A stage-specific preparation to study the Ca2+-triggered fusion steps of exocytosis: Rationale and perspectives

Despite groundbreaking work to identify numerous proteins and to focus atte ntion on molecular interactions, the mechanism of calcium-triggered membran e fusion remains unresolved. A major difficulty in such research has been t he many overlapping and interacting membrane trafficking steps in the secre tory pathway, including those of membrane retrieval. Identifying the specif ic role(s) of a given protein, beyond its general involvement in exocytosis , has therefore proven problematic. Furthermore, the power of time-resolved optical and electrophysiological assays can be best applied to testing the function of known proteins rather than to the identification of unknown, c ritical membrane components. The identification of essential membrane const ituents requires combined biochemical (molecular) and functional (physiolog ical) analyses. A fully functional, stage-specific physiological membrane p reparation would be one direct approach to dissecting the calcium-triggered fusion steps of regulated exocytosis. Herein we review our use of specific minimal membrane preparations consisting of fully primed and docked secret ory vesicles, or the isolated vesicles themselves, and characterize the lat e events of exocytosis, with an aim towards identification of essential mol ecular components. We have established a functional definition of the fusio n complex and its activation by calcium, based on our kinetic analyses. Tog ether with a variety of biochemical and alternate functional assays, we hav e tested whether the SNARE core complex that is present in our vesicle memb ranes satisfies the criteria of the Functionally defined fusion complex. Ra ther than a direct fusogenic role, the SNARE complex may promote the calciu m sensitivity of Fusion, possibly by defining or delimiting a localized, fo cal membrane fusion site that ensures rapid and efficient exocytosis in viv o. (C) 2000 Society francaise de biochimie et biologie moleculaire / Editio ns scientifiques et medicales Elsevier SAS.