Figure 6. The phosphomimetic Eps8 (Eps8-SETE) mutant has a 10-fold reduced barbed-end capping activity with respect to Eps8 WT or phosphoimpaired Eps8 (Eps8-SATA) mutant when in complex with Abi1.

(A) The indicated, increasing concentrations of equimolar amounts of purified His–Eps8 WT, SATA, and SETE (Eps8, SATA, and SETE) and GST-Abi1 were incubated with 2.5 µM G-actin (10% pyrenyl-labeled) in polymerization buffer. Actin polymerization was initiated by spectrin–actin seeds as described in Materials and Methods. GST was used as a control. (B) The relative inhibition of barbed-end polymerization rate at the indicated concentrations of the Eps8-WT:Abi1, Eps8-SATA:Abi1, and Eps8-SETE:Abi1 complexes is shown. Under suboptimal, nonsaturating conditions (100 nM concentrations), the Eps8-SETE:Abi1 complex is less effective in inhibiting barbed-end growth than Eps8 WT or Eps8-SATA:Abi1 complexes. Three independent experiments were performed. Error bars indicate SEM. (C) The relative inhibition of barbed-end polymerization at the various, indicated concentrations of the WT Eps8:Abi1 or Eps8-SETE:Abi1 complexes. The concentrations of Eps8-WT:Abi1 and Eps8-SETE:Abi1 at which half-maximal inhibition (IC₅₀) was observed were 60 and 200 nM, respectively, which correspond to a concentration of the respective complexes of: Eps8(WT):Abi1 = 0.15 nM; Eps8-SETE:Abi1 = 1.5 nM (based on calculated thermodynamic constant of association between Eps8 and Abi1 (K _d = 35 µM [72]). The data shown are representative of four independent experiments with similar results. (D) Eps8 phosphomutants bind Abi1 similar to Eps8 WT. Lysates of cells expressing GFP-Eps8 WT or SATA or SETE (indicated at the top) were incubated with immobilized GST, or GST-PIN1, used as negative control, or GST-Abi1 (asterisk [*] in the figure). The lower bands on the GST-Abi1 lanes are likely premature termination or degradative products of the Abi1 moiety of the fusion protein. Lysates and bound proteins were immunoblotted with the indicated antibodies (on the right). IVB, in vitro bound.