The actin-binding protein hisactophilin binds in vitro to partially charged membranes and mediates actin coupling to membranes.

Abstract

The interaction of the actin-binding protein hisactophilin from Dictyostelium discoideum amoebae to partially charged lipid membranes composed of mixtures of L-alpha-dimyristoylphosphatidylcholine (DMPC) with L-alpha-dimyristoylphosphatidylglycerol (DMPG) and L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is studied by film balance experiments, microfluorescence, and lateral diffusion measurements at low ionic strengths (approximately 20 mM). Excess surface concentrations and adhesion energies of the protein are evaluated by the application of Gibbs law of surface excess as a function of charged lipid content. Protein expressed in E. coli lacking a myristic acid chain (EC-HIS) and natural protein with a fatty acid (DIC-HIS) isolated from Dictyostelium cells are compared. For mixtures of DMPG and DMPC, protein binding leads to an increase in lateral pressure of the monolayer (at constant area) and causes strong lipid immobilization pointing to partial penetration of the protein into the lipid layer. The natural protein causes a much stronger immobilization than does EC-HIS. For a given bulk concentration, the adsorbed protein/lipid molar ratio increases with the molar fraction chi PG of charged lipid but saturates at about 50 mol% of DMPG. Natural hisactophilin (DIC-HIS) binding to PIP2-containing monolayers is purely electrostatic at low bulk concentration cb, and protein penetration dominates only at cb > 68 nM. Fluorescence experiments demonstrate that the natural protein (DIC-HIS) can mediate the binding of monomeric actin or very small oligomers to membranes, showing that the adsorbed protein remains functional. In contrast, the recombinant hisactophilin (EC-HIS) can mediate only the membrane coupling of larger actin structures.