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Methods Mol Biol. 2017;1538:93-105.

Reconstitution of Synaptic SNAREs into Large Liposomes with Reduced Curvature Stress.

Author information

1
Department of Structural Biochemistry, Max Planck Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44202, Dortmund, Germany. matias.hernandez@mpi-dortmund.mpg.de.

Abstract

Liposomes constitute a convenient biochemical model system to investigate mechanistic aspects of the membrane fusion of synaptic vesicles. The proteins responsible for mediating fusion are the SNAREs that belong to a highly conserved family of transmembrane proteins. Reconstituting SNAREs into liposomes using detergents has become a common approach not only to understand how SNAREs work, but also how fusion is regulated by the vast array of accessory proteins present at the presynapse. However, a concern has been that the high curvature stress of the small liposomes (diameters of ~40 nm) frequently used in many studies renders them prone to spontaneous fusion, bringing into question whether the measurements obtained faithfully represent SNARE-mediated fusion. By systematically varying the detergent concentration and characterizing the SNARE-liposome size distributions by light scattering, we describe a detailed procedure to reconstitute SNAREs into large liposomes with considerably reduced curvature stress.

KEYWORDS:

Exocytosis; Liposome; Membrane fusion; Neurotransmitter release; SNARE

PMID:
27943186
DOI:
10.1007/978-1-4939-6688-2_8
[Indexed for MEDLINE]

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