Phosphatidylinositol (4, 5)-bisphosphate targets double C2 domain protein B to the plasma membrane.

Double C2 domain protein B (DOC2B) is a high-affinity Ca2+ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)-bisphosphate [PI(4, 5)P2 ] and target SNAREs (t-SNAREs). Here, we show that PI(4, 5)P2 hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t-SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P2 in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P2 -binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P2 are required for the DOC2B-PM association and revealing multiple PI(4, 5)P2 -C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP-independent and occurs in a diffusion-like manner. Our data suggest that the Ca2+ -triggered translocation of DOC2B is diffusion-driven and aimed at PI(4, 5)P2 -containing membranes.