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Fig. 8. Localization mutants display disrupted protein-protein and protein-lipid intramolecular interactions. (A) Results of bimolecular binding assay. Bead-immobilized GST-longin domains were mixed with a Triton-X-100 cell lysate prepared from PC12 cells overexpressing either lipidated (Myc-rYkt6 residues 137-198) or non-lipidated (Myc-rYkt6 residues 137-193) SNARE motif and incubated at 4°C for 1 hour. Beads were washed, boiled in SDS sample buffer, subjected to SDS-PAGE and immunoblotted to detect the bound SNARE motif. The lipidated (residues 137-198) and non-lipidated (residues 137-193) Myc-SNARE motifs are indicated by arrows. (B) Equivalent protein loading to the beads. Proteins bound to 1.25 µl bed volume of glutathione-Sepharose beads were resolved on a 15% gel by SDS-PAGE and stained with Coomassie blue. (C) Equivalent amount of SNARE motif was used for the binding assay. 10% of the PC12 extracts containing the SNARE motifs used in the binding assay were analysed by SDS-PAGE and immunoblotting. (D) Myc-rYkt6-137-193 actively assembles with ER/Golgi SNAREs. Cell lysate containing Myc-rYkt6-137-193 was incubated with buffer alone or with purified, recombinant, ER/Golgi SNAREs (2 µM each), followed by Superose-12 gel filtration chromatography. Column fractions were immunoblotted with anti-Ykt6 antibody. Elution positions of molecular weight standards are indicated above.
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