Abstract
Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.
Highlights
Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs)
The CD spectra of WT and clogged Munc18-1 proteins were very similar (Fig. 2E), suggesting that clogged Munc18-1 was properly folded. These results demonstrated that clogged Munc18-1 is defective in promoting trans-SNARE zippering, providing a molecular explanation for its inability to accelerate the kinetics of SNARE-dependent membrane fusion
A major discovery of our previous reconstitution studies was that the SM protein Munc18-1 promotes SNARE-dependent membrane fusion, and its stimulatory function requires binding to the N-peptide motif of syntaxin-1 [18, 36, 37]
Summary
In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc, a SM protein involved in synaptic exocytosis in neurons. In genetic studies using cultured neurons or whole animals, mutations in either the syntaxin N-peptide or the N-peptide– binding pocket of Munc strongly inhibited synaptic exocytosis (46 –48). We took advantage of a “clogged” Munc protein in which an ectopic copy of the syntaxin N-peptide motif is directly fused to Munc to block its N-peptide– binding pocket [37]. The clogged Munc protein is unable to bind the native N-peptide motif on syntaxin and fails to stimulate SNARE-dependent membrane fusion in vitro [37]. Consistent with our reconstitution data, these genetic results demonstrated that the N-peptide– binding mode is indispensable to Munc function in synaptic exocytosis
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