Liposome Fusion Assay to Monitor Intracellular Membrane Fusion Machines

Fluorescence resonance energy transfer (FRET)-based lipid-mixing assays are used to study a variety of membrane-fusion events. Lipid-mixing assays are used to characterize the fusogenic properties of enveloped viruses by fusing viruses with liposomes. The successful application of lipid-mixing assays to viral fusion and the identification of many of the proteins involved in intracellular transport led to the choice of a fluorescence-dequenching assay to analyze the eukaryotic machinery of intracellular membrane fusion. This chapter introduces a well-established FRET-based lipid-mixing assay to measure fusion driven by reconstituted soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins. The application of this assay requires the functional reconstitution of a few to several membrane proteins along with diagnostic lipids. This modified assay has been used successfully to reconstitute fusion driven by seven different sets of SNARE proteins. The availability of a relatively simple and extremely robust assay to study SNARE-mediated membrane fusion permits in vitro analysis of many events that precede and regulate the fusion event. Currently reconstituted yeast post-Golgi transport reactions are being utilized to analyze the role of regulatory proteins, such as Sec1p and Sec4p in the assembly and function of the SNARE complex.