ABSTRACT
Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.
Subject(s)
Exocytosis , Membrane Proteins/physiology , Nerve Tissue Proteins/metabolism , Vesicular Transport Proteins , Action Potentials , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Animals , Binding, Competitive , Carrier Proteins/pharmacology , Cell Membrane/metabolism , Cloning, Molecular , DNA, Complementary/metabolism , Decapodiformes/metabolism , Dose-Response Relationship, Drug , Drosophila , Electrophysiology , Kinetics , Membrane Proteins/pharmacology , Microscopy, Electron , Models, Biological , Molecular Sequence Data , Nerve Tissue Proteins/physiology , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Qa-SNARE Proteins , Rats , Recombinant Proteins/metabolism , SNARE Proteins , Sequence Homology, Amino Acid , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Time FactorsABSTRACT
The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.
Subject(s)
Decapodiformes/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neurotransmitter Agents/metabolism , Synapses/metabolism , Vesicular Transport Proteins , Amino Acid Sequence , Animals , Botulinum Toxins/pharmacology , Cloning, Molecular , Membrane Fusion , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Peptide Fragments/metabolism , Presynaptic Terminals/drug effects , Presynaptic Terminals/ultrastructure , Protein Binding/drug effects , Qa-SNARE Proteins , SNARE Proteins , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Synapses/drug effects , Synapses/ultrastructure , Synaptosomal-Associated Protein 25ABSTRACT
A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.
Subject(s)
Calcium-Binding Proteins , Carrier Proteins/metabolism , Membrane Fusion/drug effects , Membrane Proteins/metabolism , Synaptic Vesicles/metabolism , Tetanus Toxin/pharmacology , Vesicular Transport Proteins , Adenosine Triphosphate/metabolism , Animals , Botulinum Toxins/pharmacology , Brain/metabolism , Exocytosis/drug effects , Hydrolysis , Macromolecular Substances , Membrane Glycoproteins/pharmacology , Membrane Proteins/drug effects , N-Ethylmaleimide-Sensitive Proteins , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/pharmacology , Neurotransmitter Agents/antagonists & inhibitors , Qa-SNARE Proteins , R-SNARE Proteins , Rats , SNARE Proteins , Sodium Dodecyl Sulfate/pharmacology , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins , Synaptosomal-Associated Protein 25 , SynaptotagminsABSTRACT
The clostridial neurotoxin, tetanus toxin, is a Zn(2+)-dependent protease which inhibits neurotransmitter exocytosis by selective cleavage of the synaptic vesicle protein, synaptobrevin. Synaptobrevin is thought to serve as a receptor for two neuronal plasma membrane proteins, syntaxin and SNAP-25, which in the presence of non-hydrolyzable ATP analogs form a 20 S fusion complex with the soluble fusion proteins NSF and alpha-SNAP. Here we show that synaptobrevin, when in this 20 S complex, or its 7 S precursor, is protected against proteolysis by the enzymatically active tetanus toxin light chain. Our data define distinct pools of synaptobrevin, which provide markers of different steps of vesicle/plasma membrane interaction.
