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1.
Science ; 336(6088): 1581-4, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22653732

ABSTRACT

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.


Subject(s)
Lipid Bilayers/metabolism , Liposomes , Membrane Fusion , SNARE Proteins/metabolism , Animals , Lipid Bilayers/chemistry , Liposomes/chemistry , Liposomes/metabolism , Protein Binding , Protein Conformation , Rats , SNARE Proteins/chemistry , Vesicle-Associated Membrane Protein 2/metabolism
2.
Proc Natl Acad Sci U S A ; 106(44): 18575-80, 2009 Nov 03.
Article in English | MEDLINE | ID: mdl-19843696

ABSTRACT

Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.


Subject(s)
Liposomes/metabolism , Membrane Fusion , Neurons/metabolism , SNARE Proteins/metabolism , Animals , Kinetics , Liposomes/chemistry , Rats , Spectrometry, Fluorescence , Time Factors
3.
EMBO J ; 26(1): 9-18, 2007 Jan 10.
Article in English | MEDLINE | ID: mdl-17159904

ABSTRACT

SNARE proteins mediate membrane fusion in eukaryotic cells. They contain conserved SNARE motifs that are usually located adjacent to a C-terminal transmembrane domain. SNARE motifs spontaneously assemble into four helix bundles, with each helix belonging to a different subfamily. Liposomes containing SNAREs spontaneously fuse with each other, but it is debated how the SNAREs are distributed between the membranes. Here, we report that the SNAREs mediating homotypic fusion of early endosomes fuse liposomes in five out of seven possible combinations, in contrast to previously studied SNAREs involved in heterotypic fusion events. The crystal structure of the early endosomal SNARE complex resembles that of the neuronal and late endosomal complexes, but differs in surface side-chain interactions. We conclude that homotypic fusion reactions may proceed with multiple SNARE topologies, suggesting that the conserved SNARE structure allows for flexibility in the initial interactions needed for fusion.


Subject(s)
Endosomes/metabolism , Liposomes/chemistry , SNARE Proteins/chemistry , Amino Acid Sequence , Animals , Circular Dichroism , Crystallography, X-Ray , Endosomes/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrometry, Fluorescence
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