title: 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.
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Jihong
authors.initials: J
authors.lastname: Tong
authors.forename: Lam
authors.initials: L
authors.lastname: Nguyen
authors.forename: Adriana
authors.initials: A
authors.lastname: Vidal
authors.forename: Sidney A
authors.initials: SA
authors.lastname: Simon
authors.forename: J H Pate
authors.initials: JH
authors.lastname: Skene
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McIntosh
mesh_terms: D019922:GAP-43 Protein / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D011485:Protein Binding; D053835:Unilamellar Liposomes / Q000737:chemistry*