Membrane fusion reactions limited by defective SNARE zippering or stiff lipid fatty acyl composition have distinct requirements for Sec17, Sec18, and adenine nucleotide.

Intracellular membrane fusion is catalyzed by SNAREs, Rab GTPases, SM proteins, tethers, Sec18/NSF and Sec17/SNAP. Membrane fusion has been reconstituted with purified vacuolar proteins and lipids to address 3 salient questions: whether ATP hydrolysis by Sec18 affects its promotion of fusion, whether fusion promotion by Sec17 and Sec18 is only seen with mutant SNAREs or can also be seen with wild-type SNAREs, and whether Sec17 and Sec18 only promote fusion when they work together or whether they can each work separately. Fusion is driven by two engines, completion of SNARE zippering (which does not need Sec17/Sec18) and Sec17/Sec18-mediated fusion (needing SNAREs but not the energy from their complete zippering).

After their membrane assembly, Sec18 (NSF) and Sec17 (SNAP) promote membrane fusion.

The energy that drives membrane fusion can come from either complete SNARE zippering, from Sec17 and Sec18, or both. Sec17 and Sec18 initially form a complex which binds membranes. Sec17, Sec18, and the apolarity of a loop on the N-domain of Sec17 are required for their interdependent membrane association.

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts.

SNARE proteins drive membrane fusion as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/ α-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains.

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17.

MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion.

Yeast vacuolar HOPS tethers membranes, catalyzes -SNARE assembly between R- and Q-SNAREs, and shepherds SNAREs past early inhibition by Sec17. After partial SNARE zippering, fusion is driven slowly by either completion of SNARE zippering or by Sec17/Sec18, but rapid fusion needs zippering and Sec17/Sec18. Using reconstituted-vacuolar fusion, we find that MARCKS Effector Domain (MED) peptide, a lipid ligand, blocks fusion reversibly at a late reaction stage.

Efficient fusion requires a membrane anchor on the vacuolar Qa-SNARE.

As a prelude to fusion, the R-SNARE on one membrane zippers with Qa-, Qb-, and Qc-SNAREs from its apposed fusion partner, forming a four-helical bundle that draws the two membranes together. Because Qa- and Qb-SNAREs are anchored to the same membrane and are adjacent in the 4-SNARE bundle, their two anchors might be redundant. Using the recombinant pure protein catalysts of yeast vacuole fusion, we now report that the specific distribution of transmembrane (TM) anchors on the Q-SNAREs is critical for efficient fusion.

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.

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.

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.

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 -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.

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.

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.

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.

Asymmetric Rab activation of vacuolar HOPS to catalyze SNARE complex assembly.

Intracellular membrane fusion requires Rab-family GTPases, their effector tethers, soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, and SNARE chaperones of the Sec1/Munc18 (SM), Sec17/α-SNAP, and Sec18/NSF families. We have developed an assay using fluorescence resonance energy transfer to measure SNARE complex formation in real time. We now show that yeast vacuolar SNAREs assemble spontaneously into RQaQbQc complexes when the R- and Qa-SNAREs are concentrated in the same micelles or in on the same membrane.

HOPS recognizes each SNARE, assembling ternary -complexes for rapid fusion upon engagement with the 4th SNARE.

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.

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.

Tethering guides fusion-competent -SNARE assembly.

R-SNAREs (soluble -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 -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.

Assembly of intermediates for rapid membrane fusion.

Membrane fusion is essential for intracellular protein sorting, cell growth, hormone secretion, and neurotransmission. Rapid membrane fusion requires tethering and Sec1-Munc18 (SM) function to catalyze R-, Qa-, Qb-, and Qc-SNARE complex assembly in , as well as SNARE engagement by the SNARE-binding chaperone Sec17/αSNAP. The hexameric vacuolar HOPS (motypic fusion and vacuole rotein orting) complex in the yeast tethers membranes through its affinities for the membrane Rab GTPase Ypt7.

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

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

Whereas SNARE (soluble -ethylmaleimide-sensitive factor attachment protein receptor) heptad-repeats are well studied, SNAREs also have upstream N-domains of indeterminate function. The assembly of yeast vacuolar SNAREs into complexes for fusion can be studied in chemically defined reactions. Complementary proteoliposomes bearing a Rab:GTP and either the vacuolar R-SNARE or one of the three integrally anchored Q-SNAREs were incubated with the tethering/SM protein complex HOPS and the two other soluble SNAREs (lacking a transmembrane anchor) or their SNARE heptad-repeat domains.

A cascade of multiple proteins and lipids catalyzes membrane fusion.

Recent studies suggest revisions to the SNARE paradigm of membrane fusion. Membrane tethers and/or SNAREs recruit proteins of the Sec 1/Munc18 family to catalyze SNARE assembly into -complexes. SNARE-domain zippering draws the bilayers into immediate apposition and provides a platform to position fusion triggers such as Sec 17/α-SNAP and/or synaptotagmin, which insert their apolar "wedge" domains into the bilayers, initiating the lipid rearrangements of fusion.

HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex.

Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains.

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

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion.

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

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.