FIGURE 2.

Pyd PDZ2 interacts with PtdIns(4,5)P₂in vivo and in vitro. A, Pyd PDZ2 fused to eYFP is enriched at the plasma membrane mainly at sites of cell-cell contacts (left) and at PtdIns(4,5)P₂-rich membranes of macropinosomes induced by Arf6-Q67L (right) as shown by confocal micrographs. B, the membrane enrichment of eYFP-Pyd PDZ2 is lost upon ionomycin-induced PtdIns(4,5)P₂ breakdown (wide field micrographs). C, recombinant Pyd PDZ2 (50 μm) elutes as a dimer (30 kDa) from an analytical size exclusion column. D, urea-induced unfolding of Pyd PDZ2 F194W, as followed by fluorescence at 10 μm protein concentration. The observed transition was fitted to a standard two-state model. E, double reference-subtracted sensorgrams of Pyd PDZ2 perfused over biotin-C6-PtdIns(4,5)P₂ (40 RU) at different concentrations (as indicated) (also see Table 1). F, the interaction between Pyd PDZ2 (12 μm) and biotin-PtdIns(4,5)P₂ can be competed by the lipid headgroup (IP₃) with a Hill slope of −1.5, as determined by fitting the dose-response dependence. G, Pyd PDZ2 interacts with all seven PtdInsPs species in vitro (5% PtdInsPs in DOPC liposomes) with the highest apparent affinity for PtdIns(3,4,5)P3 (black dotted line and black triangles) and the lowest for the monophosphorylated species (gray lines and symbols). H, binding isotherms of Pyd PDZ2 with PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ in the background of composite liposomes intended to mimic the lipid composition of the plasma membrane (30% PC, 20% PS, 40% PE, 5% PtdIns, and 5% PtdInsPs). The binding isotherm of Pyd PDZ2 to 20% PS in DOPC liposomes is also indicated for comparison.