Fig. 1. Yeast B subunit binds F-actin. F-actin was diluted to 800 nmol l^–1 and stabilized with 5 µmol l^–1 phalloidin, mixed with various concentrations of Vma2p-MBP, and subjected to ultracentrifugation. Pellets and supernatants were collected, separated by SDS-PAGE and stained with Coomassie Brilliant Blue (A). M, markers (top to bottom; 110 kDa, 67 kDa, 43 kDa); lane 1, no actin, 2 µmol l^–1 Vma2p-MBP; lane 2, 0.8 µmol l^–1 F-actin, 0.4 µmol l^–1 Vma2p-MBP; lane 3, 0.8 µmol l^–1 F-actin, 0.8 µmol l^–1 Vma2p-MBP; lane 4, 0.8 µmol l^–1 F-actin, 1.2 µmol l^–1 Vma2p-MBP; lane 5, 0.8 µmol l^–1 F-actin, 1.6 µmol l^–1 Vma2p-MBP; lane 6, 0.8 µmol l^–1 F-actin, 2.0 µmol l^–1 Vma2p-MBP. The pellets were loaded with twice the relative amount of protein compared with supernatants. (B) Actin was polymerized and diluted into solutions containing phalloidin (5 µmol l^–1) and yeast B-MBP as described under Materials and methods. The final actin concentration, indicated by the broken line, was 800 nmol l^–1. Following incubation and centrifugation, the supernatants and pellets were collected and the amount of fusion protein present was determined by densitometry of Coomassie-stained gels. (C) Varying amounts of Vma2p-MBP were added to F-actin and subjected to high speed centrifugation as described under Materials and methods. Pellets and supernatants were analyzed by SDS-PAGE and densitometry was used to determine the amount of protein in the Coomassie-stained gels. The K_d was calculated based on the finding that the fusion protein binding saturated at 1 mol of fusion protein per mol of actin filament subunit (established in A and B).