A mutant impaired in SNARE complex dissociation identifies the plasma membrane as first target of synaptobrevin 2 (original) (raw)
Related papers
Structural determinants for SNARE-mediated neurosecretion
2010
14 synaptobrevin2) and the synaptic plasma membrane proteins syntaxin1A and SNAP-25 [44-47]. A molecular model of SNARE-mediated vesicle exocytosis has emerged within the last 30 years [2, 48] (Figure 4). This model of regulated exocytosis consists of a series of transition steps that are controlled by additional late regulatory proteins including synaptotagmin, complexin, tomosyn, Munc-13, syntaphilin and snapin [49-55]. The process begins when syntaxin1A and SNAP-25, which are organized in clusters at the plasma membrane [56, 57], assemble together to form a binary complex called acceptor complex [58-60]. The acceptor complex provides a binding interface for the vesicular SNARE VAMP2, thus forming a ternary complex. Figure 4. Model for SNARE-mediated neuronal exocytosis. The neuronal t-SNAREs SNAP-25 and syntaxin1A (labelled in green and red, respectively), assemble together to form the acceptor complex followed by binding of the v-SNARE partner VAMP2 (in blue). The three SNARE proteins form the trans-SNARE complex that brings opposing membranes into close proximity awaiting a Ca 2+ signal. Additional proteins such as synaptotagmin (purple) and complexin (pink) bind to this trans-SNARE complex with possibly distinct outcomes. Ca 2+ entry triggers membrane fusion, followed by the generation of cis-SNARE complexes, which are disassembled by NSF and α-SNAP upon ATP-hydrolysis This ternary complex proceeds from a loose state (in which only the Nterminal part is assembled) as studied in vivo [61-63] and in vitro [64] to a tight Ca + Calcium influx Membrane fusion Synaptobrevin/Vamp
The EMBO Journal, 1998
In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod~14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.
Proceedings of the …, 1998
SNARE [soluble NSF (N-ethylmaleimidesensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four ␣-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.
Membrane-directed molecular assembly of the neuronal SNARE complex
Journal of Cellular and Molecular Medicine, 2011
Since the discovery and implication of N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full-length recombinant t-SNAREs and v-SNARE present in opposing liposomes, leading to the assembly of a t-/v-SNARE ring complex. Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution. Similar to the t-/v-SNARE ring complex formed when 50 nm v-SNARE liposomes meet a t-SNARE-reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t-SNARE-reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membranedirected molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.
Membrane-directed molecular assemblyof the neuronal SNARE complexWon
2011
Since the discovery and implication of N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full-length recombinant t-SNAREs and v-SNARE present in opposing liposomes, leading to the assembly of a t-/v-SNARE ring complex. Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution. Similar to the t-/v-SNARE ring complex formed when 50 nm v-SNARE liposomes meet a t-SNARE-reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t-SNARE-reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membranedirected molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.
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.