Fig. 2.

Synaptotagmin facilitates SNARE complex assemblyin vitro. A, Recombinant his6-syntaxin, his6-SNAP-25B, and his6-synaptobrevin were incubated in the presence and absence of recombinant synaptotagmin in 2 mm EGTA (−Ca²⁺) or 1 mm Ca²⁺(+Ca²⁺) for 0, 5, or 15 min at room temperature. SDS-resistant 7S SNARE-complex formation was assayed by subjecting the samples to SDS-PAGE, without previous boiling (except where indicated), and immunoblotting with anti-syntaxin antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence. 7S denotes an SDS-resistant complex consisting of syntaxin, SNAP-25, and synaptobrevin.dimer denotes the trace formation of disulfide-bonded SNARE complex dimers that form under these conditions.B, The optical densities of 7S complexes from the +Ca²⁺ lanes in A are plotted versus time of incubation. C, Assembly experiments were performed as described in A for 2 hr but in the absence of DTT, except where indicated. Assembly reactions were conducted with (+) or without (−) synaptotagmin in either 2 mm EGTA, 1 mm Mg²⁺, 1 mmCa²⁺, or 1 mm Mg²⁺plus 1 mm Ca²⁺. As controls, samples were prepared that lacked either synaptobrevin, syntaxin, or SNAP-25. As a further control, samples were boiled before analysis. Samples were analyzed by immunoblotting with a mixture of anti-synaptobrevin and anti-synaptotagmin antibodies. Ca²⁺ and synaptotagmin enhanced the formation of SDS-resistant dimers. These dimers are disulfide-linked and are dissociated by DTT. SDS-resistant 7S complex formation only occurs in the presence of all three SNAREs, and complexes are dissociated by boiling.