title: Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay.
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.
authors: Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany.
authors: Yvette
authors: Y
authors: Schollmeier
authors: Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany.
authors: Jean Michel
authors: JM
authors: Krause
authors: Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany.
authors: Susanne
authors: S
authors: Kreye
authors: Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany.
authors: Jörg
authors: J
authors: Malsam
authors: Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany. Electronic address: thomas.soellner@bzh.uni-heidelberg.de.
authors: Thomas H
authors: TH
authors: Söllner
mesh_terms: D000818:Animals; D004247:DNA / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008954:Models, Biological; D051938:Munc18 Proteins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry*; D013696:Temperature
chemical_list: D008055:Lipids; D008081:Liposomes; D051938:Munc18 Proteins; D010455:Peptides; D050600:SNARE Proteins; D004247:DNA