Bni5 recruits Myo1 to the septin hourglass via HR1. (A) Diagram of putative and demonstrated interactions among Bni5, Myo1, and Iqg1. The mTD1 and targeting domain 2 (TD2) in the tail of Myo1 mediate its direct or indirect interactions with Bni5 and Iqg1, respectively (Fang et al., 2010). Motor, IQ, and CC refer to the motor domain, IQ motifs, and coiled-coil domain, respectively. (B) Myo1 accumulation at the budding site depends on the HR1 domain of Bni5. Montages were created from selected frames of time-lapse series taken with 2.5-min intervals. Strains used: YEF11029 (bni5Δ MYO1-mScarlet [GFP-BNI5(FL)]), YEF11033 (bni5Δ MYO1-mScarlet [GFP]), and YEF11027 (bni5Δ MYO1-mScarlet [GFP-bni5(41-448)]). (C) The HR1-coding sequence of BNI5 is essential for suppression of the bni5Δ hof1Δ synthetic lethality. Growth on 5-FOA plates tested the ability of GFP-Bni5-ΔHR1 to support viability in the absence of HOF1. Strains used: YEF12280 (hof1Δ GFP-BNI5(FL) [HOF1-GFP URA3 CEN]), YEF12267 (hof1Δ GFP-bni5(41-448) [HOF1-GFP URA3 CEN]), and YEF12330 (hof1Δ bni5Δ [HOF1-GFP URA3 CEN]). (D) Localization of HR1 at the bud neck during cytokinesis depends on Myo1 and its mTD1. Montages of GFP-Bni5-HR1 in WT, myo1Δ, and myo1-mTD1Δ backgrounds were created from time-lapse series taken with 2.5-min intervals. Strains used: YEF11071 (CDC3-mCherry [GFP-bni5(1–40)]), YEF11114 (myo1Δ CDC3-mCherry [GFP-bni5(1–40)]), and YEF11757 (myo1-mTD1Δ CDC3-mCherry [GFP-bni5(1–40)]). (E) Artificial tethering of HR1 to the septin hourglass restores Myo1 localization at the bud neck in the absence of FL Bni5. Arrowheads indicate the Myo1-mScarlet signal at the bud neck mediated by HR1-GBP and Cdc11-GFP interaction. Strains used: YEF11212 (CDC11-GFP MYO1-mScarlet bni5(1–40)-GBP) (bni5(1–40)-GBP replaced the endogenous BNI5 ORF), YEF11211 (CDC11-GFP MYO1-mScarlet bni5Δ::GBP) (GBP replaced the endogenous BNI5 ORF), and YEF11088 (CDC11-GFP MYO1-mScarlet). (F) The HR1 domain of Bni5 interacts directly with the mTD1 of Myo1 in vitro. Top left: SDS-PAGE stained with Coomassie blue depicting amounts of each indicated purified protein used as the input for the in vitro–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 MBP-mTD1. Green boxes represent GST and GST-tagged Bni5 fragments described top right. Right: Immunoblotted membrane with antibody against MBP; kDa = kiloDalton. This experiment was repeated three times, and a representative immunoblot is shown. Source data are available for this figure: SourceData F6.
Figure 6.

Bni5 recruits Myo1 to the septin hourglass via HR1. (A) Diagram of putative and demonstrated interactions among Bni5, Myo1, and Iqg1. The mTD1 and targeting domain 2 (TD2) in the tail of Myo1 mediate its direct or indirect interactions with Bni5 and Iqg1, respectively (Fang et al., 2010). Motor, IQ, and CC refer to the motor domain, IQ motifs, and coiled-coil domain, respectively. (B) Myo1 accumulation at the budding site depends on the HR1 domain of Bni5. Montages were created from selected frames of time-lapse series taken with 2.5-min intervals. Strains used: YEF11029 (bni5Δ MYO1-mScarlet [GFP-BNI5(FL)]), YEF11033 (bni5Δ MYO1-mScarlet [GFP]), and YEF11027 (bni5Δ MYO1-mScarlet [GFP-bni5(41-448)]). (C) The HR1-coding sequence of BNI5 is essential for suppression of the bni5Δ hof1Δ synthetic lethality. Growth on 5-FOA plates tested the ability of GFP-Bni5-ΔHR1 to support viability in the absence of HOF1. Strains used: YEF12280 (hof1Δ GFP-BNI5(FL) [HOF1-GFP URA3 CEN]), YEF12267 (hof1Δ GFP-bni5(41-448) [HOF1-GFP URA3 CEN]), and YEF12330 (hof1Δ bni5Δ [HOF1-GFP URA3 CEN]). (D) Localization of HR1 at the bud neck during cytokinesis depends on Myo1 and its mTD1. Montages of GFP-Bni5-HR1 in WT, myo1Δ, and myo1-mTD1Δ backgrounds were created from time-lapse series taken with 2.5-min intervals. Strains used: YEF11071 (CDC3-mCherry [GFP-bni5(1–40)]), YEF11114 (myo1Δ CDC3-mCherry [GFP-bni5(1–40)]), and YEF11757 (myo1-mTD1Δ CDC3-mCherry [GFP-bni5(1–40)]). (E) Artificial tethering of HR1 to the septin hourglass restores Myo1 localization at the bud neck in the absence of FL Bni5. Arrowheads indicate the Myo1-mScarlet signal at the bud neck mediated by HR1-GBP and Cdc11-GFP interaction. Strains used: YEF11212 (CDC11-GFP MYO1-mScarlet bni5(1–40)-GBP) (bni5(1–40)-GBP replaced the endogenous BNI5 ORF), YEF11211 (CDC11-GFP MYO1-mScarlet bni5Δ::GBP) (GBP replaced the endogenous BNI5 ORF), and YEF11088 (CDC11-GFP MYO1-mScarlet). (F) The HR1 domain of Bni5 interacts directly with the mTD1 of Myo1 in vitro. Top left: SDS-PAGE stained with Coomassie blue depicting amounts of each indicated purified protein used as the input for the in vitro–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 MBP-mTD1. Green boxes represent GST and GST-tagged Bni5 fragments described top right. Right: Immunoblotted membrane with antibody against MBP; kDa = kiloDalton. This experiment was repeated three times, and a representative immunoblot is shown. Source data are available for this figure: SourceData F6.

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