PE Lipids in Single SNARE Vesicle Fusion Assay on Supported Membranes Promote Docking and Reduce the Number of SNARE Complexes Required for Fast Fusion

SNARE proteins are the core of the membrane fusion machinery in mammalian cells. Both in vitro and in vivo studies focus mostly on neuronal SNAREs (syntaxin1A, SNAP25, synaptobrevin2), which are involved in Ca2+ regulated exocytosis of synaptic vesicles. A recently developed single vesicle SNARE-mediated fusion assay in planar supported bilayers allows us to study individual docking and fusion events with a millisecond time resolution in a well-controlled lipid environment. Although, the fusion time in this assay is differently defined than in cellular settings, various parameters describing SNARE-mediated membrane fusion can be obtained, including the efficiency of vesicle docking and the probability of fusion after docking. Additionally, the number of particles in the fusion site and their activation rates can be determined from modeling of the fusion kinetics data.To better mimic the composition of the plasma membrane, we added various concentrations of PE and PS to our standard PC/Chol mixture. We observed a 1.5- to 3-fold increase in docking efficiency when the PE content was raised from 0 to 30%. In contrast, the fusion probability decreased 3- to 6-fold compared to PC/Chol membranes. Interestingly, including PE only in the vesicle membrane was sufficient to cause these effects. A detailed analysis of the fusion kinetics revealed that the fusion rate did not change significantly while the number of SNARE complexes that drive fast membrane fusion decreased with 3 complexes constituting the minimal fusion site. These results are rationalized with a model, in which fewer SNAREs are required to overcome the lower activation energy barrier in the presence of negative curvature-promoting PE than the higher activation energy that is likely present in the absence of PE.