Identification of yeast cofilin residues specific for actin monomer and PIP2 binding

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long a-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this a-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this a-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P-2 binding site by using a collection of cofilin mutants. The PI(4,5)P-2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneoulsy with more than one PI(4,5)P-2 molecule. The PI(4,5)P-2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P-2. The biological roles of actin and PI(4,5)P-2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P-2 binding.