Selective Activation of Cognate SNAREpins by Sec1/Munc18 (SM) Proteins

Full-length mouse VAMP2-His 6 (pTW2) and the t-SNARE complex between mouse His 6-SNAP-25 and rat syntaxin 1A (pTW34) were expressed and purified as previously described (Melia et al., 2002; Weber et al., 2000). Rat syntaxin 4 and SNAP-23 expression vectors were generated in a similar way as neuronal SNAREs. The proteins were eluted into an OG buffer (25mM HEPES/KOH, pH 7.4, 400 mM KCl, 10% glycerol, 1% OG and 1mM DTT) containing a gradient of 50-500mM imidazole. Human lysosomal/late endosomal SNAREs-syntaxin7, mTlg1/syntaxin8, Vti1b and VAMP8-were expressed as previously described (Antonin et al., 2000; Paumet et al., 2005). Human VAMP3, VAMP4, VAMP7 and yeast Snc2p were expressed in a similar way as VAMP8. Thrombin-cleavable SNAREs were described previously (Parlati et al., 1999). Rat Munc18-1/nSec1 (from GST-Munc18-1 template, gift of T. Sollner) was subcloned into a pET28a-based SUMO vector to obtain a construct encoding a