Role of GAP-43 in Sequestering Phosphatidylinositol 4,5-Bisphosphate to Raft Bilayers

Abstract

The lipid phosphatidylinositol 4,5-bisphosphate (PIP 2) is critical for a number of physiological functions, and its presence in membrane microdomains (rafts) appears to be important for several of these spatially localized events. However, lipids like PIP 2 that contain polyunsaturated hydrocarbon chains are usually excluded from rafts, which are enriched in phospholipids (such as sphingomyelin) containing saturated or monounsaturated chains. Here we tested a mechanism by which multivalent PIP 2 molecules could be transferred into rafts through electrostatic interactions with polybasic cytoplasmic proteins, such as GAP-43, which bind to rafts via their acylated N-termini. We analyzed the interactions between lipid membranes containing raft microdomains and a peptide (GAP-43P) containing the linked N-terminus and the basic effector domain of GAP-43. In the absence or presence of nonacylated GAP-43P, PIP 2 was found primarily in detergent-soluble membranes thought to correspond to nonraft microdomains. However, when GAP-43P was acylated by palmitoyl coenzyme A, both the peptide and PIP 2 were greatly enriched in detergent-resistant membranes that correspond to rafts; acylation of GAP-43P changed the free energy of transfer of PIP 2 from detergent-soluble membranes to detergent-resistant membranes by −1.3 kcal/mol. Confocal microscopy of intact giant unilamellar vesicles verified that in the absence of GAP-43P PIP 2 was in nonraft microdomains, whereas acylated GAP-43P laterally sequestered PIP 2 into rafts. These data indicate that sequestration of PIP 2 to raft microdomains could involve interactions with acylated basic proteins such as GAP-43.