Identification and functional analyses of both in vivo and in vitro Elm1- and Gin4-dependent phospho-sites in Bni5, and analysis of the differential effects of mitotic exit on the bud neck localization of Gin4, Elm1, and Bni5. Related to Fig. 5. (A) Time-lapse imaging analysis of Elm1- and Gin4-dependent phosphorylation site mutants. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates Bni5 variants. Strains used: YEF11600 [mScarlet-TUB1 GFP-bni5(41-448)], YEF12109 [mScarlet-TUB1 GFP-bni5(41-448)-5A], and YEF12112 [mScarlet-TUB1 GFP-bni5(41-448)-5DE]. (B) Top: In vitro kinase assay. Bni5 was incubated with or without GST-Elm1 in the presence of ATP, followed by SDS-PAGE and Coomassie Blue staining. Boxed regions were excised for mass spectrometry analysis. Bottom: Schematic of Bni5 protein showing domain boundaries and identified phosphorylation sites (blue vertical lines) determined by mass spectrometry. These sites were mutated to generate phospho-deficient (bni5-5A) and phospho-mimic (bni5-5D) alleles. (C) Time-lapse imaging analysis of Elm1-catalyzed phosphorylation site mutants. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates Bni5 variants. Strains used: YEF11600 [mScarlet-TUB1 GFP-bni5(41-448)], YEF12240 [mScarlet-TUB1 GFP-bni5(41-448)-5A], and YEF12241 [mScarlet-TUB1 GFP-bni5(41-448)-5D]. (D) Top: In vitro kinase assay. Bni5 was incubated with or without 6xHis-SUMO-Gin4 in the presence of ATP, followed by SDS-PAGE and Coomassie Blue staining. Boxed regions were excised for mass spectrometry. Bottom: Schematic of Bni5 as described above (B). (E) Time-lapse imaging analysis of Gin4-catalyzed phosphorylation site mutants. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates Bni5 variants. Strains used: YEF11546 [mScarlet-TUB1 GFP-BNI5], YEF12440 [mScarlet-TUB1 GFP-bni5-1A], YEF12441 [mScarlet-TUB1 GFP-bni5-1D], YEF12442 [mScarlet-TUB1 GFP-bni5-3A], and YEF12441 [mScarlet-TUB1 GFP-bni5-3D]. (F) Time-lapse imaging of GFP-Bni5 and Gin4-mScarlet during cytokinesis. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. The strain used: YEF12427 (bni5∆ GFP-BNI5 GIN4-mScarlet). (G) Representative images of GFP-Bni5, Gin4-GFP, and Elm1-GFP in cdc15-2 cells at the restrictive temperature. Cells were grown to exponential growth phase at 25°C in YM-1 medium and shifted to 37°C for 2.5 h before imaging. Strains used: YEF12227 [cdc15-2mScarlet-TUB1 GFP-BNI5(FL)], YEF12402 (cdc15-2mScarlet-TUB1 GIN4-GFP), and YEF12412 (cdc15-2 mScarlet-TUB1ELM1-GFP).
Figure S4.

Identification and functional analyses of both in vivo and in vitro Elm1- and Gin4-dependent phospho-sites in Bni5, and analysis of the differential effects of mitotic exit on the bud neck localization of Gin4, Elm1, and Bni5. Related to Fig. 5. (A) Time-lapse imaging analysis of Elm1- and Gin4-dependent phosphorylation site mutants. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates Bni5 variants. Strains used: YEF11600 [mScarlet-TUB1 GFP-bni5(41-448)], YEF12109 [mScarlet-TUB1 GFP-bni5(41-448)-5A], and YEF12112 [mScarlet-TUB1 GFP-bni5(41-448)-5DE]. (B) Top: In vitro kinase assay. Bni5 was incubated with or without GST-Elm1 in the presence of ATP, followed by SDS-PAGE and Coomassie Blue staining. Boxed regions were excised for mass spectrometry analysis. Bottom: Schematic of Bni5 protein showing domain boundaries and identified phosphorylation sites (blue vertical lines) determined by mass spectrometry. These sites were mutated to generate phospho-deficient (bni5-5A) and phospho-mimic (bni5-5D) alleles. (C) Time-lapse imaging analysis of Elm1-catalyzed phosphorylation site mutants. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates Bni5 variants. Strains used: YEF11600 [mScarlet-TUB1 GFP-bni5(41-448)], YEF12240 [mScarlet-TUB1 GFP-bni5(41-448)-5A], and YEF12241 [mScarlet-TUB1 GFP-bni5(41-448)-5D]. (D) Top: In vitro kinase assay. Bni5 was incubated with or without 6xHis-SUMO-Gin4 in the presence of ATP, followed by SDS-PAGE and Coomassie Blue staining. Boxed regions were excised for mass spectrometry. Bottom: Schematic of Bni5 as described above (B). (E) Time-lapse imaging analysis of Gin4-catalyzed phosphorylation site mutants. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Asterisk indicates Bni5 variants. Strains used: YEF11546 [mScarlet-TUB1 GFP-BNI5], YEF12440 [mScarlet-TUB1 GFP-bni5-1A], YEF12441 [mScarlet-TUB1 GFP-bni5-1D], YEF12442 [mScarlet-TUB1 GFP-bni5-3A], and YEF12441 [mScarlet-TUB1 GFP-bni5-3D]. (F) Time-lapse imaging of GFP-Bni5 and Gin4-mScarlet during cytokinesis. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. The strain used: YEF12427 (bni5∆ GFP-BNI5 GIN4-mScarlet). (G) Representative images of GFP-Bni5, Gin4-GFP, and Elm1-GFP in cdc15-2 cells at the restrictive temperature. Cells were grown to exponential growth phase at 25°C in YM-1 medium and shifted to 37°C for 2.5 h before imaging. Strains used: YEF12227 [cdc15-2mScarlet-TUB1 GFP-BNI5(FL)], YEF12402 (cdc15-2mScarlet-TUB1 GIN4-GFP), and YEF12412 (cdc15-2 mScarlet-TUB1ELM1-GFP).

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