F-actin-like ATPase Activity in a Polymerization-defective Mutant Yeast Actin (V266G/L267G)

Abstract

Polymerization increases a low level G-actin ATPase activity yielding ADP-Pi F-actin and then ADP F-actin following release of P i. By monitoring Pi release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val266 and Leu267, were mutated to Gly. Although GG-actin does not polymerize by itself in vitro, GG cells are viable. We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form. A plot of Pi release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of ∼8 μ for Mg2+ GG-actin and 11 μ for Ca2+ GG-actin. In contrast to WT-actin, Pi release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region. Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers. Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.