Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

Anders S Munch; Girish H Kedar; Jan R T van Weering; Sonia Vazquez-Sanchez; Enqi He; Timon André; Thimo Braun; Thomas H Söllner; Matthijs Verhage; Jakob B Sørensen

doi:10.1523/jneurosci.0007-16.2016

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

Anders S Munch, Girish H Kedar, Jan R T van Weering, Sonia Vazquez-Sanchez, Enqi He, Timon André, Thimo Braun, Thomas H Söllner, Matthijs Verhage, Jakob B Sørensen

Neuronal Signalling

21 Citations (Scopus)

Abstract

Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324 – 339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil– coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324 –339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca²⁺ dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.

Original language	English
Journal	The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume	36
Issue number	26
Pages (from-to)	6881-6891
Number of pages	11
ISSN	0270-6474
DOIs	https://doi.org/10.1523/jneurosci.0007-16.2016
Publication status	Published - 29 Jun 2016

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title = "Extension of Helix 12 in Munc18-1 Induces Vesicle Priming",

abstract = "Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324 – 339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil– coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324 –339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca2+ dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.",

author = "Munch, {Anders S} and Kedar, {Girish H} and {van Weering}, {Jan R T} and Sonia Vazquez-Sanchez and Enqi He and Timon Andr{\'e} and Thimo Braun and S{\"o}llner, {Thomas H} and Matthijs Verhage and S{\o}rensen, {Jakob B}",

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year = "2016",

month = jun,

day = "29",

doi = "10.1523/jneurosci.0007-16.2016",

language = "English",

volume = "36",

pages = "6881--6891",

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T1 - Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

AU - Munch, Anders S

AU - Kedar, Girish H

AU - van Weering, Jan R T

AU - Vazquez-Sanchez, Sonia

AU - He, Enqi

AU - André, Timon

AU - Braun, Thimo

AU - Söllner, Thomas H

AU - Verhage, Matthijs

AU - Sørensen, Jakob B

PY - 2016/6/29

Y1 - 2016/6/29

N2 - Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324 – 339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil– coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324 –339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca2+ dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.

AB - Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324 – 339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil– coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324 –339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca2+ dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.

U2 - 10.1523/jneurosci.0007-16.2016

DO - 10.1523/jneurosci.0007-16.2016

M3 - Journal article

C2 - 27358447

SN - 0270-6474

VL - 36

SP - 6881

EP - 6891

JO - The Journal of neuroscience : the official journal of the Society for Neuroscience

JF - The Journal of neuroscience : the official journal of the Society for Neuroscience

IS - 26

ER -

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

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