Topological arrangement of the intracellular membrane fusion machinery

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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SNARE zippering requires activation by SNARE-like peptides in Sec1/Munc18 proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802645115 ◽

2018 ◽

Vol 115 (36) ◽

pp. E8421-E8429 ◽

Cited By ~ 7

Author(s):

Haijia Yu ◽

Chong Shen ◽

Yinghui Liu ◽

Bridget L. Menasche ◽

Yan Ouyang ◽

...

Keyword(s):

Membrane Fusion ◽

Lipid Bilayers ◽

Fusion Reaction ◽

Coiled Coil ◽

Fusion Reactions ◽

Sm Proteins ◽

Close Proximity ◽

Protein Receptors ◽

Sm Protein

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE–SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE–SLP intermediate must form before SNAREs can drive efficient vesicle fusion.

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SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

The Journal of Cell Biology ◽

10.1083/jcb.201003148 ◽

2010 ◽

Vol 190 (1) ◽

pp. 55-63 ◽

Cited By ~ 68

Author(s):

Jingshi Shen ◽

Shailendra S. Rathore ◽

Lavan Khandan ◽

James E. Rothman

Keyword(s):

Membrane Fusion ◽

Complete Removal ◽

Snare Complex ◽

Sm Proteins ◽

Membrane Bilayer ◽

Core Domain ◽

Target Membrane ◽

Sm Protein ◽

Typical Configuration ◽

Intracellular Membrane Fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved Habc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the Habc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin Habc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Qbc configuration. We found that neither the linker nor the Qbc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.

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Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice

F1000Research ◽

10.12688/f1000research.15707.1 ◽

2018 ◽

Vol 7 ◽

pp. 1649

Author(s):

Agathe Verraes ◽

Beatrice Cholley ◽

Thierry Galli ◽

Sebastien Nola

Keyword(s):

Membrane Protein ◽

Membrane Fusion ◽

Hela Cells ◽

Intracellular Membrane ◽

Wild Type ◽

Attachment Protein ◽

Sensitive Factor ◽

Protein Receptors ◽

Brain Extracts ◽

Intracellular Membrane Fusion

VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and immunocytochemistry staining profiles and determine the extent of the antibodies’ specificity. Using this method, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.

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Protein–protein interactions in intracellular membrane fusion

Current Opinion in Structural Biology ◽

10.1016/s0959-440x(00)00151-2 ◽

2000 ◽

Vol 10 (6) ◽

pp. 662-671 ◽

Cited By ~ 20

Author(s):

Kira MS Misura ◽

Andrew P May ◽

William I Weis

Keyword(s):

Membrane Fusion ◽

Protein Interactions ◽

Intracellular Membrane ◽

Protein Protein Interactions ◽

Intracellular Membrane Fusion

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Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913985116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23573-23581

Author(s):

Youngsoo Jun ◽

William Wickner

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Protein Complex ◽

Negative Regulation ◽

Rab Gtpases ◽

Sm Proteins ◽

Rab Family ◽

Sm Protein

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

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