Abstract
Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.
Highlights
From the ‡Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, Health Sciences Centre, Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada and the ‡‡Department of Molecular and Cellular Biology and Biochemistry, Brown University, Providence, Rhode Island 02912
Other evidence suggests that the SNARE proteins are not the minimal, essential machinery required for Ca2ϩ-triggered fusion of native membranes [11,12,13,14,15] and instead indicate that SNAREs function at a critical stage of exocytosis that is upstream of membrane fusion
Using established methods, including blockade of protein interactions (8, 24 –27) and protease treatments [15], the analysis presented here firmly places the roles of identified SNARE interactions at a stage of exocytosis upstream of triggered membrane fusion
Summary
Potheses [5,6,7,8] This model suggests that the SNARE proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25) interact to form a heterotrimeric core complex consisting of a 4-stranded helical bundle [7, 9, 10], which is proposed to “zipper-up,” and thereby drive or catalyze the fusion of apposed membranes. By effectively isolating the triggered steps of fusion from the other concurrent and overlapping stages of the exocytotic pathway, this system permits rigorous coupling of the functional and biochemical analyses necessary to effectively dissect this molecular mechanism [11, 15, 18] This preparation allows access to the entire membrane surface of CV, ameliorating issues of access to essential proteins at fully docked sites.
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