Article Figures & Data
Figures
- Fig 1
Localization and degradation of a septin-Swe1p fusion. (A) Schematic of the Cdc3-GFP-Swe1p fusion protein. (B) Localization of Cdc3-GFP-Swe1p to the neck is independent of Hsl1p and Hsl7p. GFP-Swe1p is shown as a control. Cells were grown overnight at 30°C. Representative cells are shown, and over 200 cells of each strain were scored for bud neck signals. Strains containing GFP-Swe1p (with genotypes indicated in parentheses) were DLY12320 (wild type [WT]), DLY12369 (hsl7Δ), DLY12237 (hsl1Δ), and DLY12229 (hsl1Δ hsl7Δ), and strains containing Cdc3-GFP-Swe1p were DLY12321 (wild type), DLY12238 (hsl1Δ), DLY12370 (hsl7Δ), and DLY12230 (hsl1Δ hsl7Δ). (C) Swe1p and septin-Swe1p levels rise and fall in parallel as cells traverse the cell cycle. Cells containing both Swe1p-12×myc and septin-Swe1p-12×myc (DLY12321) were arrested in G1 phase with pheromone and released into fresh medium at 30°C to traverse the cell cycle. Pheromone was reintroduced 60 min after release to rearrest cells at the next G1 phase. Samples taken at 15-min intervals were analyzed by Western blotting with anti-myc antibody (Cdc11 as a loading control) and quantitated (graph). (D) Synchrony parameters (budding and nuclear division) for the experiment depicted in panel C were scored for >200 cells per sample. (E) Swe1p degradation kinetics are reproducible between experiments. Five replicates of the experiment depicted in panel C were quantified, and an average degradation profile for wild-type Swe1p is plotted as a dotted black line. (F) Degradation of septin-Swe1p occurs independent of wild-type Swe1p. A single-cycle synchrony experiment, as described above for panel C, was performed with swe1Δ strain DLY15563. (G) Synchrony parameters (budding and nuclear division) for the experiment depicted in panel F were scored for >200 cells per sample.
- Fig 2
Hsl1p and Hsl7p are required for degradation of septin-tethered Swe1p in G2/M phase. (A) Swe1p and septin-Swe1p are both stabilized upon the deletion of HSL1. A single-cycle synchrony experiment, as described in the legend of Fig. 1C, was performed with hsl1Δ strain DLY12238. (B) Synchrony parameters (budding and nuclear division) for the experiment depicted in panel A were scored for >200 cells per sample. (C) Swe1p and septin-Swe1p are both stabilized upon the deletion of HSL7. A single-cycle synchrony experiment, as described in the legend of Fig. 1C, was performed with hsl7Δ strain DLY12370. (D) Synchrony parameters (budding and nuclear division) for the experiment depicted in panel C were scored for >200 cells per sample.
- Fig 3
Interaction between Hsl1p and Hsl7p is not required for degradation of septin-tethered Swe1p. (A) Schematic of Hsl1p. a.a., amino acids. (B) Swe1p is stabilized upon deletion of Hsl1p region 8, but septin-Swe1p is not. A single-cycle synchrony experiment, as described in the legend of Fig. 1C, was performed with hsl1Δ8 strain DLY15224. (C) Synchrony parameters (budding and nuclear division) for the experiment depicted in panel B were scored for >200 cells per sample.
- Fig 4
Hsl1p kinase activity is required for degradation of septin-tethered Swe1p. (A) Schematic of Hsl1p. The K110R point mutation in the kinase domain is noted by an asterisk. (B) Septin-Swe1p is localized to the bud neck in hsl1K110R cells, but GFP-Swe1p is not. Cells of strains DLY12973 and DLY15597 were grown at 30°C and photographed. (C) Swe1p and septin-Swe1p are both stabilized in a strain harboring the kinase-dead hsl1K110R mutant. A single-cycle synchrony experiment, as described in the legend of Fig. 1C, was performed with hsl1K110R strain DLY12973. (D) Synchrony parameters (budding and nuclear division) for the experiment depicted in panel C were scored for >200 cells per sample.
- Fig 5
Interaction between Swe1p and Hsl7p is required for degradation of septin-tethered Swe1p. (A) Schematic of Swe1p. (B) Septin-tethered Swe1pΔ1 is localized to the bud neck. Cells of strain DLY13156 were grown at 30°C and photographed. (C) Swe1pΔ1 and septin-Swe1pΔ1 are both stable in G2/M phase. A single-cycle synchrony experiment, as described in the legend of Fig. 1C, was performed with strain DLY13156. (D) Synchrony parameters (budding and nuclear division) for the experiment in depicted panel C were scored for >200 cells per sample.
Tables
- Table 1
Yeast strains used in this study
Strain Genotypea DLY12229 a bar1 CDC28E12K SWE1myc:HIS2 hsl1::URA3 hsl7::URA3 GFP-SWE1-12×myc:TRP1 DLY12230 a bar1 CDC28E12K SWE1myc:HIS2 hsl1::URA3 hsl7::URA3 CDC3-GFP-SWE1-12×myc:TRP1 DLY12237 a bar1 CDC28E12K SWE1myc:HIS2 hsl1::URA3 GFP-SWE1-12×myc:TRP1 DLY12238 a bar1 CDC28E12K SWE1myc:HIS2 hsl1::URA3 CDC3-GFP-SWE1-12×myc:TRP1 DLY12320 a bar1 CDC28E12K SWE1myc:HIS2 GFP-SWE1-12×myc:TRP1 DLY12321 a bar1 CDC28E12K SWE1myc:HIS2 CDC3-GFP-SWE1-12×myc:TRP1 DLY12369 a bar1 CDC28E12K SWE1myc:HIS2 hsl7::URA3 GFP-SWE1-12×myc:TRP1 DLY12370 a bar1 CDC28E12K SWE1myc:HIS2 hsl7::URA3 CDC3-GFP-SWE1-12×myc:TRP1 DLY12973 a bar1 CDC28E12K SWE1myc:HIS2 hsl1K110R CDC3-GFP-SWE1-12×myc:TRP1 DLY13156 a bar1 CDC28E12K swe1::LEU2 SWE1Δ1myc:URA3 CDC3-GFP-SWE1Δ1-12×myc:TRP1 DLY15224 a bar1 CDC28E12K SWE1myc:HIS2 hsl1Δ8::URA3 CDC3-GFP-SWE1-12×myc:TRP1 DLY15563 a bar1 CDC28E12K swe1::LEU2 CDC3-GFP-SWE1-12×myc:TRP1 DLY15597 a bar1 CDC28E12K SWE1myc:HIS2 hsl1K110R GFP-SWE1-12×myc:TRP1 ↵a Single colons in genotypes indicate that the gene is integrated at a particular locus without disrupting it, and double colons indicate that the gene is disrupted by the marker.
