Molecular Mechanisms of Membrane Fusion

In the central nervous system, regulated exocytosis is characterized by a rapid synchronized fusion of synaptic vesicles with the plasma membrane. This process is triggered by an influx of Ca2+ into the presynaptic cell. Like fusion in other systems, this process involves the participation of a large number of soluble and membrane associated protein components, including the SNAREs. The SNAREs are membrane proteins which assemble into a coiled-coil complex. This complex bridges the vesicle and plasma membranes and drives the two together. Although SNAREs have been proposed to constitute a minimal machinery required for fusion, Ca2+ does not appear to directly bind and regulate the SNAREs. Synaptotagmin is a membrane protein associated with the synaptic vesicle that is believed to act as the Ca2+ sensor for neuronal exocytosis. There is compelling evidence that Ca2+ triggers fusion by binding to the two C2 domains of synaptotagmin.

At the present time, the molecular role of synaptotagmin is not understood. It is not known whether synaptotagmin triggers fusion by binding to membranes, SNAREs, or both. There have been numerous studies on soluble fragments of synaptotagmin and the SNAREs, but relatively little data is available on the configurations of these proteins or their interactions at the membrane surface. Numerous genetic studies on synaptotagmin have examined the effects of mutations on function, but virtually no information is available on how these mutations alter the position of the C2 domains at the membrane interface.

In collaboration with Lukas Tamm (Physiology, UVa) and Reinhard Jahn (Max-Planck Institute, Gottingen) we currently have an NIH funded program project grant directed at determining the mechanism of pre-synaptic membrane fusion from a biophysical perspective. Our role in this program project is to determine the configuration of synaptotagmin and segments of the SNAREs when associated with the membrane interface, and to test models of the role of synaptotagmin in fusion. The binding of the polyphosphoinositide, PI(4,5)P2, to these proteins will also be determined because it is a lipid that has been shown to be critical for fusion. The overall goal of the proposed work is to define the molecular mechanisms that trigger fusion in the central nervous system.