Structural Basis for Target Specificity in Intracellular Membrane Fusion

The fusion of one biological membrane is required for neurotransmitter release, intracellular movement of proteins and other materials, fertilization, and viral infection. Our work focuses on the proteins which mediate intracellular membrane fusion between vesicles and either intracellular compartments or the plasma membrane. At least three roles can be envisioned for such proteins:

1. targeting - the cell's internal architecture depends on the specificity of fusion, in that a vesicle with cargo intended for a given destination must not fuse with inappropriate target membranes;
2. fusion proper - once docked at the proper target, vesicles fuse; fusion itself, which is not spontaneous between biological membranes, is presumably mediated by a protein or protein complex; and
3. regulation - neurotransmitter release, for example, which entails the fusion of synaptic vesicles with the plasma membrane at axon terminals, is tightly coupled to the calcium influx which results from an action potential. Calcium sensor proteins presumably regulate fusion in this context.

Biochemical and genetic approaches in a number of laboratories have led to the identification of NSF/Sec18 and SNAP/Sec17, soluble proteins required for the fusion step in intracellular transport and, probably, for neurotransmission. Using these reagents, Rothman and colleagues discovered a family of proteins termed SNAREs which, in addition to serving as membrane receptors for NSF and SNAP, appear to play a key role in determining target specificity: the 'SNARE hypothesis' proposes that an intracellular vesicle finds and docks to the proper target membrane via a specific protein-protein complex between a vesicle-associated SNARE and a target membrane-associated SNARE. This SNARE complex, then, is recognized and bound by NSF and SNAP, leading somehow to membrane fusion. Substantial in vivo and in vitro evidence supports this hypothesis.

We have overexpressed and purified cytoplasmic domains of SNAREs involved in neurotransmitter release. We have initiated biophysical and structural characterization of these SNAREs and complexes among them. Our primary goal is x-ray crystallographic structure determination of SNAREs and SNARE complexes.