ABSTRACT
The interaction between the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (Sx) and regulatory partner Sec/Munc18 (SM) protein is a critical step in vesicle fusion. The exact role played by SM proteins, whether positive or negative, has been the topic of much debate. High-resolution structures of the SM:Sx complex have shown that SM proteins can bind syntaxin in a closed fusion incompetent state. However, in vitro and in vivo experiments also point to a positive regulatory role for SM proteins that is inconsistent with binding syntaxin in a closed conformation. Here we present protocols we used for the expression and purification of the SM proteins Munc18a and Munc18c and syntaxins 1 and 4 along with procedures used for small-angle X-ray and neutron scattering that showed that syntaxins can bind in an open conformation to SM proteins. We also describe methods for chemical cross-linking experiments and detail how this information can be combined with scattering data to obtain low-resolution structural models for SM:Sx protein complexes.
Subject(s)
Munc18 Proteins/metabolism , Protein Binding , Qa-SNARE Proteins/metabolism , Scattering, Small Angle , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , Deuterium/chemistry , Mass Spectrometry/instrumentation , Mass Spectrometry/methods , Membrane Fusion , Munc18 Proteins/chemistry , Munc18 Proteins/isolation & purification , Neutron Diffraction , Protein Structure, Tertiary , Qa-SNARE Proteins/chemistry , Qa-SNARE Proteins/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , X-Ray DiffractionABSTRACT
The fusion of intracellular vesicles with target membranes is mediated by two classes of conserved molecules-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAP receptors or SNAREs) and Sec1/Munc18 (SM) proteins. A conserved function of SM proteins is to recognize their cognate trans-SNARE complexes and accelerate fusion kinetics. Here, we describe a physiologically relevant reconstitution system in which macromolecular crowding agents are included to recapitulate the crowded intracellular environment. Through this system, we elucidate the molecular mechanisms by which SNAREs and SM proteins drive vesicle fusion.
Subject(s)
Cytoplasmic Vesicles/metabolism , Membrane Fusion , Munc18 Proteins/metabolism , SNARE Proteins/metabolism , Exocytosis , Glucose Transporter Type 4/metabolism , Kinetics , Munc18 Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SNARE Proteins/isolation & purificationABSTRACT
Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.
Subject(s)
Arachidonic Acid/pharmacology , Munc18 Proteins/drug effects , Syntaxin 1/drug effects , Amino Acid Sequence , Animals , Brain Chemistry , Cell Membrane/chemistry , Cell Membrane/metabolism , Dissociative Disorders , Molecular Sequence Data , Munc18 Proteins/isolation & purification , Munc18 Proteins/metabolism , Protein Interaction Mapping , Qa-SNARE Proteins/chemistry , Qa-SNARE Proteins/metabolism , Rats , SNARE Proteins/drug effects , SNARE Proteins/isolation & purification , SNARE Proteins/metabolism , Synaptosomal-Associated Protein 25/drug effects , Synaptosomal-Associated Protein 25/isolation & purification , Synaptosomal-Associated Protein 25/metabolism , Syntaxin 1/isolation & purification , Syntaxin 1/metabolismABSTRACT
Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.
