Bni5 interacts with septin filaments and Elm1 via overlapping regions at its C terminus. (A) Diagram of Bni5 structural features. Top: AlphaFold-predicted structure of Bni5. The HR1, IDR, Ext, HR2, and HR3 regions are color-coded in brown, gray, purple, blue, and orange, respectively. Bottom: IUPred3 prediction of intrinsic disorder. Scores above the dotted line indicate a higher probability of disorder. Shaded boxes correspond to the same regions as in the structural model. (B) Time-lapse analysis of Bni5 fragment co-localization with septins at the budding site. Montages of GFP-tagged Bni5 fragments with respect to Cdc3-mCherry were created from time-lapse series taken with 2.5-min intervals. Arrowhead indicates the onset of GFP-HR2-HR3 accumulation. Asterisk indicates variants of Bni5. Strains used: YEF11053 (bni5Δ CDC3-mCherry [GFP-BNI5(FL)]), YEF11181 (bni5Δ CDC3-mCherry [GFP-bni5(306-339)]), YEF11040 (bni5Δ CDC3-mCherry [GFP-bni5(340-448)]), and YEF11039 (bni5Δ CDC3-mCherry [GFP-bni5(306-448)]). (C) Bni5 binds to septin filaments via Ext-HR2. Left: SDS-PAGE stained with Coomassie blue, showing purified proteins used as input for in vitro–binding assay. Right: In vitro–binding assay results (Coomassie blue–stained gel) for indicated GST-tagged proteins co-sedimented with septin filaments by ultracentrifugation; kDa = kiloDalton. Green boxes represent GST-fusion proteins. Asterisk indicates variants of Bni5. A representative result from three independent experiments is shown. See also Fig. S2, B and C. (D) Localization of GFP-Ext-HR2 in WT and septin mutants. Cells in the exponential growth phase were imaged. Strains used: YEF11207 (BNI5 CDC3-mCherry [GFP-BNI5(306-393)]), YEF11819 (BNI5 cdc11Δ CDC3-mCherry [GFP-BNI5(306-393)]), YEF11811 (BNI5 shs1Δ CDC3-mCherry [GFP-BNI5(306-393)]), and YEF11812 (BNI5 cdc10Δ CDC3-mCherry [GFP-BNI5(306-393)]). See also Fig. S2 A. (E) Localization of Bni5 fragments in bni5Δ elm1Δ cells. Cells in the exponential growth phase were imaged. Strains used: YEF11190 (bni5Δ elm1Δ CDC3-mCherry [GFP-BNI5(FL)]), YEF11197 (bni5Δ elm1Δ CDC3-mCherry [GFP-bni5(306-393)]), YEF11194 (bni5Δ elm1Δ CDC3-mCherry [GFP-bni5(340-448)]), and YEF11193 (bni5Δ elm1Δ CDC3-mCherry [GFP-bni5(306-448)]). (F) HR2-containing fragments of Bni5 interact with Elm1. Top left: SDS-PAGE stained with Coomassie blue, showing purified proteins used in binding assay. Bottom left: In vitro–binding assay results (Coomassie blue–stained gel) for the indicated GST-tagged proteins bound to glutathione resin and their ability to pull down His-SUMO-Elm1(421-640). Green boxes represent GST and GST-tagged Bni5 fragments shown at top right. Bottom right: Immunoblotted membrane with antibody against His; kDa = kiloDalton. Asterisk indicates variants of Bni5. A representative result from three independent experiments is shown. (G) Quantification of Bni5 fragment localization at the bud neck. Left: Representative images of cells in the exponential growth phase. Right: Quantification of GFP signal intensities at the bud neck in cells with a small or medium bud (S/G2 cells). Green circles, blue bars, and gray bars represent individual data points, mean values, and SD, respectively. Asterisk indicates variants of Bni5. P values were determined using a two-sided Mann–Whitney U test. Strains used: YEF11821 [GFP-BNI5(FL)], YEF11823 [GFP-bni5(306-393)], YEF11824 [GFP-bni5(340-448)], and YEF11825 [GFP-bni5(306-448)]. (H) Dosage suppression of the cdc12-6 septin mutant by Bni5 fragments. Left: Summary diagram showing suppression (blue) and non-suppression (black) by the indicated BNI5 alleles. Bold lines and dashed lines represent the presence and absence of Bni5 regions, respectively. Right: Spot assay result after 3 days of incubation at 25°C or 32°C. Strains were generated by introducing the pUG36-Bni5* plasmid series (Table S2) into YEF11247 (cdc12-6 bni5Δ). Source data are available for this figure: SourceData F3.