Figure 6. Bil1 unmasks Bud6(L) stimulatory effects on Bnr1. (A) Quantitative GST pull-down assays using indicated concentrations of purified immobilized GST-Bil1 and soluble Bud6(L) or soluble Bud6(S). Data shown were pooled from three independent experiments. The line in A is a fit to the equation B = a × c/(c + K), in which B is the fraction of Bud6 bound, a is the amplitude of the binding interaction, c is the molar concentration of GST-Bil1, and K is the binding affinity (i.e., Kd). (B) Bulk actin assembly assays. 2 µM monomeric actin was assembled in the presence of 5 µM yeast profilin and 2 nM C-Bnr1. Where indicated, 100 nM Bud6(L) and/or 500 nM Bil1 was included. The data shown are one representative example of four independent experiments. AU, arbitrary unit. (C) Concentration-dependent effects of Bil1 on Bnr1-mediated actin assembly in the presence of either Bud6(L) or Bud6(S). Data shown were pooled from four independent experiments. The line in C is a fit to the equation B = a × c/(c + K), in which B is the percent increase in actin assembly, c is the molar concentration of Bil1, a is the percent increase in actin assembly at saturating concentrations of Bil1, and K is the concentration of Bil1 at which B = 0.5 × a (i.e., the Kapp). (D) Actin filament densities observed using TIRF-M for reactions containing 0.5 µM monomeric actin and 0.2 nM C-Bnr1. Where indicated, 100 nM Bud6(L) and/or 500 nM Bil1 was included. The data shown are one representative example of two independent experiments. Bar, 20 µm. (E) Quantification of filament densities observed by TIRF-M under conditions described in D. Each bar represents a mean of nine fields of view from three independent experiments; error bars show SEM. (F) Mean elongation rates of filaments observed in D, calculated as in Fig. 4 F. Data shown are from two independent experiments, where for each condition in each experiment >15 filaments were measured; error bars show SEM. (G and H) Working model for Bud6 regulation of actin assembly by Bni1 (G) and Bnr1 (H). Bud6 dimers (yellow), which are bound to actin monomers (gray), interact with the Bud6 binding site (BBS) on the formin (green). Because of differences in the position of the BBS in Bni1 versus Bnr1, the interaction of Bud6 leads to distinct functional effects. In the case of Bni1, Bud6 binding results in enhanced actin nucleation, whereas in the case of Bnr1, Bud6 binding obstructs nucleation as a result of the proximity of the regulatory (R) region of Bud6 to the FH2 domain. Binding of Bil1 (blue) to the R region of Bud6 relieves the obstruction, triggering enhanced nucleation. Profilin (orange) is also required for the Bud6 stimulatory effects on Bnr1, although the mechanistic basis for this requirement is still unclear.
Figure 6.

Bil1 unmasks Bud6(L) stimulatory effects on Bnr1. (A) Quantitative GST pull-down assays using indicated concentrations of purified immobilized GST-Bil1 and soluble Bud6(L) or soluble Bud6(S). Data shown were pooled from three independent experiments. The line in A is a fit to the equation B = a × c/(c + K), in which B is the fraction of Bud6 bound, a is the amplitude of the binding interaction, c is the molar concentration of GST-Bil1, and K is the binding affinity (i.e., Kd). (B) Bulk actin assembly assays. 2 µM monomeric actin was assembled in the presence of 5 µM yeast profilin and 2 nM C-Bnr1. Where indicated, 100 nM Bud6(L) and/or 500 nM Bil1 was included. The data shown are one representative example of four independent experiments. AU, arbitrary unit. (C) Concentration-dependent effects of Bil1 on Bnr1-mediated actin assembly in the presence of either Bud6(L) or Bud6(S). Data shown were pooled from four independent experiments. The line in C is a fit to the equation B = a × c/(c + K), in which B is the percent increase in actin assembly, c is the molar concentration of Bil1, a is the percent increase in actin assembly at saturating concentrations of Bil1, and K is the concentration of Bil1 at which B = 0.5 × a (i.e., the Kapp). (D) Actin filament densities observed using TIRF-M for reactions containing 0.5 µM monomeric actin and 0.2 nM C-Bnr1. Where indicated, 100 nM Bud6(L) and/or 500 nM Bil1 was included. The data shown are one representative example of two independent experiments. Bar, 20 µm. (E) Quantification of filament densities observed by TIRF-M under conditions described in D. Each bar represents a mean of nine fields of view from three independent experiments; error bars show SEM. (F) Mean elongation rates of filaments observed in D, calculated as in Fig. 4 F. Data shown are from two independent experiments, where for each condition in each experiment >15 filaments were measured; error bars show SEM. (G and H) Working model for Bud6 regulation of actin assembly by Bni1 (G) and Bnr1 (H). Bud6 dimers (yellow), which are bound to actin monomers (gray), interact with the Bud6 binding site (BBS) on the formin (green). Because of differences in the position of the BBS in Bni1 versus Bnr1, the interaction of Bud6 leads to distinct functional effects. In the case of Bni1, Bud6 binding results in enhanced actin nucleation, whereas in the case of Bnr1, Bud6 binding obstructs nucleation as a result of the proximity of the regulatory (R) region of Bud6 to the FH2 domain. Binding of Bil1 (blue) to the R region of Bud6 relieves the obstruction, triggering enhanced nucleation. Profilin (orange) is also required for the Bud6 stimulatory effects on Bnr1, although the mechanistic basis for this requirement is still unclear.

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