Michael Zick

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

Unravelling the SM-SNARE conundrum So-called SNARE proteins mediate and lend specificity to the fusion between different intracellular membranes. The SM proteins are universally required for intracellular vesicle fusion, yet their mechanism of action has long been enigmatic. Baker et al. have solved a piece of the puzzle by “capturing” SNAREs in the process of assembling into fusogenic complexes on the surface of an SM protein. The findings suggest exactly how and why SM proteins help vesicular fusion during intracellular membrane trafficking. Science, this issue p. 1111 Structures of a Sec1/Munc18 protein engaging two SNARE motifs reveal a key mechanism in cellular membrane fusion. 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.

Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis

The fusion of biological membranes entails a drastic rearrangement of the lipid bilayer. New assays that distinguish fusion from lysis were developed to study an in vitro reconstitution of the yeast vacuolar fusion machinery. These assays revealed that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and by lysis.

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

A lipid-anchored SNARE supports membrane 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.

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

Large tethering complexes play an essential role in many intracellular membrane fusion events, yet their mode of action is poorly understood. A new function of the HOPS complex is uncovered in facilitating vacuolar fusion, the specific recruitment of the soluble SNARE Vam7 for the formation of fusogenic trans-SNARE complexes.

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

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. 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: Sec17L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17F21S,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 Sec17F21S,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.

A distinct tethering step is vital for vacuole membrane fusion

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. DOI: http://dx.doi.org/10.7554/eLife.03251.001

Phosphorylation of the Effector Complex HOPS by the Vacuolar Kinase Yck3p Confers Rab Nucleotide Specificity for Vacuole Docking and Fusion

The Rab GTPase Ypt7p and its effector complex HOPS participate in catalyzing the fusion of yeast vacuoles. The role of the vacuolar kinase Yck3p in this relation is examined. It is shown how the regulatory ability of the Rab GTPase cycle is enforced only by posttranslational modification of the effector complex HOPS.

Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion.

Acidic lipids act as coreceptors with Ypt7p to bind the HOPS complex to support membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.

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

In vitro reconstitution is a powerful approach to deciphering membrane fusion. However, current reconstitutions do not adequately mimic the physiological process. This study takes a big step toward overcoming those shortcomings, achieving fusion with SNARE densities comparable to the native membrane.