Figure 3.

Activities of a cdc12p profilin-binding mutant in vitro. (A) Coomassie-stained SDS-PAGE gel of purified wild-type and ΔPBD mutant FH1FH2 proteins. (B) The effects of cdc12p wild-type or ΔPBD His₆ -FH1FH2 fusion proteins and S. pombe profilin (cdc3p) on actin polymerization, using a pyrene actin assembly assay. Actin (4 μM; 5% pyrene-labeled) was incubated with indicated concentrations of either wild-type or ΔPBD cdc12 FH1FH2p and/or cdc3p. F-actin accumulation was represented by the increase in pyrene fluorescence (arbitrary units) over time (s). (C) Filament elongation assay. Effects of wild-type and ΔPBD cdc12 FH1FH2 proteins on barbed-end elongation of pre-existing actin filaments in the presence or absence of profilin. Monomeric actin (0.5 μM, 10% pyrene labeled) was polymerized at the barbed ends of mechanically sheared actin filament seeds (333 nM) in the presence of indicated concentrations of cdc12 FH1FH2p and/or cdc3p.