Abstract
Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphatases / metabolism
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Adenosine Triphosphatases / ultrastructure
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Amino Acid Sequence
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Animals
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Binding Sites
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Carrier Proteins / chemistry
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Carrier Proteins / metabolism
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Carrier Proteins / ultrastructure*
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Endopeptidases / ultrastructure
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Freeze Etching / methods
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Membrane Proteins / chemistry
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Membrane Proteins / metabolism
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Membrane Proteins / ultrastructure*
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Microscopy, Electron / methods
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Models, Structural
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N-Ethylmaleimide-Sensitive Proteins
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Nerve Tissue Proteins / chemistry
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Nerve Tissue Proteins / metabolism
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Nerve Tissue Proteins / ultrastructure*
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Peptide Fragments / chemistry
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Polymerase Chain Reaction
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Protein Binding
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Protein Conformation*
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Qa-SNARE Proteins
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R-SNARE Proteins
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Rats
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Recombinant Proteins / ultrastructure
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Synaptosomal-Associated Protein 25
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Vesicular Transport Proteins*
Substances
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Carrier Proteins
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Membrane Proteins
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Nerve Tissue Proteins
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Peptide Fragments
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Qa-SNARE Proteins
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R-SNARE Proteins
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Recombinant Proteins
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Snap25 protein, rat
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Synaptosomal-Associated Protein 25
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Vesicular Transport Proteins
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Endopeptidases
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Adenosine Triphosphatases
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N-Ethylmaleimide-Sensitive Proteins
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Nsf protein, rat