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The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis.


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.

SUBMITTER: Shen C 

PROVIDER: S-EPMC6254354 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis.

Shen Chong C   Liu Yinghui Y   Yu Haijia H   Gulbranson Daniel R DR   Kogut Igor I   Bilousova Ganna G   Zhang Chen C   Stowell Michael H B MHB   Shen Jingshi J  

The Journal of biological chemistry 20181001 47


Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to <i>N</i>-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 <i>in vitro</i> 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  ...[more]

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