![Figure 3. Bud6(S) stimulates C-Bni1 and C-Bnr1 in actin assembly assays. (A and C) 2 µM monomeric actin was polymerized in the presence of 10 nM C-Bni1 (A) or 2 nM C-Bnr1 (C), and the indicated concentrations of Bud6(S) 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. (B and D) Concentration-dependent effects of Bud6(S) on C-Bni1 (B) or C-Bnr1 (D) actin assembly activity, performed as in A and C. Filled triangles indicate reactions performed in the presence of 5 µM yeast profilin; hollow triangles indicate identical reactions lacking yeast profilin. Data shown are from one representative example of four independent experiments. See Materials and methods for calculation of rates. Both lines in B and the solid line in D are fits to the equation B = a × c/(c + K), in which B is the percent increase in formin activity, c is the molar concentration of Bud6(S), a is the percent increase in formin activity at saturating concentrations of Bud6(S), and K is the concentration of Bud6(S) at which B = 0.5 × a (i.e., the Kapp). The dashed line in D is drawn to guide the eyes. (E and F) Quantitative GST pull-down assays performed as in Fig. 2 (B and C) using indicated concentrations of immobilized GST-Bud6(S) and 1.0 µM of soluble C-Bni1 (E) or C-Bnr1 (F). The data in each panel were pooled from two independent experiments. The lines in E and F are fits to the equation B = a × c/(c + K), in which B is the fraction of C-Bni1 (E) or C-Bnr1 (F) bound, a is the amplitude of the binding interaction, c is the molar concentration of GST-Bud6(S), and K is the binding affinity (i.e., Kd). (G) Quantitative binding assays using 150 nM pyrene-labeled actin (100% labeling efficiency) and indicated amounts of either Bud6(L) or Bud6(S). The lines in G are fits to the equation B = ([c + a + k] − [(c + a + k)2 − 4 × c × a]0.5)/(2 × a), in which B is the fraction of G-actin bound, c is the molar concentration of Bud6, a is the molar concentration of G-actin (170 nM in all cases), and k is the binding affinity (i.e., Kd). AU, arbitrary unit.](https://cdn.rupress.org/rup/content_public/journal/jcb/201/4/10.1083_jcb.201212059/5/jcb_201212059_fig3.jpeg?Expires=1767669813&Signature=rOVTslfVqzcMqnYpxSXV4ltO609M0BJUHcqHVshR2U1dhSKHQmhSyZf-XvQEE2N6e6Oi7cjk7Ux-OftNmfajx-sb1-xiX-LIZmjpGCh1B4MF3KF9YbxN5au9UAOLBDjl1hJ65o0WbE2ql7o2RY-EfC8HMEYNmNQvwo58D4rdR4FXL5o~uYG52q~7yQj2lYQTBNiLylSFYDtvw01rx4kKP1O78sK8TpoC0EDCuzcMHPqFixQIdQLogwk-IWdeW-QcT-OL9ltq2hufiif6ztNbJeIW-Ejh2ekTP0lpbV8W-qvT2SJU2qHNlamR6-qOCQSknqKWKYRtpphlF8bVfQfjsg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Bud6(S) stimulates C-Bni1 and C-Bnr1 in actin assembly assays. (A and C) 2 µM monomeric actin was polymerized in the presence of 10 nM C-Bni1 (A) or 2 nM C-Bnr1 (C), and the indicated concentrations of Bud6(S) 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. (B and D) Concentration-dependent effects of Bud6(S) on C-Bni1 (B) or C-Bnr1 (D) actin assembly activity, performed as in A and C. Filled triangles indicate reactions performed in the presence of 5 µM yeast profilin; hollow triangles indicate identical reactions lacking yeast profilin. Data shown are from one representative example of four independent experiments. See Materials and methods for calculation of rates. Both lines in B and the solid line in D are fits to the equation B = a × c/(c + K), in which B is the percent increase in formin activity, c is the molar concentration of Bud6(S), a is the percent increase in formin activity at saturating concentrations of Bud6(S), and K is the concentration of Bud6(S) at which B = 0.5 × a (i.e., the Kapp). The dashed line in D is drawn to guide the eyes. (E and F) Quantitative GST pull-down assays performed as in Fig. 2 (B and C) using indicated concentrations of immobilized GST-Bud6(S) and 1.0 µM of soluble C-Bni1 (E) or C-Bnr1 (F). The data in each panel were pooled from two independent experiments. The lines in E and F are fits to the equation B = a × c/(c + K), in which B is the fraction of C-Bni1 (E) or C-Bnr1 (F) bound, a is the amplitude of the binding interaction, c is the molar concentration of GST-Bud6(S), and K is the binding affinity (i.e., Kd). (G) Quantitative binding assays using 150 nM pyrene-labeled actin (100% labeling efficiency) and indicated amounts of either Bud6(L) or Bud6(S). The lines in G are fits to the equation B = ([c + a + k] − [(c + a + k)2 − 4 × c × a]0.5)/(2 × a), in which B is the fraction of G-actin bound, c is the molar concentration of Bud6, a is the molar concentration of G-actin (170 nM in all cases), and k is the binding affinity (i.e., Kd). AU, arbitrary unit.
