Fig. 1. Mutagenesis of lipid binding sites in vinculin. (A,B) Ribbon and space-filling representations of the vinculin tail (Vt, amino acids 885-1066); data from Bakolitsa et al. (Bakolitsa et al., 1999). Solvent-accessible surfaces of wild-type (wt) in A and mutant vinculin tail in B are coloured according to electrostatic potential, with basic patches in blue and acidic patches in red. The two lipid-binding sites (basic collar and basic ladder) are indicated by dotted lines. In mutant vinculin tail (B), point mutations of the C-terminus (CT) and helix 3 (H3) are circled. Vinculin mutants are referred to as CT and H3, and as lipid-binding-deficient mutant (LD) for the combination of both, respectively. (C) Pull-down assay to analyse the lipid-binding capacity of Vt mutants Vt(CT, H3, LD) and Vt(1052), in comparison to the Vt wild type. Large unilamellar vesicles of the phospholipids PC, PS and PIP₂ were incubated with Vt proteins and sedimented. Pairs of pellet (P) and supernatant (S) fractions were detected on Coomassie-stained gels and analysed by densitometry. Columns represent the proportions of vesicle-bound Vt protein (means±s.e.; n=3). Notice the decreased binding of Vt mutants to acidic phospholipids. Average residual binding of all Vt proteins to pure PC vesicles is indicated (dashed line). Identical molar amounts of Vt protein were used for all assays. (D) The interaction of Vt proteins with Vh was analysed by GST pull-down assay. GST-Vh(D1) fusion protein bound to glutathione-Sepharose beads was incubated with Vt protein. Pellet fractions were analysed on Coomassie-stained gels. (E) Interaction of Vt mutants with actin filaments was analysed in a high-speed cosedimentation assay. Pellet and supernatant fractions were analysed by densitometry. Differences in actin binding between the Vt mutants were observed at a molar ration of actin:Vt of 2:1. Columns represent the proportions of actin-bound Vt protein (means±s.e.; n=3). (F) Expression of full-length GFP-vinculin in stably transfected B16-F1 mouse melanoma cells was compared with endogenous vinculin levels by western blotting (WB) using anti-vinculin antibodies. (G) Proportions of exogenous vinculin (means±s.e.; n=3).