Figure 2. Biochemical activities of purified Bud6(L) on yeast formins. (A) Domain layout of Bud6. Bud6(L) (residues 489–788) and Bud6(S) (residues 550–788) each contain the dimeric “core” domain (yellow; residues 550–688) that binds Bni1 and the “flank” domain that binds G-actin (blue; residues 689–788). Bud6(L) additionally contains a short N-terminal sequence of unknown function (red). (B and C) Quantitative binding assays using supernatant depletion analysis. The indicated concentrations of GST-Bud6(L) immobilized on glutathione beads were incubated with 1.0 µM C-Bni1 (B) or C-Bnr1 (C). For each data point, an equivalent reaction was performed using GST alone immobilized on an equal volume of glutathione beads to correct for nonspecific binding. Coomassie-stained gels below each plot show representative reactions for each of the datasets collected. Each band represents formin remaining in the supernatant after incubation with immobilized protein and glutathione agarose precipitation. The data in each panel were pooled from two independent experiments. In the top gel of C, intervening lanes were removed for presentation purposes (white line), but all bands are from the same gel. The lines in B and C are fits to the equation B = a × c/(c + K), in which B is the fraction of C-Bni1 (B) or C-Bnr1 (C) bound, a is the amplitude of the binding interaction, c is the molar concentration of GST-Bud6(L), and K is the binding affinity (i.e., Kd). (D and E) 2 µM monomeric actin was polymerized in the presence of 10 nM C-Bni1 (D) or 2 nM Bnr1 (E), and the indicated concentrations of Bud6(L) in the absence (left) or presence (right) of 5 µM profilin. Data shown in each panel are from one representative example of four independent experiments. (F and G) Concentration-dependent effects of Bud6(L) on C-Bni1 or C-Bnr1 in the absence (F) or presence (G) of 5 µM profilin. The data in each panel were pooled from four independent experiments. The green lines in F and G are fits to the equation B = a × c/(c + K), in which B is the fold change in formin activity, c is the molar concentration of Bud6(L), a is the fold change in formin activity at saturating concentrations of Bud6(L), and K is the concentration of Bud6(L) at which B = 0.5 × a (i.e., the Kapp). The orange lines in F and G are drawn to guide the eyes. See Materials and methods for calculation of rates. AU, arbitrary unit.
Figure 2.

Biochemical activities of purified Bud6(L) on yeast formins. (A) Domain layout of Bud6. Bud6(L) (residues 489–788) and Bud6(S) (residues 550–788) each contain the dimeric “core” domain (yellow; residues 550–688) that binds Bni1 and the “flank” domain that binds G-actin (blue; residues 689–788). Bud6(L) additionally contains a short N-terminal sequence of unknown function (red). (B and C) Quantitative binding assays using supernatant depletion analysis. The indicated concentrations of GST-Bud6(L) immobilized on glutathione beads were incubated with 1.0 µM C-Bni1 (B) or C-Bnr1 (C). For each data point, an equivalent reaction was performed using GST alone immobilized on an equal volume of glutathione beads to correct for nonspecific binding. Coomassie-stained gels below each plot show representative reactions for each of the datasets collected. Each band represents formin remaining in the supernatant after incubation with immobilized protein and glutathione agarose precipitation. The data in each panel were pooled from two independent experiments. In the top gel of C, intervening lanes were removed for presentation purposes (white line), but all bands are from the same gel. The lines in B and C are fits to the equation B = a × c/(c + K), in which B is the fraction of C-Bni1 (B) or C-Bnr1 (C) bound, a is the amplitude of the binding interaction, c is the molar concentration of GST-Bud6(L), and K is the binding affinity (i.e., Kd). (D and E) 2 µM monomeric actin was polymerized in the presence of 10 nM C-Bni1 (D) or 2 nM Bnr1 (E), and the indicated concentrations of Bud6(L) in the absence (left) or presence (right) of 5 µM profilin. Data shown in each panel are from one representative example of four independent experiments. (F and G) Concentration-dependent effects of Bud6(L) on C-Bni1 or C-Bnr1 in the absence (F) or presence (G) of 5 µM profilin. The data in each panel were pooled from four independent experiments. The green lines in F and G are fits to the equation B = a × c/(c + K), in which B is the fold change in formin activity, c is the molar concentration of Bud6(L), a is the fold change in formin activity at saturating concentrations of Bud6(L), and K is the concentration of Bud6(L) at which B = 0.5 × a (i.e., the Kapp). The orange lines in F and G are drawn to guide the eyes. See Materials and methods for calculation of rates. AU, arbitrary unit.

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