FIGURE 4.

Sec6 does not affect the rate of SNARE assembly. The cytoplasmic domain of Sso1 and the SNAP-25 domain of Sec9 (WT or Sec9-142) were incubated together at 10 μm for 0–72 h, with or without equimolar Sec6, and resolved on a histidine-MOPS polyacrylamide native gel (pH 6.6). The free Sso1 band was quantified and fit to an integrated second-order rate equation, assuming equal concentrations of reactants as described previously (11). A, representative native gels showing the assembled SNARE complex band and free Sso1 band, which was quantified. The asterisk denotes the non-resolved Sec9 band and nonspecific contaminants from the Sec9-142 prep, and two asterisks denote a contaminant from the Sec9 prep. B, quantification of the native gel assays fit to the integrated second-order rate equation assuming equal concentration of reactants. Data from four replicates were fit globally, and the rate constants are reported as mean ± S.E. of the fit (GraphPad Prism).