Article Figures & Data
Figures
- FIG. 1.
Cdc28 phosphorylates Swi6 at serine 160. (A) Modular structure of Swi6 with amino acid residues numbered showing protein fragments used as substrates for Cdc28 kinase. Phosphorylation is indicated by + or −. The arrows indicate the location of serine 160 in a flexible linker region and an RXL motif at residues 256 to 258. T, transcriptional activation region; ANK, ankyrin repeat domain. (B) Upper panel, autoradiogram showing phosphorylation of the substrates indicated by low-salt, cyclin-proficient preparations of Cdc28. Lower panel, Coomassie blue staining of input proteins. BSA, bovine serum albumin; H1, histone H1. (C) Phosphorylation of the Swi6-60k fragment and histone H1 by wild-type (wt) Cdc28 and thermosensitive (ts) Cdc28-13 at 25°C and 37°C. (D) Comparison of phosphorylation by Cdc28 of wild-type and mutant forms of Swi6-60k (left panel) and full-length Swi6 (right panel). S→A, serine 160 mutated to alanine; S→D, serine 160 mutated to aspartate; RXL, R256A L257A L258A triple mutant. Protein stain shows input protein substrates visualized by Coomassie blue staining.
- FIG. 2.
Phosphorylation of Swi6 at serine 160 depends on Clb6. (A) High-salt, cyclin-depleted preparations of Cdc28 were tested for phosphorylation of full-length Swi6 and histone H1 in the absence or presence of additional cyclins or Cks1 as indicated. (B) High-salt, cyclin-depleted preparations of Cdc28 were tested for phosphorylation of wild-type and mutant forms of full-length Swi6 and the Swi6-60k fragment in the absence or presence of additional Clb6. Abbreviations are as described in the legend for Fig. 1D. The protein stain shows input protein substrates visualized by Coomassie blue staining.
- FIG. 3.
Clb5 and Clb6 are needed for optimal phosphorylation of Swi6. (A) Low-salt, cyclin-containing preparations of GST-Cdc28 were made from wild-type and Δclb5, Δclb6, Δclb5 Δclb6, and clb2 MGY2 yeast cells as indicated. Two concentrations of Cdc28 preparations were used to ensure that assays were performed under nonsaturating conditions. The amounts of input affinity-purified Cdc28 were between 25 and 150 ng. Protein staining indicates input substrate visualized by Coomassie blue staining. (B) Quantification of phosphorylation of Swi6 compared to histone H1. The level of phospholabeling of Swi6 by each kinase preparation was normalized against the level of phosphorylation of histone H1 by phosphorimaging; 100% represents the ratio of Swi6 to histone H1 labeling from wild-type extracts.
- FIG. 4.
Swi6 interacts with Clb6. (A) Full-length Swi6 was assayed for binding to glutathione-Sepharose beads carrying GST, GST-Clb2, GST-Clb4, and GST-Clb6. (B) Deletion mapping of Swi6 for binding to Clb6. The fragments of Swi6 diagrammed in the line drawing were assayed for binding to glutathione-Sepharose beads carrying GST and GST-Clb6. T, transcriptional activation region; ANK, ankyrin repeat domain. Bound proteins were visualized by immunoblotting with polyclonal anti-Swi6 antibodies. Nonspecific bands are indicated by the arrow.
- FIG. 5.
Clb6 is required for nuclear export of Swi6. Immunolocalization of Swi6-myc in wild-type cells (A) and Δclb6 cells (B) after release from α-factor arrest. Numbers denote minutes after release from arrest. DNA was visualized by DAPI staining. (C) DNA content of cells following release from α-factor arrest, determined by flow cytometry.
- FIG. 6.
Dephosphorylation and nuclear import of Swi6 after Cdc14 activity. (A) Radioactive Swi6 labeled at serine 160 was treated with the phosphatase indicated and the remaining radioactivity was visualized by autoradiography. AP, alkaline phosphatase. (B) Immunolocalization of Swi6-13myc and nuclear staining in cdc14-1 SWI6-13myc cells at 23°C or after 165 min of incubation at 37°C. (C) SWI6-GFP cells carrying pCDC14yexEMBL, expressing Cdc14 under the control of the GAL1,10 galactose-inducible promoter, were grown for 2 h in 2% sucrose and nocodazole-containing medium to produce a metaphase arrest (time zero). The culture was divided and incubation was continued a further 2 h with nocodazole in the presence of dextrose or galactose to repress or induce Cdc14 expression. Inset values quantify the percentage of cells of the culture as shown in the images (n > 100).
- FIG. 7.
Scheme for cell cycle regulation of nuclear export and import of Swi6. Swi6 is represented by grey shading in the lower cartoon. The broken line indicates reduction in MBF and SBF activity by loss of Swi6 from the nucleus. See the Discussion for further explanation.
