Gin4-catalyzed phosphorylation of Bni5 promotes its dissociation from the septin hourglass. (A) Left: Diagram showing Gin4-catalyzed phosphorylation sites in Bni5 identified by in vitro kinase assay. Right: Summary of phospho-deficient and -mimic bni5 alleles and their respective mutation sites. (B) Time-lapse analysis of GFP-tagged Bni5 phospho-mutants at the bud neck. Left: Dissociation kinetics of GFP-Bni5 and its phospho-mutants were quantified over time. Absolute values (left) and normalized data (right) are shown. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates variants of Bni5. Strains used: YEF11546 [mScarlet-TUB1 GFP-BNI5(FL)], YEF12444 (mScarlet-TUB1 GFP-bni5-6A), and YEF12445 (mScarlet-TUB1 GFP-bni5-6DE). (C) Time-lapse analysis of the septin HDR transition in WT and Bni5 phospho-mutants. Top: Montages of Cdc10-mScarlet created from time-lapse series taken with 1.5-min intervals. Bottom: The dissociation kinetics of Cdc10-mScarlet during the HDR transition in indicated strains are shown. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Strains used: YEF11772 (CDC10-mScarlet Venus-TUB1 GFP-BNI5(FL)-6A), YEF12456 (CDC10-mScarlet Venus-TUB1 GFP-BNI5(FL)-6A), and YEF12457 (CDC10-mScarlet Venus-TUB1 GFP-BNI5(FL)-6DE). (D) In vitro binding of Bni5-Ext-HR2 variants to septin filaments. Left: SDS-PAGE stained with Coomassie blue, showing purified proteins used as input. Input septin complexes contained five septins: Cdc10, His6-Cdc12, Cdc3, Cdc11, and Shs1. Right: Results of binding assays, showing Coomassie blue stained of GST-tagged proteins co-sedimented with septin filaments by ultracentrifugation. Asterisk indicates variants of Bni5-Ext-HR2. kDa = kiloDalton. A representative result from three independent experiments is shown. Source data are available for this figure: SourceData F5.