Three v-SNAREs and Two t-SNAREs, Present in a Pentameric cis-SNARE Complex on Isolated Vacuoles, Are Essential for Homotypic Fusion (original) (raw)
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Journal of Biological …, 2003
FIG. 5. Accumulation of trans-SNARE complexes in the absence of the Vam3 TMD. A, authentic trans-SNARE complexes of Vam3-CCIIM vacuoles. Vacuoles (65 g each) from BJnyv1⌬ containing Vam3-wt or Vam3-CCIIM were incubated with DKYvam3⌬ at 26°C in a 600-l reaction with or without ATP in the presence of cytosol, CoA, Sec18, and the indicated docking inhibitor Gdi1 (30 g/ml) or fusion inhibitor GTP␥S (2 mM) for 40 min. Protein complexes were analyzed by coimmunoprecipitations with antibodies to Vam3 as described under "Experimental Procedures." B, accumulation of trans-SNARE complexes of Vam3-CCIIM triggered by Sec18 addition. The experiment was done as described in the legend to B. Sec18 (3 g/ml) was added where indicated. At the times indicated samples were set on ice before processing for coimmunoprecipitation. C, quantification of trans-SNARE complexes. trans-SNARE complex accumulating in the presence of Sec18 after 40 min were quantified by laser densitometry (n ϭ 8). Ratios of Nyv1 and Vam3 signals of each precipitation with Vam3-CCIIM were set to 100%.
Journal of Biological Chemistry, 2015
Capsule Background: Tethering complexes such as HOPS bind SNAREs to coordinate fusion. Results: HOPS binds the Vam3 H abc domain and the SNARE complex, which is required for membrane fusion. Conclusion: Binding of the Vam3 H abc domain prepositions HOPS for optimal fusion support. Significance: Our data reveal that HOPS coordinates SNARE assembly and fusion via two distinct SNARE binding sites. Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the H abc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 H abc domain, assembled Q-SNAREs and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the H abc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the H abc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE-binding prevents recognition of the H abc domain. In agreement, membranes carrying Vam3∆H abc fuse poorly unless an excess of HOPS is provided. These data suggest that the H abc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion.
One SNARE complex is sufficient for membrane fusion
Nature Structural & Molecular Biology, 2010
In eukaryotes, most intracellular membrane fusion reactions are mediated by the interaction of SNARE proteins that are present in both fusing membranes. However, the minimal number of SNARE complexes needed for membrane fusion is not known. Here, we show unambiguously that one SNARE complex is sufficient for membrane fusion. We performed controlled in vitro Förster resonance energy transfer (FRET) experiments and found that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes, or with purified synaptic vesicles. Furthermore, we demonstrate that multiple SNARE complexes do not act cooperatively, showing that synergy between several SNARE complexes is not needed for membrane fusion. Our findings shed new light on the mechanism of SNARE-mediated membrane fusion and ask for a revision of current views of fusion events such as the fast release of neurotransmitters.
Molecular Cell, 1999
Sacher et al., 1998). Additional general factors acting on SNAREs are NSF (NEM-sensitive factor; Block New York, New York 10021 † Department of Molecular Biophysics et al., 1988) and SNAPs (soluble NSF attachment proteins; Clary et al., 1990), which alter the conformation and Biochemistry Yale University of SNAREs and disassemble v-t SNARE complexes, thereby regenerating separate v-and t-SNAREs for re-New Haven, Connecticut 06520 peated use. Most SNARE proteins possess a single transmembrane domain at their extreme carboxy terminus and Summary are predicted to have a high propensity to form coiledcoil structures. Assembled cytosolic domains of SNARE The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to proteins form very stable structures in all cases that have been closely examined (Hayashi et al., 1994; Yang do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins et al., 1999), likely due to their coiled-coil nature. Furthermore, electron microscopic (Hanson et al., 1997b; Hohl and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline et al., 1998) and biophysical (Lin and Scheller, 1997; Poirier et al., 1998) analysis of assembled full-length residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, neuronal SNARE complexes revealed that the transmembrane domains of both the v-SNARE VAMP/synap-and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a tobrevin (Trimble et al., 1988; Baumert et al., 1989; Sü dhof et al., 1989) and the t-SNARE syntaxin 1A (Bennett flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately et al., 1992, 1993) emerge at the same end of the 7S or 20S particle establishing a parallel orientation of the affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. assembled SNARE proteins. These characteristics suggested that SNARE proteins are also directly responsible Together, these results rule out a requirement for helical continuity and suggest that distance is a critical for membrane fusion (Hanson et al., 1997a, 1997b; Lin and Scheller, 1997; Hohl et al., 1998), and this has now factor for membrane fusion. been directly demonstrated with artificial liposomes and isolated SNAREs (Weber et al., 1998) and confirmed with Introduction permeabilized cells (Chen et al., 1999). The recently obtained three-dimensional structure of Members of the SNARE (SNAP receptor) protein family the neuronal SNARE complex (Poirier et al., 1998; Sutton (Sö llner et al., 1993a, 1993b) are key components in et al., 1998) has provided additional information to guide the process of transport vesicle docking and fusion. structure-function studies aimed at clarifying the bio-Individual SNARE family members are maintained in disphysical mechanisms involved in fusion. The crystal crete locations throughout the secretory pathway prostructure confirms the previous biophysical predictions viding a roadmap of vesicle flow patterns (Hay and of coiled-coil structure and provides a structural frame-Scheller, 1997; Linial, 1997; Advani et al., 1998; Nichols work to explain the intrinsic stability of the assembled and Pelham, 1998; Steegmaier et al., 1998). The assem-SNARE complex. Another striking feature of the core bly of trans-SNARE complexes (SNAREpins) between SNARE complex structure is its overall similarity to the membranes is likely the underlying principle of lipid biproposed fusogenic cores of a variety of virally encoded layer fusion (Weber et al., 1998; Chen et al., 1999), and fusion proteins (Skehel and Wiley, 1998). This similarity as such, it must be highly regulated in many cell types.
SNAREs Contribute to the Specificity of Membrane Fusion
Neuron, 2000
Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information
The EMBO journal, 2001
Activated fatty acids stimulate budding and fusion in several cell-free assays for vesicular transport. This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identi-®ed. We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion. Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion. Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only. Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18pand ATP-dependent priming. During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane. We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion.
Self-interaction of a SNARE Transmembrane Domain Promotes the Hemifusion-to-fusion Transition
Journal of Molecular Biology, 2006
SNARE proteins mediate intracellular fusion of eukaryotic membranes. Some SNAREs have previously been shown to dimerise via interaction of their transmembrane domains. However, the functional significance of these interactions had remained unclear. Here, we show that mutating alternate faces of the transmembrane helix of the yeast vacuolar Q-SNARE Vam3p reduces the ability of the full-length protein to induce contents mixing in yeast vacuole fusion to different extents. Examination of liposome fusion induced by synthetic transmembrane domains revealed that inner leaflet mixing is delayed relative to outer leaflet mixing, suggesting that fusion transits through a hemifusion intermediate. Interestingly, one of the mutations impaired inner leaflet mixing in the liposome system. This suggests that the defect seen in vacuolar contents mixing is due to partial arrest of the reaction at hemifusion. Since covalent dimerisation of this mutant recovered wild-type behaviour, homodimerisation of a SNARE transmembrane domain appears to control the transition of a hemifusion intermediate to complete lipid mixing.
The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion
The EMBO journal, 2003
The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes during priming. Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified. Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion. This subreaction is controlled by a Sec17-independent function of Sec18. Our data indicate that Ykt6 presents Pal-CoA via its N-terminal longin domain to Vac8, while transfer to Vac8's SH4 domain occurs spontaneously and not enzymatically. The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events.