Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: an electrostatic model and experimental results.

Direct fluorescence digital imaging microscopy observations demonstrate that a basic peptide corresponding to the effector region of the myristoylated alanine-rich C kinase substrate (MARCKS) self-assembles into membrane domains enriched in the acidic phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). We show here that pentalysine, which corresponds to the first five residues of the MARCKS effector region peptide and binds to membranes through electrostatic interactions, also forms domains enriched in PS and PIP2. We present a simple model of domain formation that represents the decrease in the free energy of the system as the sum of two contributions: the free energy of mixing of neutral and acidic lipids and the electrostatic free energy. The first contribution is always positive and opposes domain formation, whereas the second contribution may become negative and, at low ionic strength, overcome the first contribution. Our model, based on Gouy-Chapman-Stern theory, makes four predictions: 1) multivalent basic ligands, for which the membrane binding is a steep function of the mole fraction of acidic lipid, form domains enriched in acidic lipids; domains break up at high concentrations of either 2) basic ligand or 3) monovalent salt; and 4) if multivalent anionic lipids (e.g., PIP2) are present in trace concentrations in the membrane, they partition strongly into the domains. These predictions agree qualitatively with experimental data obtained with pentalysine and spermine, another basic ligand.

Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.