FIGURE 7:

When wild-type (anchored) Qb levels are reduced to limit QaQb complex, fusion remains insensitive to the Jx-domain swap. R(JxQa) + Qa(JxR)Qb proteoliposomes and R + QaQb proteoliposomes give comparable fusion, even as the level of anchored Qb is steadily reduced.  R and R(JxQa) proteoliposomes as well as QaQb and Qa(JxR)Qb proteoliposomes were prepared with Ypt7 as described in Materials and Methods. The molar ratio of Qb:lipid was varied from 1:16,000 to 1:128,000 by successive twofold dilutions of the initial stock of purified Qb. Fusion was assayed as described in Materials and Methods except that the liposome pairs were nucleotide exchanged together in a 10 µl volume, and the remaining soluble components were prepared as one mix, also contributing 10 µl of volume to the total 20 µl reaction. Concentrations of each component in 20 µl remain as described in Materials and Methods, with 50 nM HOPS and 100 nM Qc but no Sec17 or Sec18. Proteoliposomes had wild-type R and Qa (open bars) or swapped JxR and JxQa (black). Mean and SD values of fusion after 20 min are presented for triplicate experiments. See Supplemental Figure S3 for typical kinetic data.