Munc18-1 binds directly to the neuronal SNARE complex

Abstract

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.exocytosis ͉ membrane fusion ͉ neurotransmitter release ͉ Sec1/Munc18-like proteins ͉ synapse E very eukaryotic cell relies on the precise and regulated trafficking of proteins, membranes, and other types of cargo between cellular compartments. The molecular machinery responsible for membrane traffic is partly conserved to provide for basic membrane fusion and fission reactions and is partly cell type-and compartment-specific to meet the unique requirements of a particular cellular locale (1). Neurons have arguably the most complex membrane trafficking of all cells, primarily because synaptic transmission requires continuous (but at the same time tightly regulated) synaptic membrane traffic. At a synapse, neurotransmitter release is effected by fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptic vesicle fusion requires two conserved protein families that are universally involved in membrane fusion reactions: SNARE proteins, which are thought to pull membranes together by forming tight ''SNARE complexes,'' and Sec1/Munc18-like proteins (SM proteins), which perform an unknown but essential role in fusion and interact with SNARE proteins (reviewed in refs. 2-4).Synaptic vesicle exocytosis involves one SM protein (Munc18-1) and three SNARE proteins (synaptobrevin/VAMP on synaptic vesicles, and SNAP-25 and syntaxin-1A/1B on the plasma membrane; syntaxin-1A and -1B are highly homologous and thought to have identical functions). All SNARE proteins contain a conserved 60-to 70-residue sequence, the SNARE motif, that assembles into a four-helical bundle during SNARE complex formation (5). Synaptobrevin/VAMP and SNAP-25 are ''minimal SNARE proteins'' that are composed of one (synaptobrevin) or two (SNAP-25) SNARE motifs and a membraneanchor sequence. In syntaxin-1, however, the SNARE motif and transmembrane region occupy less than half of the sequence, with the...

Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

“…The binding mode resembles that reported for Sly1p/Sed5p, indicating a conserved molecular mechanism. Based on comparison with the Munc18-1 structure and Stx4 and Stx1a N-peptide sequences, a similar N-peptide binding mode can be expected to form between Munc18-1 and Stx1a, consistent with recent reports (21,22). We also used a structural bioinformatics approach to predict the presence or absence of an N-peptide binding mode in all yeast and mammalian SM/Stx pairs.…”

“…1). Furthermore, evidence is emerging that Munc18-1 can interact with SNARE complexes (21,22,26,27) and that the Stx1a N terminus is important for this interaction (21,22). We surmised that an N-peptide interaction can occur in Munc18-1 and that this would explain the requirement for the N-peptide in SNARE complex interactions with Munc18-1.…”

trans-SNARE complex assembly and yeast vacuole membrane fusion

“…Recent studies of the binding of Sec1/Munc18 proteins, complexin, and synaptotagmin to the synaptic SNARE complex suggest intriguing parallels to our results. Sec1/Munc18 proteins bind to SNARE complexes (28,29). Complexin interacts with and stabilizes synaptic SNARE complexes next to the membrane proximal region (10).…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…There are six mammalian family members including nSec1A and B, munc-18b (muSec1) and munc-18c and munc13-1, 13-2 and 13-3. 97 nSec1 binds with high affinity to syntaxin 1A and to the SNARE complex, 98 stabilizing its closed conformation which antagonizes priming 96,[99][100][101] by preventing binding to SNARE partners and as mentioned earlier, syntaxin 1A-mediated inhibition of N-type channels. 61,67 nSec1 is a critical regulator of the exocytotic machinery with null mutants exhibiting no neurotransmission.…”