FIGURE 3:

SNARE complex formation depends on HOPS and the R- or Qa–integral SNARE. Liposomes were prepared as described with or without Ypt7 and with one or no integral SNAREs, incubated with HOPS and the other soluble SNAREs, isolated by flotation, and assayed by immunoblot for bound (A) sR, (B) sQa, (C) sQb, (D) Qc, and (E) HOPS as measured by its Vps16 subunit. The complete binding reaction (black bars), containing RPLs with Ypt7 and one integrally bound SNARE plus the three remaining sSNAREs and HOPS, is compared with reactions without Ypt7 (gray bars), without HOPS (striped bars), without Ypt7 or HOPS (white bars), or with singly omitted sSNAREs as indicated. Integrally bound SNARE inputs were ∼5 pmol each, soluble protein inputs were 8 pmol of sR, sQb, and Qc, 2 pmol of sQa, and 0.5 pmol of HOPS. Data were analyzed by immunoblot as described in Materials and Methods and represent the average and SD of three independent experiments.