Detection of F-box protein-substrate interactions by the refined two-hybrid system. (A) The refined two-hybrid system allows the detection of Cdc4-dF-Sic1 interaction. By using the filter assay, we examined the expression of reporter LacZ in wild-type (WT) and cdc4–1 mutants harboring the indicated combinations of bait and prey constructs. (B) The refined two-hybrid system identified interactions of Cdc4-dF with Gcn4, Swi5, Rcn1, and Spo74. (C) Stability of AD- and HA-tagged proteins expressed from the GAL1 promoter after the addition of cycloheximide was compared in the indicated cells. Aliquots were taken at 10 min intervals and immunoblotted with anti-HA (12CA5) and anti-PSTAIRE antibodies. Cdc28 is shown as a loading control. (*, Nonspecific signals detected in this experiment.)

Swi5 is a physiological substrate of the SCF^Cdc4 complex. (A and B) Swi5 is stabilized and accumulates in nuclei of cells with functionally defective SCF^Cdc4 complex. Log phase cultures of TK769 (GAL-CDC20 SWI5-Myc) and TK770 (GAL-CDC20 SWI5-Myc cdc4–1) were arrested in metaphase in YPR medium. Cells were released from the metaphase arrest by the addition of galactose and further cultured at 30°C (TK769) or 37°C (TK770). Aliquots were taken at 20 min intervals for protein analysis (A) and for determination of subcellular localization (B). (A) Protein levels of Swi5-Myc, Clb2, and Cdc28 were analyzed by immunoblotting. (B) Swi5-Myc was visualized by anti-Myc antibody (9E10). DAPI (4′6-diamidino-2-phenylindole) was used to visualize nuclei. Numbers in parentheses represent the numbers of strains. (C) Swi5-ST8A-HH is stabilized in vivo. Cultures of TK713 (GAL-CDC20 SWI5-HH) or TK-714 (GAL-CDC20 SWI5-ST8A-HH) cells were processed as described in Fig. 2a at 30°C. Aliquots were taken at the time indicated and immunoblotted. (D) Cells arrested at G₁ phase by α factor were treated with galactose for 20 min (to induce expression of Swi5-Myc or Swi5-ST8A-Myc) and then with cycloheximide. Aliquots were taken at the preselected time, and degradation of Swi5-Myc or Swi5-ST8A-Myc was monitored by immunoblotting. Bands were quantified, and Swi5-Myc signals were normalized against Cdc28 signals from the same blot probed with anti-PSTAIRE antibody. Values are shown as a percentage of the signal at 0 min time point: Swi5-Myc in wild-type cells (○) or in srb10Δ mutants (●); Swi5-ST8A-Myc in wild-type cells (□) or in srb10Δ mutants (■). (E) Ubiquitination of Swi5 in vivo. (Top) pdr5Δ mutants (lanes 1–10) or cdc4–1 pdr5Δ mutants (lanes 11 and 12) in the absence (lanes 1, 3, 5, 7, 9, and 11) or presence (lanes 2, 4, 6, 8, 10, and 12) of MG132, with the use of the following combinations of plasmids: YC33G (control plasmid) and pCUP-Ubi^HIS-MYC-RA (lanes 1 and 2); YC33G-SWI5-HA and pCUP-Ubi^HIS-MYC-RA (lanes 3, 4, 9, and 10); YC33G-SWI5-HA and pCUP-Ubi^MYC-RA (lanes 5 and 6); YC33G-SWI5-ST8A-HA and pCUP-Ubi^HIS-MYC-RA (lanes 7 and 8); and pYES2-SWI5-HA and pCUP-Ubi^HIS-MYC-RA (lanes 11 and 12). We used pYES2-SWI5-HA (a high-copy plasmid for expression of SWI5-HA from GAL1 promoter) because expression of Swi5-HA from YC33G-SWI5-HA was low in cdc4–1 pdr5Δ mutants. Extracts were incubated with Ni-NTA beads, and bead-bound proteins were immunoblotted with anti-HA (12CA5) antibody. (*, Nonspecific signals detected in this experiment.) (Middle and Bottom) Total extracts were analyzed by immunoblotting to examine the expression of Swi5-HA (Middle) and Cdc28 as loading control (Bottom).

SCF^Cdc4-dependent degradation of Swi5 contributes to proper progression into S phase. (A) Serial 3-fold dilutions of TK713 (GAL-CDC20 SWI5-HH), TK714 (GAL-CDC20 SWI5-ST8A-HH), TK832 (GAL-CDC20 SWI5-HH sic1Δ), and TK835 (GAL-CDC20 SWI5-ST8A-HH sic1Δ) were spotted on YPRG plates (containing raffinose and galactose as carbon sources) and incubated at 30°C for 38 h. (B) Arrest of metaphase in TK713 and TK714 cells was released by the addition of galactose, and aliquots were taken at the indicated time points for FACS analysis. (C) Arrest of metaphase in TK832 and TK835 cells was released by the addition of galactose, and aliquots were taken at the indicated time points for FACS analysis. (D) Degradation of Swi5 is required to terminate SIC1 transcription. TK713 (GAL-CDC20 SWI5-HH) or TK714 (GAL-CDC20 SWI5-ST8A-HH) cells were grown as described in Fig. 2A. Total RNA was extracted and analyzed with SWI5, SIC1, and ACT1 (for loading control) probes as described in ref. 18. (E) Sic1 hyperaccumulated in cells expressing Swi5-ST8A. Arrest of metaphase in TK779 (GAL-CDC20 SWI5-HH SIC1-HA) and TK780 (GAL-CDC20 SWI5-ST8A-HH SIC1-HA) cells was released by the addition of galactose. Aliquots were taken at the indicated time points, and the protein level of Sic1-HA was compared by immunoblotting. Cdc28 is shown as a loading control. Numbers in parentheses represent the numbers of strains.

Schematic diagram of regulation of S-phase entry by the SCF^Cdc4 complex. SCF^Cdc4 complex regulates S-phase entry by degrading not only Sic1 at late G₁ phase but also Swi5 at early G₁. Sic1 degradation is strictly required for S-phase entry, whereas Swi5 degradation is required to proper progression into S phase through termination of SIC1 transcription at early G₁ phase. Phosphorylation ensures timing of degradation of Swi5 and Sic1. Srb10 triggers degradation of Swi5 at early G₁, whereas G₁-CDKs trigger degradation of Sic1 at late G₁.