Figure 4. Sec17 allows partially-zipped SNARE complexes to drive fusion with or without Sec18.

The chemically defined RPL fusion system is diagrammed in Figure 4—figure supplement 1. RPLs bearing Ypt7-GTP (Rab), and either the Qa- and Qb-SNARES, or the R-SNARE, were incubated with HOPS (100 nM) and the indicated Qc-SNAREs (250 nM). Reactions were performed in the absence or presence of Sec17 and Sec18, as indicated. 1 mM ATP and HOPS were present under all conditions. The Rab Ypt7 was present on both vesicle populations and was loaded with GTP (Materials and methods). On the vertical axes, 100% fusion indicates complete association of the FRET probes encapsulated within the two vesicle populations, as determined in control reactions. Each data point shows the mean content mixing signal ± s.e.m. for three independent experiments. The lines show nonlinear best-fits of a second-order kinetic model.

Figure 4—figure supplement 1. RPL fusion assay system.

(A) The RPL fusion assay simultaneously monitors lipid and content mixing using orthogonal FRET pairs (Zucchi and Zick, 2011). Lipid mixing is monitored by FRET between phosphatidylethanolamine (PE) lipid derivitized with either NBD or Marine Blue. Content mixing is monitored through the association of encapsulated biotin and avidin conjugates (to phycoerythrin and Cy5 fluorophores, respectively). Although both lipid and content mixing signals were collected in the experiments shown, only content mixing signals are presented because the two signals were highly correlated, and because content mixing is the reaction's biologically relevant endpoint. (B) The RPLs used in this study contained a lipid mixture approximating that of the yeast vacuole membrane (Zick et al., 2014), native purified Ypt7-GTP, and either Qa and Qb, or R-SNAREs. The asymmetric SNARE topology allows docking and fusion without any prior requirement for Sec18-mediated cis-SNARE complex disassembly.