Fig. 3

Formation of FAT:LD2/4:vinculin tail (Vin-T) ternary complexes observed by sedimentation velocity analytical ultracentrifugation (svAUC). (A-D) Continuous sedimentation coefficient (S) distribution curves are shown for samples indicated in the legends. Peaks are labeled with likely species they contain based on their size. Labelling is according to the key in panel (F). (E) Superposition of S distribution curves from samples containing the tripartite FAT:LD2/4:Vin-T ternary complex (species 6 in panel F) together with the LD2/4 + Vin-T sample (orange dotted curve). Note that compared to FAT-WT, ternary complex formation is increased for FAT-H14 and decreased for FAT-H23 and only for the latter the LD2/4:Vin-T binary complex (species 5 in panel F) remains detectable. (F) List of complexes and free proteins species potentially present in svAUC samples together with their schematic illustration and key used in panels A-E. (G) GST-pulldown experiments were performed with GST-fused Vin-T, LD2/4 and His-tagged FAT to confirm ternary complex formation. In agreement with AUC experiments, complex formation resulting in detection of His-FAT is promoted by H14 and reduced by H23 mutations. His-FAT-H14 is only pulled down in the presence of the LD2/4 peptide. A representative experiment is shown in the left panel and the averaged quantification from 3 experiments is plotted in the right panel. For quantifications signals were normalized to FAT-WT pulled down with GST-Vin-T. Error bars represent SEM from three independent experiments