THE ACTIN-BINDING PROTEIN HISACTOPHILIN BINDS IN-VITRO TO PARTIALLY CHARGED MEMBRANES AND MEDIATES ACTIN COUPLING TO MEMBRANES

The interaction of the actin-binding protein hisactophilin from Dictyo stelium discoideum amoebae to partially charged Lipid membranes compos ed of mixtures of L-alpha-dimyristoylphosphatidylcholine (DMPG) with L -alpha-dimyristoylphosphatidylglycerol (DMPG) and L-alpha-phosphatidyl inositol 4,5-bisphosphate (PIP2) is studied by film balance experiment s, microfluorescence, and lateral diffusion measurements at low ionic strengths (similar to 20 mM). Excess surface concentrations and adhesi on energies of the protein are evaluated by the application of Gibbs l aw of surface excess as a function of charged lipid content. Protein e xpressed 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) a nd 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 bull; concentra tion, the adsorbed protein/lipid molar ratio increases with the molar fraction X(PG) Of charged lipid but saturates at about 50 mol% of DMPG . Natural hisactophilin (DIG-HIS) binding to PIP2-containing monolayer s is purely electrostatic at low bulk concentration c(b), and protein penetration dominates only at c(b) >68 nM. Fluorescence experiments de monstrate that the natural protein (DIG-HIS) can mediate the binding o f monomeric actin or very small oligomers to membranes, showing that t he adsorbed protein remains functional. In contrast, the recombinant h isactophilin (EC-HIS) can mediate only the membrane coupling of larger actin structures.