18, Sec17, and lipid, helps assemble the fusion machinery.

Fusion with wild-type SNARE domains is controlled by juxtamembrane domains, transmembrane anchors, and Sec17.

Membrane fusion requires tethers, SNAREs of R, Qa, Qb, and Qc families, and chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. SNAREs have N-domains, SNARE domains that zipper into 4-helical RQaQbQc coiled coils, a short juxtamembrane (Jx) domain, and (often) a C-terminal transmembrane anchor. We reconstitute fusion with purified components from yeast vacuoles, where the HOPS protein combines tethering and SM functions. The vacuolar Rab, lipids, and R-SNARE activate HOPS to bind Q-SNAREs and catalyze trans-SNARE associations. With SNAREs initially disassembled, as they are on the organelle, we now report that R- and Qa-SNAREs require their physiological juxtamembrane (Jx) regions for fusion. Swap of the Jx domain between the R- and Qa-SNAREs blocks fusion after SNARE association in trans. This block is bypassed by either Sec17, which drives fusion without requiring complete SNARE zippering, or transmembrane-anchored Qb-SNARE in complex with Qa. The abundance of the trans-SNARE complex is not the sole fusion determinant, as it is unaltered by Sec17, Jx swap, or the Qb-transmembrane anchor. The sensitivity of fusion to Jx swap in the absence of a Qb transmembrane anchor is inherent to the SNAREs, because it remains when a synthetic tether replaces HOPS.

Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS.

Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion.

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.

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

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.

A Rab prenyl membrane-anchor allows effector recognition to be regulated by guanine nucleotide.

Membrane fusion is catalyzed by conserved proteins R, Qa, Qb, and Qc SNAREs, which form tetrameric RQaQbQc complexes between membranes; SNARE chaperones of the SM, Sec17/αSNAP, and Sec18/NSF families; Rab-GTPases (Rabs); and Rab effectors. Rabs are anchored to membranes by C-terminal prenyl groups, but can also function when anchored by an apolar polypeptide. Rabs are regulated by GTPase-activating proteins (GAPs), activating the hydrolysis of bound GTP. We have reconstituted fusion with pure components from yeast vacuoles including SNAREs, the HOPS (homotypic fusion and vacuole protein sorting) tethering and SNARE-assembly complex, and the Rab Ypt7, bound to membranes by either C-terminal prenyl groups (Ypt7-pr) or a recombinant transmembrane anchor (Ypt7-tm). We now report that HOPS-dependent fusion occurs with Ypt7 anchored by either means, but only Ypt7-pr requires GTP for activation and is inactive either with bound GDP or without bound guanine nucleotide. In contrast, Ypt7-tm is constitutively active for HOPS-dependent fusion, independent of bound guanine nucleotide. Fusion inhibition by the GAP Gyp1-46 is not limited to Ypt7-tm with bound GTP, indicating that this GAP has an additional mode of regulating fusion. Phosphorylation of HOPS by the vacuolar kinase Yck3 renders fusion strictly dependent on GTP-activated Ypt7, whether bound to membranes by prenyl or transmembrane anchor. The binding of GTP or GDP constitutes a selective switch for Ypt7, but with Ypt7-tm, this switch is only read by HOPS after phosphorylation to P-HOPS by its physiological kinase Yck3. The prenyl anchor of Ypt7 allows both HOPS and P-HOPS to be regulated by Ypt7-bound guanine nucleotide.

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

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

Tethering guides fusion-competent trans-SNARE assembly.

R-SNAREs (soluble N-ethylmaleimide-sensitive factor receptor), Q-SNAREs, and Sec1/Munc18 (SM)-family proteins are essential for membrane fusion in exocytic and endocytic trafficking. The yeast vacuolar tethering/SM complex HOPS (homotypic fusion and vacuole protein sorting) increases the fusion of membranes bearing R-SNARE to those with 3Q-SNAREs far more than it enhances their trans-SNARE pairings. We now report that the fusion of these proteoliposomes is also supported by GST-PX or GST-FYVE, recombinant dimeric proteins which tether by binding the phosphoinositides in both membranes. GST-PX is purely a tether, as it supports fusion without SNARE recognition. GST-PX tethering supports the assembly of new, active SNARE complexes rather than enhancing the function of the fusion-inactive SNARE complexes which had spontaneously formed in the absence of a tether. When SNAREs are more disassembled, as by Sec17, Sec18, and ATP (adenosine triphosphate), HOPS is required, and GST-PX does not suffice. We propose a working model where tethering orients SNARE domains for parallel, active assembly.

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

At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.

Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18.

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.

Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion.

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. DOI: http://dx.doi.org/10.7554/eLife.01879.001.

Fusion proteins and select lipids cooperate as membrane receptors for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Vam7p.

Vam7p, the vacuolar soluble Qc-SNARE, is essential for yeast vacuole fusion. The large tethering complex, homotypic fusion and vacuole protein sorting complex (HOPS), and phosphoinositides, which interact with the Vam7p PX domain, have each been proposed to serve as its membrane receptors. Studies with the isolated organelle cannot determine whether these receptor elements suffice and whether ligands or mutations act directly or indirectly on Vam7p binding to the membrane. Using pure components that are active in reconstituted vacuolar fusion, we now find that Vam7p binds to membranes through its combined affinities for several vacuolar membrane constituents: HOPS, phosphatidylinositol 3-phosphate, SNAREs, and acidic phospholipids. Acidic lipids allow low concentrations of Vam7p to suffice for fusion; without acidic lipids, the block to fusion is partially bypassed by high concentrations of Vam7p.

Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion.

The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle-Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)-this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.

Phosphoinositides function asymmetrically for membrane fusion, promoting tethering and 3Q-SNARE subcomplex assembly.

Phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are essential for rapid SNARE-dependent fusion of yeast vacuoles and other organelles. These phosphoinositides also regulate the fusion of reconstituted proteoliposomes. The reconstituted reaction allows separate analysis of phosphoinositide-responsive subreactions: fusion with SNAREs alone, with the addition of the HOPS tethering factor, and with the further addition of the SNARE complex disassembly chaperones Sec17p and Sec18p. Using assays of membrane tethering, trans-SNARE pairing, and lipid mixing, we found that PI(3)P and PI(4,5)P(2) have distinct functions that are asymmetric with respect to R-SNARE (Nyv1p) and the 3Q-SNAREs (Vam3p, Vti1p, and Vam7p). Fusion reactions with the Q-SNAREs and R-SNARE on separate membranes showed that PI(3)P has two distinct functions. PI(3)P on Q-SNARE proteoliposomes promoted Vam7p binding and association with the other two Q-SNAREs. PI(3)P on R-SNARE proteoliposomes was recognized by the PX domain of Vam7p on Q-SNARE proteoliposomes to promote tethering, although this function could be supplanted by the tethering activity of HOPS. PI(4,5)P(2) stimulated fusion when it was on R-SNARE proteoliposomes, apposed to Q-SNARE proteoliposomes bearing PI(3)P. These functions are essential for the phosphoinositide-dependent synergy between HOPS and Sec17p/Sec18p in promoting rapid fusion.

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

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.

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components.

Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.

Phosphoinositides and SNARE chaperones synergistically assemble and remodel SNARE complexes for membrane fusion.

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)P(2)]. 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)P(2) can activate SNARE chaperones for the recruitment of Vam7p into fusion-competent SNARE complexes; and either PI(3)P or PI(4,5)P(2) 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.

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones.

The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.

HOPS proofreads the trans-SNARE complex for yeast vacuole fusion.

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.