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. 2019 Sep 23;181(3):1096–1113. doi: 10.1104/pp.19.00919

Figure 4.

Figure 4.

The cytosolic domain KC167–91 associates differentially with the cognate SNAREs in binary combination and in complex. A, Tagged proteins were expressed and purified from E. coli using His-affinity and size-exclusion chromatography before incubation in vitro overnight at 4°C. Shown is one of three independent pulldown experiments with Coomassie-stained SDS-PAGE analysis following separation with Strep-Tactin resin-immobilized KC167–91-Flag3-StrepII. Lane sets are the SDS-PAGE results of pulldowns with a 3-fold excess of SYP121ΔC and VAMP721ΔC with competing additions of 0, 0.3-, 1-, and 3-fold excess of VAMP721ΔC SNAP33, as indicated (above). Also shown are the pulldown results on incubation of all three SNAREs with the K+ channel peptide (right). SNAREs were incubated at 4°C overnight with Strep-Tactin-immobilized K+ channel peptide-Flag3-StrepII. Fixed concentrations (5 μm) of SYP121ΔC and VAMP721ΔC were used in each case. Similar results for KAT11–63 are shown in Supplemental Figure S4. B, Bound fraction analysis of KC161–97 and KAT11–63 pulldowns from all six independent experiments. Data are means ± se of the ratios of SNAREs to K+ channel peptide. Lowercase letters indicate statistical differences at P < 0.05. C, Comigration of KC167–91 peptide-SNARE complex on gel filtration. The KC167–91 peptide-SNARE complex assembly reactions were started either by comixing the channel peptide with SYP121ΔC, SNAP33, and VAMP721ΔC (above) or by adding the channel peptide following incubation of SYP121ΔC, SNAP33, and VAMP721ΔC to preform the SNARE complex. The SNARE complex was formed by incubation overnight at 4°C and purified by gel filtration chromatography, and the homo-oligomeric peak of SNARE complex was then mixed with KC167–91 peptide. Shown are the SDS-PAGE analyses of gel filtration fractions with the SNAREs and channel peptides, with molecular weights (MW) of standards indicated above each column. Note that the channel peptide remained associated with the SNARE complex when comixed with the SNAREs (Combined) but not with the preformed SNARE complex (Preassembled).