Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay

• Verfasst von: Schollmeier, Yvette [VerfasserIn]
• Verfasst von: Krause, Jean Michel [VerfasserIn]
• Verfasst von: Kreye, Susanne [VerfasserIn]
• Verfasst von: Malsam, Jörg [VerfasserIn]
• Verfasst von: Söllner, Thomas [VerfasserIn]
• Titel: Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay
• Verf.angabe: Yvette Schollmeier, Jean Michel Krause, Susanne Kreye, Jörg Malsam, and Thomas H. Söllner (from the Heidelberg University Biochemistry Center)
• E-Jahr: 2011
• Jahr: [2 September 2011]
• Umfang: 9 S.
• Illustrationen: Diagramme
• Fussnoten: Gesehen am 25.10.2022
• Titel Quelle: Enthalten in: The journal of biological chemistry
• Ort Quelle: Bethesda, Md. : ASBMB Publications, 1905
• Jahr Quelle: 2011
• Band/Heft Quelle: 286(2011), 35 vom: Sept., Seite 30582-30590
• ISSN Quelle: 1083-351X
• DOI: doi:10.1074/jbc.M111.269886
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: Exocytosis
• Sach-SW: Membrane Fusion
• Sach-SW: Membrane Reconstitution
• Sach-SW: Secretion
• Sach-SW: SNAP-25
• Sach-SW: Syntaxin 1
• Sach-SW: VAMP2
• Sach-SW: VAMP8
• Sach-SW: Vesicles
• K10plus-PPN: 1819862844

Abstract

Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1·SNAP-25) and v-/t-SNARE (VAMP2·syntaxin 1·SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.