CLB2 overexpression enhances the growth defect in cdc15-2, cdc14-1, dbf2-2, and tem1-3 mutants. Mutant strains containing GAL-CLB2 were generated by crossing to ADR58. Progeny from tetratype spores were grown to midlog phase in YP/raffinose at 23°C, serially diluted fivefold, and spotted onto YP/dextrose or YP/galactose-raffinose plates. Plates were incubated for 2 d at 30°C or 3 d at 23°C.

The late mitotic mutants arrest with low APC activity toward cyclin. (A) Wild-type and mutant strains were transformed with a plasmid carrying CDC27HA under the control of its own promoter. Cells were grown to midlog phase at 23°C, cultures were divided, and half were shifted to 37°C for 4 h. The second set of cultures was arrested for 3.5 h at 23°C with 1 μg/ml α-factor and then shifted to 37°C in the presence of α-factor for an additional hour. The Cdc27HA subunit of the APC was immunoprecipitated from 500 μg of cell lysate with 12CA5, and conjugation of ubiquitin to the ¹²⁵I-labeled amino terminus of sea urchin cyclin B1 was assessed as described in MATERIALS AND METHODS. Ubiquitin conjugates were observed at ∼8-kDa intervals above the unconjugated cyclin B1 fragment. The asterisk indicates a nonspecific background band observed in the presence of cyclin substrate alone (far left lane). Note that APC activity in wild-type asynchronous cells is normally lower than that in G1-arrested cells (Charles et al., 1998); in this experiment, the high activity in asynchronous cells is due to the relatively high protein levels in these samples (see anti-Cdc28 Western blot in B). (B) Lysates (∼35 μg) from the experiment in panel (A) were subjected to Western blotting with polyclonal antibodies against Clb2 (top) and Cdc28 (bottom). (C) Histone H1 kinase activity was measured in anti-Clb2 immunoprecipitates from 100 μg of cell lysate. (D) Cell lysates (100 μg) were subjected to immunoblotting with affinity-purified polyclonal antibodies against Sic1.

Cdc15 protein levels and kinase activity are constant during the cell cycle. (A) A cdc15::CDC15HA3 strain (SLJ23) was arrested for 3 h at 30°C with 1 μg/ml α-factor, released from the arrest, and allowed to grow at 30°C. Cells were harvested at the indicated times, and lysates (100 μg) were analyzed by Western blotting with 12CA5 (top) or anti-Clb2 antibodies (bottom). (B) Wild-type cells carrying CDC15HA3 on a 2μ plasmid were arrested for 3 h at 30°C with 1 μg/ml α-factor, released from the arrest, and allowed to progress through the cell cycle at 30°C. Cells were harvested at the indicated times, and lysates (35 μg) were analyzed by Western blotting with 12CA5 (top) or anti-Clb2 antibodies (second from top). Cdc15HA3 was immunoprecipitated from 250 μg of lysate and tested for its ability to phosphorylate itself (third from top) or MBP (bottom).

High-copy suppressors of cdc15-2. Four of the cdc15-2 high copy suppressors (GAL-CDC15, GAL-SIC1, GAL-SPO12, and GAL-YAK1ΔN) were retransformed into a cdc15-2 strain (SLJ02), grown in YP/raffinose to midlog phase, serially diluted fivefold, and spotted onto YP/dextrose or YP/galactose-raffinose plates. Plates were incubated at 23 or 37°C for 2.5 d.

Clb2, but not Pds1, is stabilized in late mitotic mutants. (A) Wild-type or mutant strains in which the endogenous copy of Pds1 was replaced with Pds1HA (SLJ423–429) were grown in YPD to midlog phase at 23°C. Cultures were divided and either grown as asynchronous cultures or arrested as indicated for 3.5 h. During the last 30 min of the nocodazole and α-factor arrests, these cultures were also shifted to 37°C. Cell lysates were subjected to immunoblotting with the anti-HA antibody 12CA5 to detect Pds1 (which normally migrates as a doublet; left panels) or with anti-Clb2 antibodies (right panels). (B) GAL-PDS1HA or GAL-CLB2HA was integrated at the TRP1 locus of cdc15-2 to create SLJ272 and SLJ269, respectively. Midlog phase YP/raffinose cultures were arrested in 1 μg/ml α-factor, in 15 μg/ml nocodazole, or at 37°C for 3.5 h. During the last 30 min, α-factor- and nocodazole-arrested cultures were shifted to 37°C. Galactose was added to a final concentration of 2% to induce expression of Pds1HA or Clb2HA. After 30 min of induction, transcription and translation were repressed by addition at time zero of 2% dextrose and 10 μg/ml cycloheximide, and cells were harvested at the indicated times. Cell lysates were subjected to Western blotting with 12CA5 antibodies.

CDC15 encodes a protein kinase. (A) A version of CDC15 carrying a carboxyl-terminal triple HA tag was used to replace the endogenous CDC15 gene (SLJ23; lane 2) or was cloned onto a 2μ plasmid (pSJ103; lane 4). Lysates from the indicated asynchronous cultures (120 μg in lanes 1 and 2, 35 μg in lanes 3–5) were subjected to Western blotting with 12CA5 antibodies. (B) 12CA5 immunoprecipitates from 1 mg (lanes 1 and 2) or 250 μg (lanes 3–5) of cell extract were tested for their ability to phosphorylate MBP in a standard kinase reaction. A protein the size of Cdc15HA3 was also labeled in these immunoprecipitates. In other experiments with singly tagged Cdc15HA, this band migrates slightly faster, indicating that it represents the Cdc15 protein itself (our unpublished data). In lane 5, the kinase reaction was performed with a version of Cdc15 (pSJ59) carrying a point mutation (K54L) that is predicted to abolish kinase activity.

Model of regulatory pathways governing Cdc28 activity in late mitosis. Late mitotic gene products stimulate mitotic cyclin destruction and may also induce increased levels of Sic1 (see DISCUSSION). This model also accommodates evidence that Cdc28 inhibits APC activity (Amon, 1997) and also inhibits SIC1 transcription and Sic1 stability (Moll et al., 1991; Toyn et al., 1996; Verma et al., 1997), resulting in a feedback system that triggers rapid and complete Cdc28 inactivation when Cdc28 activity is reduced to some threshold. For simplicity, this diagram does not include an additional feedback loop suggested by the observation that Cdc28–Clb complexes stimulate CLB transcription (Amon et al., 1993).