Abstract
This chapter discusses the purification of actin and actin-associated proteins from yeast and presents updated methods for isolating yeast actin and actin-associated proteins that may facilitate the biochemical analyses of the yeast actin cytoskeleton. It also presents a detailed outline of the DNase I affinity method for purifying yeast actin. This method is based on the reconstitution of actin assembly in soluble yeast extracts and allows a one-step isolation of a semi-intact actin cytoskeleton. Some of the most common uses for purified yeast actin are (1) testing the ability of a protein to cosediment with filamentous actin, (2) testing the effects of an actin-binding protein on actin assembly and/or actin disassembly kinetics, (3) determining the structures and/or activities of mutant actins. Using additional chromatography steps, various components can be isolated, providing the opportunity to study the activities of native actin-associated proteins from wild-type and mutant strains.