Fig. 3.
t-SNARE ΔPep12p/ΔTlg1p,ΔVti1p remains specific for Snc1p/Snc2p v-SNAREs. (A) Characterization of the t-SNARE ΔPep12p/ΔTlg1p,ΔVti1p fusion capacity. GST-ΔPep12p, GST-ΔTlg1p, and GST-ΔVti1p were preincubated to form a complex and then reconstituted into acceptor liposomes. After flotation, the GST moiety was thrombin-cleaved from the liposomes. Donor and cleaved acceptor liposomes were mixed (5:45 μl) in a microtiter plate with Snc2-C-pept plus buffer, Snc2p cytosolic domain plus buffer, or Snc2-C-pept plus Snc2p cytosolic domain as indicated on the figure. The plate was transferred to a 37°C fluorescent plate reader, and the converted fluorescence (rounds of fusion) was plotted. (B and C) Membrane fusion between ΔPep12p/ΔTlg1p,ΔVti1p t-SNARE liposomes and potential v-SNARE liposomes in the absence or presence of Snc2p-C-pept. t-SNARE ΔPep12p/ΔTlg1p,ΔVti1p acceptor liposomes were incubated with each v-SNARE donor liposome (45:5) in the absence (B) or presence (C) of peptide. The fluorescence signal was monitored over2hat37°C. As in Fig. 2, the extent of fusion at 2 h was normalized to the amount of cognate v-SNARE fusion in the presence of peptide (Snc2p, black bars in C). Fusion with protein-free liposomes was subtracted from all of the fusion results. The maximal extent of fusion (100%) with Snc2p donor liposomes and snc2-C-pept was 0.925 rounds. This experiment is representative of at least three different assays.