Figure 5. Requirements for fusion in the absence of C-terminal SNARE domain zippering.
(A) Fusion is lost without SNARE zippering unless ATP is replaced by ATPγS. The fusion of proteoliposomes with Ypt7:GTP and either the R-SNARE or QaQb-SNAREs in the presence of Sec17 and Sec18 was assayed with ATP or ATPγS, with wild-type Vam7 or Vam7-3Δ and with or without HOPS, as indicated, as described in Materials and Methods. The final concentration of MgCl2 was 0.69 mM. (B) With incomplete SNARE zippering, fusion requires HOPS, Sec17, and Sec18. Fusion reactions had ATPγS, and either wild-type Qc or Qc-3Δ as well as Sec17, Sec18, and HOPS. Fusion reactions with wild-type Qc are depicted with open symbols, those with Qc-3Δ are filled symbols. Fusion with Vam7-3Δ was lost upon omission of HOPS (filled black diamonds), Sec17 (solid triangles), or Sec18 (solid squares). Fusion reactions without any Qc are in gray symbols, in the presence of HOPS, Sec17 and Sec18 (circles) or without Sec17/Sec18 (squares), as described in Materials and methods. (C) Arrested SNARE zippering requires Sec17, Sec18, and ATPγS for fusion. Fusion of Ypt7(GTP):R-SNARE proteoliposomes with Ypt7(GTP):QaQb-SNARE proteoliposomes was performed as described in Materials and Methods, but with HOPS and the C-terminally truncated Qc SNARE Vam7-3Δ and with either ATPγS (open symbols) or glucose/hexokinase (filled symbols). Incubations had Sec17 and Sec18 (circles), Sec18 (squares), Sec17 (triangles), or neither (diamonds). The final concentration of MgCl2 was 0.69 mM.
DOI: http://dx.doi.org/10.7554/eLife.26646.016
Figure 5—figure supplement 1. Average and standard deviations of fusion after 30 min for triplicate assays as in Figure 5, relative to the maximal fusion condition.
