Mutations of conserved residues in the FH2 domain impair formin activity in vitro and in vivo. (A) Schematic of the budding yeast Bni1(FH2) “tethered dimer” structure (pdb ly64; ). The lasso, linker, knob, coiled-coil, and post from one hemidimer (purple) are labeled. Mutations in conserved residues in the lasso–post actin-binding (blue and green), lasso–post dimerization and actin-binding (orange), and knob actin-binding (red) regions are indicated. (B, D, and F) Spontaneous assembly of 20% pyrene-labeled 2.5 μM Mg-ATP actin monomers. Dependence of the actin-assembly rate (slope) on the concentration of the indicated (B) MBP-Cdc12(FH2), (D) MBP-Fus1(FH2), or (F) MBP-For3(FH2) constructs. (C, E, and G) Complementation of mutant fission yeast formin strains by expression of medium-strength p572-41X-formin(full length)-GFP constructs containing the indicated FH2 domain point mutations. Error bars specify SD of triplicate experiments. (C) Percent of temperature-sensitive mutant cdc12-112 cells (strain KV427) with one, two, or more than nuclei following 16 h at 36°C in Edinburgh minimal media (EMM). (E) Percentage of h90 fus1Δ cells (strain EG999; ) that have mated and then formed tetrads (Tetrads) or not (Conjugated) following 36 h in ME mating medium at 25°C. (G) Average morphology (cell length/width) of for3Δ cells (strain BFY9; ), following 20 h at 25°C in EMM.