Resolving the Function of Distinct Munc18-1/SNARE Protein Interaction Modes in a Reconstituted Membrane Fusion Assay*
| Autor: | Jörg Malsam, Jean Michel Krause, Susanne Kreye, Yvette Schollmeier, Thomas H. Söllner |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Vesicle-associated membrane protein 8
Vesicle fusion Protein Conformation Biology Biochemistry Membrane Fusion Models Biological Munc18 Proteins Membrane Biology Protein Interaction Mapping Syntaxin Animals Lipid bilayer Molecular Biology Peripheral membrane protein Temperature Lipid bilayer fusion Cell Biology DNA Syntaxin 1 Lipids Syntaxin 3 Cell biology Rats nervous system Liposomes biological phenomena cell phenomena and immunity Peptides SNARE Proteins Protein Binding |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|