The Bud3 DH domain is necessary for the axial budding pattern. (A) Budding pattern (percentage) of haploid WT, bud3Δ, bud3ΔCR1, and bud3ΔCR2. The sequences of CR1, CR2, and CR3 are shown. Residues that are deleted or substituted are marked with lines or asterisks, respectively. A mean percentage of each pattern is shown from three independent countings (n = 300; SEM < 3%). An example of bud scar (circles) distribution of each budding pattern is shown. (B) Coimmunoprecipitation of Bud3-Myc or Bud3ΔCR2-Myc with Bud4. The Bud3-Myc (either BUD4 or bud4Δ) and Bud3ΔCR2-Myc strains were subjected to immunoprecipitation using anti-Bud4 antibodies. Proteins were analyzed by immunoblotting with anti-Myc antibody (top) or anti-Bud4 antibodies (bottom). (C) TAP pull-down assays using the AXL1-TAP strains (with either WT or a mutant BUD3 allele) and an untagged strain. After pull-down assays, proteins were analyzed by immunoblotting with anti-Bud4 antibodies. (D and E) TAP pull-down assays using the BUD5-TAP (D) and BUD5-TAP AXL1-Myc (E) strains with the indicated BUD3 alleles. The first lane shows a control with untagged BUD5 (D) or untagged AXL1 (E). Proteins were analyzed by immunoblotting with anti-Bud4 antibodies (D) or the anti-Myc antibody (E). Ab, antibody; IP, immunoprecipitation.