Figure 6: Model for alternate Golgi SNARE complexes.

(A) SNAP29 and VTI1B are GS28 substitutes. SNAP29 operates in a complex with STX5 and VAMP7, while VTI1B operates in a complex with STX5, STX8 and YKT6. (B-D) In vitro interactions between GST-STX5, SNAP29-His₆ and GFP-VAMP7. (B) Purified bacterially expressed GST-STX5 and SNAP29-His₆ were mixed in equal amounts and the complex was isolated using purification on Talon affinity column. Individual proteins and the eluate from Talon affinity resin were loaded on 4-15% SDS gradient gel and stained with Coomassie G250. (C, D) STX5/SNAP29 Q-SNARE complex is specifically pulled-down by R SNARE GFP-VAMP7. (C) Purified GST-STX5 (lane 1) and SNAP29-His₆ (lane 2) were mixed and incubated on ice for 6 h; the resulting complex was sequentially purified on Glutathione and Talon affinity columns (lanes 3, 4).The STX5/SNAP29 Q-SNARE complex was recovered with GFP-VAMP7, GFP-YKT6 or GFP immobilized on GBP (GFP binding protein)-Sepharose beads after overnight incubation at 4°C. Proteins were identified by WB and the recovery of individual SNAREs was quantified. The picture underneath lanes 5-7 depict Coomassie-stained GFP-tagged proteins. (D) Bar graphs represent relative recovery of STX5 and SNAP29 on GBP beads. Note that GFP-VAMP7 was the preferred R-SNARE partner for STX5/SNAP29 Q-SNARE complex. (E, F) STX5/VTI1B/STX8 Q-SNARE complex is specifically pulled down by GFP-YKT6 and GFP-VAMP7. (E) Purified bacterially expressed GST-STX5 (lane 1), VTI1B-His₆ (lane 2) and STX8-His₆ (lane 3) were mixed and incubated on ice for 6 h; the resulting complex was sequentially purified on Glutathione and Talon affinity columns (lanes 4, 5). Purified STX5/VTI1B/STX8 Q-SNARE complex was mixed with GFP-YKT6, GFP or GFP-VAMP7 (lanes 6-8) immobilized on GBP-Sepharose beads and incubated overnight at 4°C. Proteins were identified by WB with antibodies as indicated and the recovery of individual SNAREs was quantified. The picture underneath lanes 6-8 represents Coomassie-stained GFP-tagged proteins. (F) Bar graphs represent relative recovery of STX5, VTI1B and STX8 on GBP beads.