YEAST ACTIN - POLYMERIZATION KINETIC-STUDIES OF WILD-TYPE AND A POORLY POLYMERIZING MUTANT

Wild-type actin and a mutant actin were isolated from yeast (Saccharom yces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin, Whil e similar to the properties of skeletal muscle ai:tin, there are sever al important differences between the wild-type yeast and muscle actins . First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result f rom a difference in the steps involving formation of the nucleating sp ecies. Second, as measured with pyrene-labeled yeast actin, but not wi th intrinsic fluorescence, there is an overshoot in the fluorescence t hat has not been observed with skeletal muscle actin under the same co nditions. Third, in order to simulate the polymerization process of wi ld-type yeast actin it is necessary to assume some fragmentation of th e filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutan t actin (R177A/D179A) has also been isolated and studied, The mutation s are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elonga tion rates of the wild type and the mutant differ by only about 3-fold , and the slower polymerization of the mutant appears to result primar ily from poorer nucleation.