C-Bud6 strongly enhances Bni1-mediated actin assembly. (A, B) Actin monomers (2 μM, 5% pyrene labeled) were polymerized in the presence of 10 nM C-Bni1 and/or 200 nM C-Bud6, in either the presence (A) or the absence (B) of 5 μM profilin. (C) Rates of actin assembly were measured for reactions as before but in the presence of different concentrations of actin as indicated. Rates of assembly were determined from the slopes of the curves and averaged.

Wild-type and mutant C-Bud6 interactions with Bni1. (A) Alignment of amino acid sequences in the C-terminal halves of fungal homologues of Bud6. Residues shaded in gray are conserved; those that were mutated to create the bud6 alleles are designated with a blue A. bud6-3 and bud6-5 were combined to produce bud6-35. (B) Coomassie-stained gel of purified wild-type and mutant C-Bud6 (489–788) polypeptides. Each lane contains ∼100 ng of protein. (C) Bead pull-down assays. Beads coated with 6His-C-Bni1 (1 μM final) or empty control beads were incubated for 10 min with soluble C-Bud6 polypeptides (1 μM final) and then centrifuged. Samples of supernatants (S) and pellets (P) were analyzed on gels by Coomassie staining. (D) Percentage of C-Bud6 depleted from the supernatants by Bni1-coated beads. Each bar represents an average of two independent trials. Error bars, SD.

Model for Bud6 function as an NPF. (A) Cocrystal structure of profilin (yellow) bound to G-actin (gray) with the Bud6 binding footprint highlighted (red circle). Surfaces on actin essential for Bud6–actin interactions in two-hybrid assays (Amberg et al., 1997) are red; residues that make lesser contributions to the interaction are orange. (B) Alignment of WH2-like sequence in Bud6 homologues and WH2 sequences in other proteins. Yellow, conserved residues; red, residues mutated in bud6-8. (C) Model for Bud6 mechanism. C-Bud6 (red cylinders) uses its WH2-like domain to bind G-actin (gray) and a separate binding site to interact with the Bni1 DAD. This positions actin monomers near the FH2 (blue) to promote nucleation, catalyzing formation of actin dimers that are captured by the FH2. Bud6 dissociates, and elongation proceeds with the FH1-domain recruiting profilin (yellow)-bound actin monomers to accelerate barbed-end growth. (D) Arp2/3 complex (blue) interacts with two molecules of WH2 domain–containing WASp (red cylinders; Padrick et al., 2008), which recruit actin monomers to promote nucleation by the filament end–capturing Arp2/3 complex. Although formins and Arp2/3 complex capture opposite ends of the filament, elongation proceeds in both cases by barbed-end addition.

C-Bud6 stimulates actin nucleation but not elongation. (A) Time-lapse TIRF microscopy comparing barbed-end elongation rates. Reactions contained 1.2 μM monomeric actin, different concentrations of C-Bni1 (top, 2.5 nM; middle, 1 nM; bottom, 5 nM; explained in Results), and 200 nM C-Bud6 or 3.6 μM profilin. Time points indicated. (B) Average rates of elongation (n = 10 filaments). Colored bars correspond to reactions in A. Gray bars are controls. (C) Examples of individual filaments increasing in length over time, color coded as in B. (D) Average rates of elongation measured in seeded elongation assays (error bar for C-Bni1 + C-Bud6 is small). (E) Two-step nucleation/elongation assays. First reactions contained 2 μM actin monomers ± 20 nM C-Bni1 ± 200 nM C-Bud6. Samples were removed at 50% polymerization (dotted line, inset) and used to seed a second reaction containing 1 μM actin monomers (10% pyrene labeled). (F) Quantification of number of Bni1-generated filaments by TIRF. (G) Example images from these experiments, in which Bni1-generated filaments (red arrowhead) are distinguished from spontaneously generated filaments (white arrowhead) due their threefold-faster elongation rates and reduced fluorescent intensities (see Materials and Methods). Bottom, highlighted Bni1-generated filaments (red lines).

Wild-type and mutant C-Bud6 interactions with G-actin. (A) Polymerization of 3 μM monomeric actin (5% pyrene labeled) in the presence of 4 μM wild-type or mutant C-Bud6 polypeptide. (B) Quantification of data as in A. Assembly rates for reactions containing actin + C-Bud6 were normalized to rates for reactions containing actin alone. Rates were determined from the slopes of the curves and averaged from three independent trials. Error bars, SD.

Wild-type and mutant C-Bud6 effects on Bni1-mediated actin assembly. (A) Concentration-dependent effects of wild-type C-Bud6 on the assembly of 2 μM monomeric actin (5% pyrene labeled) by 10 nM C-Bni1. (B) Comparison of the effects of 200 nM wild-type or mutant C-Bud6 on actin assembly by 10 nM C-Bni1 as in A. (C) Concentration-dependent effects of wild-type and mutant C-Bud6 on rate of actin assembly by 10 nM C-Bni1 as in A. Rates determined from slopes of raw curves in A and in Supplemental Figure S2, A and B, and similar data sets for C-Bud6-1 and C-Bud6-6. Each data point on the graphs is an average of at least two independent trials. To calculate fold increase (y-axis), rate of actin assembly for C-Bud6 + C-Bni1 was divided by rate of actin assembly for C-Bni1 alone. (D) Summary table of data from Figures 3–5.

Effects of bud6 alleles on cell growth and actin organization in haploids. (A) Immunoblot of whole-cell extracts from haploid strains, probed with anti-hemagglutinin and anti-tubulin antibodies (loading control). The control lane is from a wild-type strain expressing Bud6 with no tag. (B) Strains were serial diluted and grown at 25, 34, and 37°C on YEPD plates. (C) The strains were grown to log phase at 25°C, fixed, and stained with Alexa 488–phalloidin. Scale bar, 10 μm. (D) Quantification of F-actin phenotypes after fixation and actin staining as in C. For each strain, >200 budded cells were scored and categorized as follows: 1) Robust cables in mother; cables sometimes visible in bud; polarized patches. 2) Fewer and thinner cables in the mother, sometimes with a disorganized appearance; cables occasionally visible in bud; polarized patches. 3) Very few visible cables in the mother; no cables in bud; polarized patches. 4) No visible cables in the mother or bud; depolarized patches.

Effects of bud6 alleles on cell growth and actin organization in diploids. (A) Diploid strains were serial diluted and grown at 25, 34, and 37°C on YEPD plates. (B, C) The strains were grown to log phase at 25°C, fixed, and stained with Alexa 488–phalloidin. Scale bars, 10 μm. (D) Quantification of F-actin phenotypes. Cells were grown to log phase at 25°C, then fixed and stained with Alexa 488–phalloidin as in B and C, or shifted to 37°C for an additional 2 h before fixation. For each strain, >200 budded cells were scored and categorized as in Figure 6D.