Figure 9. Munc18-1 helps to initiate SNARE assembly.
(A) FECs of single SNARE complexes obtained in the presence of 10 µM Munc18-1 in the solution. Black and red arrows indicate t-SNARE unfolding and de novo SNARE reassembly, respectively. The FECs #3-#5 were obtained by consecutively pulling and relaxing the same SNARE complex. (B) Percentage of de novo assembly (%) among a total number of unfolded SNARE complexes tested (N) for the wild-type (WT), NRD-removed (-NRD), and mutant (L60A and L63A) SNARE complexes in the presence (+) or absence (−) of 10 µM Munc18-1. (C) Histogram distributions of the forces associated with the Munc18-1-mediated de novoSNARE assembly. (D) FECs of the SNARE complexes crosslinked at a site N-terminal to the SNARE motifs (Figure 1—figure supplement 1, construct I) in the absence (−Munc18-1) or presence (+Munc18-1) of Munc18-1. In the absence of Munc18-1, SNAP-25 tended to dissociate from the tethered syntaxin and VAMP2 when the t-SNARE complex was unfolded (black arrows). As a result, the SNARE complex generally reassembled for no more than four rounds during repetitive pulling and relaxation (Gao et al., 2012). However, in the presence of 10 µM Munc18-1, the yield of SNARE reassembly was greatly enhanced, leading to robust SNARE disassembly and reassembly for at least 19 rounds on the single SNARE complex. For clarity, only the FECs of some rounds of pulling and relaxation (indicated by the red numbers) are shown. In this case, full SNARE reassembly (red arrows) was preceded by t-SNARE formation (blue arrows) in the force range of 12-17 pN, due to the slow and force-sensitive NTD assembly of this construct. FECs, force-extension curves; NTD, N-terminal domain.