Yeast strains and media.
The yeast strains referred to in this report are listed in Table . All media used are as described in reference 14
Yeast strains were grown to an optical density at 600 nm (OD600) of 0.3 in YPD at 23°C and then shifted to 37°C for 3 h or maintained at 23°C. Cells were lysed in 170 μl of lysis buffer (20 mM Tris-HCl [pH 7.4], 50 mM NaF, 5 mM EDTA, 100 mM NaCl, 150 mM β-glycerophosphate, 0.2% Triton X-100, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 μg of aprotinin per ml, 1 μg of leupeptin per ml) with glass beads in a Biospec bead beater. Lysates were clarified by ultracentrifugation for 10 min at 4°C. For freeze-thaw lysis, cells were incubated in spheroplast buffer (1.1 M sorbitol, 50 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 2 mM DTT) with 4 μl of Zymolase (5 mg/ml; Seikagaku Corporation) for 40 min at 23 or 37°C. Spheroplasts were washed in spheroplast buffer, lysed in hypotonic lysis buffer (20 mM HEPES-NaOH [pH 7.4], 25 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, 2 μg of aprotinin per ml, 1 μg of leupeptin per ml) by freezing on dry ice and thawing in ice water three times, and then clarified by centrifugation.
Western blotting, immunoprecipitation, and kinase assays.
Western blotting was carried out essentially as described elsewhere (6
), using rabbit anti-Cdc37 antibodies (1:1,000), rabbit anti-Cdc28 antibodies (1:1,000), or hemagglutinin (HA)-specific monoclonal antibody 12CA5 (1:1,000).
For immunoprecipitation of HA-tagged proteins, cell lysates (100 to 250 μg) were incubated with 1 μl of 12CA5, 20 μl of protein A-Sepharose beads, and yeast lysis buffer in a final volume of 300 μl for 1 to 2 h at 4°C on a rotator. Immunoprecipitates were washed three times with lysis buffer and once with 50 mM HEPES-NaOH (pH 7.4)–1 mM DTT. Histone H1 kinase assays were performed by adding to the immunoprecipitates 20 μl of histone H1 kinase mix (50 mM HEPES-NaOH [pH 7.4], 1 mM DTT, 10 mM MgCl2, 100 μM ATP, 0.25 mg of human histone H1 per ml, 0.125 mCi of [γ-32P]ATP per ml). After incubation at room temperature for 20 min, the reaction products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an 11% polyacrylamide gel.
Cak1 assays were performed as follows. Cell lysates (250 to 500 μg) were immunoprecipitated with anti-HA monoclonal antibody 12CA5 for 2 h at 4°C as described above. For direct determination of Cak1 activity, 1 μg of purified Cdc28 was added to each Cak1 immunoprecipitate in 20 μl of kinase reaction buffer (50 mM HEPES-NaOH [pH 7.4], 1 mM DTT, 10 mM MgCl2, 0.25 mCi of [γ-32P]ATP per ml, 50 μM ATP). Reaction mixtures were incubated for 20 min at 23°C and analyzed by SDS-PAGE and autoradiography. For indirect analysis of Cak1 activity, Cak1 immunoprecipitates were incubated with 0.24 μg of purified Cdc28-HA, 1 μg of purified glutathione S-transferase (GST)–Clb2, and 2 μg of HA peptide in 10 μl of activation buffer (50 mM HEPES-NaOH [pH 7.4], 1 mM DTT, 10 mM MgCl2, 1 mM ATP). Reaction mixtures were incubated for 20 min at 23°C. Activation of the Cdc28–GST-Clb2 complex was then measured by histone H1 kinase assay.
Pulse-chase labeling and analysis.
Wild-type and cdc37-1 cultures were grown at room temperature to an OD600 of 0.1 in YPD. Cells were harvested, washed twice in YNB, resuspended in YND minus methionine (YND-Met), and grown to an OD600 of 0.3 at 23°C (6 h). Cultures were split into two; one half was shifted to 37°C, and the other was left at room temperature. After 1 h, the cultures were harvested and resuspended in 3 ml of YND-Met supplemented with [35S]methionine (50 μCi/OD). Cells were incubated at room temperature or at 37°C for 15 min, harvested, and resuspended in 3 ml of YPD plus 40 μg of unlabeled methionine per ml. Samples were collected at various times thereafter and washed once in YND prior to freezing. In some experiments, cells were incubated with [35S]methionine (50 μCi/OD) for 30 min at 23°C, harvested, resuspended in 3 ml of prewarmed (37°C) YPD with 40 μg of unlabeled methionine per ml, and maintained at 37°C for the remainder of the experiment. Samples were lysed by bead beating, and lysates (100 to 250 μg) were immunoprecipitated with 12CA5. After separation of proteins by SDS-PAGE and gel fixation, gels were incubated for 30 min in Amplify (Amersham) prior to drying and autoradiography.
Protein purification. (i) Cdc28HA purification.
Sf9 insect cells infected with the Cdc28-HA virus were lysed in buffer A (20 mM HEPES-NaOH [pH 7.4], 25 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT) containing 1 mM PMSF, 1 μg of leupeptin per ml, and 2 μg of aprotinin per ml. Following ultracentrifugation (60 min, 100,000 × g, 4°C), the lysate was applied to a HiTrap Q column (Pharmacia) preequilibrated in buffer A. The HiTrap Q column flowthrough containing Cdc28-HA was then applied to a HiTrap S column (Pharmacia) preequilibrated in buffer A, and proteins were eluted with a linear gradient of 25 to 500 mM NaCl in buffer B (20 mM HEPES-NaOH [pH 7.4], 1 mM EDTA, 10% glycerol, 1 mM DTT). Fractions containing Cdc28-HA were diluted with buffer B and applied to an ATP-agarose column preequilibrated with buffer A. Cdc28-HA was eluted from the ATP-agarose column with a linear gradient of 25 to 500 mM NaCl in buffer B, and final contaminants were removed by separation on a Superose 12 sizing column (Pharmacia) equilibrated in buffer C (150 mM NaCl, 20 mM HEPES-NaOH [pH 7.4], 1 mM EDTA, 10% glycerol, 1 mM DTT).
(ii) Cdc37 purification.
Insect cells were infected with a Cdc37 virus and lysed as described above for Cdc28-HA purification. Cdc37-containing lysate was applied to a HiTrap Q column preequilibrated in buffer A, and proteins were eluted with a linear gradient of 25 to 500 mM NaCl in buffer B. Fractions containing Cdc37 (250 to 350 mM NaCl) were diluted with buffer B to below 100 mM NaCl and applied to a HiTrap SP column preequilibrated with buffer A. The SP flowthrough containing Cdc37 was concentrated with a HiTrap Q column, and the eluted Cdc37 was injected on a Superose 12 column preequilibrated with buffer C. Cdc37 migrates as a 160-kDa species on gel filtration. Cdc37-containing fractions were pooled, (NH4)2SO4 was added to 1 M, and the sample was applied to a HiTrap octyl-Sepharose 4FF column (Pharmacia) preequilibrated with 20 mM HEPES-NaOH (pH 7.4)–1 M (NH4)2SO4 and eluted with a decreasing linear gradient of 1 M to 0 M (NH4)2SO4 in 20 mM HEPES-NaOH (pH 7.4)–1 mM EDTA–10% glycerol.
(iii) His6-Cdc37 purification.
A HiTrap chelating column (Pharmacia) was prebound with 200 mM CoCl2. Bacterial lysate containing six-histidine-tagged Cdc37 (His6-Cdc37) was applied to the column in sonication buffer (50 mM sodium phosphate [pH 7.8], 300 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 mM PMSF) and eluted with a linear gradient of 7.5 to 300 mM imidazole (pH 7.8) in sonication buffer. His6-Cdc37-containing fractions were pooled and diluted to 100 mM NaCl with buffer B. Contaminating proteins were removed with a HiTrap Q column step as described for Cdc37 purification from insect cells.
Activation of Cdc28-HA or Cln2-HA3.
Insect cell lysate containing Cdc28-HA (5 μg) or Cln2-HA3 (30 μg) was mixed with increasing amounts of yeast lysate (0, 10, 50, 100, 250, and 500 μg of RD488 or RD249-2D), and reaction mixtures were incubated for 30 min at 23°C. Cdc28-HA or Cln2-HA3 was then immunoprecipitated with 12CA5, and histone H1 kinase activity was measured as described above.
Cdk complex assembly in vitro.
Purified Cdc28-HA (1 μg) was incubated with or without 1 μg of purified GST-Clb2, 1 μg of purified His6-Cdc37, or 50 ng of purified mammalian CAK (Cdk7-cyclin H-Mat1) in a final reaction volume of 10 μl (50 mM HEPES-NaOH [pH 7.4], 1 mM ATP, 1 mM DTT, 10 mM MgCl2). Reaction mixtures were incubated on ice for 15 min and then at 23°C for 15 min. To each reaction mixture was added 20 μl of histone H1 reaction mix (300 μM ATP, 0.125 mCi of [γ-32P]ATP per ml, 10 mM MgCl2, 50 mM HEPES-NaOH [pH 7.6], 0.25 mg of histone H1 per ml, 1 mM DTT). Reaction mixtures were incubated at room temperature for 20 min and analyzed by SDS-PAGE on an 11% polyacrylamide gel.
Gel filtration chromatography.
Insect cell lysate (500 μl) or yeast lysate (500 μg) was clarified by centrifugation for 30 min at 100,000 × g (4°C). Samples were applied in 500-μl aliquots to a Superose 12 sizing column (Pharmacia) preequilibrated with buffer C. Fractions (0.5 ml) were collected at 0.4 ml/min and analyzed by Western blotting. For analysis of the sizing profiles of Cdc28-HA in cdc37-1 cells and of Cak1-HA3 in wild-type and cdc37-1 cells, fractions were immunoprecipitated with 1 μl of 12CA5 and 20 μl of protein A-Sepharose beads for 1.5 h at 4°C as described above. Immunoprecipitated proteins were resolved by SDS-PAGE, and analyzed by immunoblotting with 12CA5.