Abstract
Actin binding in the β-propeller domain is a defining feature of all coronin proteins. Here, we study the actin binding surface of yeast coronin (Crn1) through an extensive mutagenesis of highly conserved charged and solvent-exposed residues. We use both biochemical assays for actin bundling and in vivo tests of cell growth defects upon overexpression and subcellular localization patterns to determine the severity of defect in actin binding for specific mutants. In addition, we analyze the effect of mutations in the coiled-coil domain on actin organizational defects in cells overexpressing coronin. Through these analyses, we have isolated three β-propeller mutants, crn1-2, crn1-15, and crn1-36, that are defective in actin binding, as well as one residue in the coiled-coil domain, L628, required for mediating overexpression defects. This has produced insight into the actin binding surface of the Crn1 β-propeller, from which additional analyses of the role of actin binding in the varied functions of coronin can be carried out. Further, these mutants have allowed us to test the importance of coronin-actin interactions in vivo for its cellular functions.