Snarepins Are Functionally Resistant to Disruption by Nsf and αSNAP

Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 06/2000; 149(5):1063-72.
Source: PubMed


SNARE (SNAP [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn't NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.

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    • "Under normal conditions, the presynaptic membrane, represented by the thin red line, contains non-productive SNARE complexes which are incapable of directly participating in membrane fusion and include syntaxin1a tetramers (Misura et al., 2001b), and syntaxin1a:SNAP25 heterodimers (Xiao et al., 2001; Misura et al., 2001a). Along with the N-ethylmaleimide-sensitive factor (NSF) and NSF adaptor proteins (Weber et al., 2000), not shown here for the sake of simplicity, STXBP1 is required for the disassembly of the non-productive complexes and the capturing of syntaxin1a monomers in their closed conformation. According to the findings of (Ma et al., 2013), these STXBP1:syntaxin1a heterodimers represent the true starting point of functional SNARE fusion complex assembly, and SNAP25 only becomes re-involved in a later step of the pathway which is not shown here. "

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    • "The liposome fusion assay was performed essentially as described [24], [25]. 45 µL of t-SNARE liposomes and 5 µL of v-SNARE liposomes were mixed and incubated at 37°C for 2 h. "
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    ABSTRACT: Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.
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    • "Given the overall similarity of topologies 5 and 6, it is surprising that topology 6 supports robust Munc18-1 activation but Topology 5 does not. We found that the reactivity of topologies 5 and 6 was not influenced by the Vc peptide or NSF/α- SNAP (Supplemental Figure S3), which are known to prime SNARE assembly (Weber et al., 2000; Melia et al., 2002; Pobbati et al., 2006) or by the variations in the lipid compositions of the liposomes (Supplemental Figure S4). Thus the fusogenicity of the topological combinations is likely dictated by the intrinsic physicochemical properties of SNAREs and SM proteins. "
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    ABSTRACT: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.
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