Topological requirements for PS, PIP₂, and syt during reconstituted membrane fusion. (A and B) PS (A) or PIP₂ (3%; B) was reconstituted into either Vr with 30 copies of syt or Tr. In A, all Tr contained 3% PIP₂; in B, all Tr contained 25% PS, and all Vr contained 15% PS. Fusion assays were performed as described in Fig 1 B. Both PS and PIP₂ are required in Tr, but not Vr. (C) 30 copies of syt were reconstituted into either Vr or Tr; syt must be present on Vr to stimulate fusion. Representative examples from three or more independent trials are shown.