Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters spin-labeled phosphatidylinositol 4,5-bisphosphate in lipid bilayers

The myristoylated alanine-rich protein kinase C substrate (MARCKS) may function to sequester phosphoinositides within the plane of the bilayer. To characterize this interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2), a novel spin-labeled derivative, proxyl-PIP2, was synthesized and characterized. In the presence of molecules known to bind PI(4,5)P-2 the EPR spectrum of this label exhibits an increase in line width because of a decrease in label dynamics, and titration of this probe with neomycin yields the expected 1:1 stoichiometry. Thus, this probe can be used to quantitate the interactions made by the PI(4,5)P-2 head group within the bilayer. In the presence of a peptide comprising the effector domain of MARCKS the EPR spectrum broadens, but the changes in line shape are modulated by both changes in label correlation time and spin-spin interactions. This result indicates that at least some proxyl-PIP2 are in close proximity when bound to MARCKS and that MARCKS associates with multiple PI(4,5)P-2 molecules. Titration of the proxyl-PIP2 EPR signal by the MARCKS-derived peptide also suggests that multiple PI(4,5)P-2 molecules interact with MARCKS. Site-directed spin labeling of this peptide shows that the position and conformation of this protein segment at the membrane interface are not altered significantly by binding to PI(4,5)P-2. These data are consistent with the hypothesis that MARCKS functions to sequester multiple PI(4,5)P-2 molecules within the plane of the membrane as a result of interactions that are driven by electrostatic forces.