Molecular analysis of SNAP-25 function in exocytosis.

It is generally accepted that the SNARE proteins form the core of the machinery for intracellular membrane fusion and that formation of a SNARE complex is crucially important. Our aim is to dissect the molecular roles of the SNARE proteins and their regulators in physiological membrane fusion during exocytosis. We have developed approaches that allow us to manipulate protein expression in model secretory cells, PC12 and adrenal chromaffin cells, and to combine this with assay of exocytosis at high-time resolution using carbon-fiber amperometry. This technique allows us to assess the extent of exocytosis and to follow the kinetics of single secretory granule release events with millisecond time resolution. We established that manipulation of proteins involved in the exocytotic machinery can lead to detectable and interpretable changes in exocytosis kinetics that have revealed novel roles in late stages of exocytosis. Using this approach we have begun to analyze the function of SNAP-25B using a mutant resistant to the Clostridial neurotoxin BoNT/E. This SNAP-25 mutant can reconstitute exocytosis in BoNT/E-treated cells. With this construct it is possible to analyze the consequences of any introduced mutation in the absence of functional endogenous protein. We review here its use in the analysis of palmitoylated cysteines of SNAP-25 and the conserved residues of the 0 layer of the SNARE complex. The data suggest an important role of the cysteines, but not the 0 layer glutamines, in triggered exocytosis.