Dynamin−SNARE interactions control trans-SNARE formation in intracellular membrane fusion (original) (raw)
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Compartmental specificity of cellular membrane fusion encoded in SNARE proteins
Nature, 2000
Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.
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
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.
Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins
… et Biophysica Acta (BBA …, 2003
SNARE proteins function at the center of membrane fusion reactions by forming complexes with each other via their coiled-coil domains. Several SNAREs have N-terminal domains (NTDs) that precede the coiled-coil domain and have critical functions in regulating the fusion cascade. This review will highlight recent findings on NTDs of syntaxins, the longin domain of VAMP proteins and SNAP-23/25 homologues in yeast. Biochemical and genetic experiments as well as the resolution of several NMR and crystal structures of SNARE NTDs shed light on their diverse function. D
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.
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.
Hemifusion in SNARE-mediated membrane fusion
Nature Structural & Molecular Biology, 2005
SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wildtype SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate.
Membrane Fusion Intermediates via Directional and Full Assembly of the SNARE Complex
Science, 2012
No More Fusion Confusion Biophysical models explain membrane fusion as a sequence of steps—including membrane contact, formation of a fusion stalk (merger of proximal monolayers), development of contact between distal monolayers that may or may not expand (hemifusion), and, finally, rupture of this diaphragm resulting in the opening of a fusion pore. Biological membrane fusion reactions are often driven by so-called SNARE proteins. By using a reconstituted membrane fusion system, Hernandez et al. (p. 1581 , published online 31 May) have now been able to correlate precisely the states of SNARE zippering with intermediate structures along the fusion pathway. The results suggest that a tightly docked state, with a membrane distance so close that no proteins fit in between them, represents a critical fusion intermediate as a consequence of SNARE zippering. This intermediate is incompatible with a SNARE-driven stalk or with a ringlike arrangement of SNAREs depicted in most current models...
v-SNARE transmembrane domains function as catalysts for vesicle fusion
eLife, 2016
Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its s...
Journal of Cell Biology, 1999
Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.