FIG. 3.

Tax causes a drastic reduction in Clb2p and Pds1p levels in S. cerevisiae. (A) Tax causes a reduction in Clb2p level. KY630 cells carrying a gene encoding CLB2-3×HA integrated at the CLB2 locus were transformed with either Gla10-ΔTax (lanes −) or Gal10-Tax (lanes +). After induction for 12 h in galactose-containing medium, cells were diluted to an A₆₀₀ of 0.25 and treated with nocodazole (12 μg/ml) for 4 or 5 h. Immunoblots for Clb2 and Tax were carried out with anti-HA and 4C5 monoclonal antibodies, respectively. Immunoblotting (WB) for the catalytic subunit of protein phosphatase 2A (PP2Ac) was carried out as an internal protein control. Clb2-associated kinase activity (histone H1 kinase) was determined with histone H1 as a substrate (see Materials and Methods). (B) Tax promotes a reduction in Clb2p level prior to the onset of mitosis. KY630/Gal10-Tax cells were cultured in SC medium containing raffinose at 30°C overnight for 12 h, then diluted to 0.25 A₆₀₀, grown to mid-log phase, and treated with hydroxyurea (0.2 M) for 4 h to arrest cells at G₁/S. The synchronized cells were divided into two parts, induced for Tax expression (Tax +) or not (Tax −) by the addition of 2% galactose or 2% glucose, and simultaneously released from the cell cycle arrest. Cell lysates were prepared at the indicated times for Clb2, protein phosphatase 2A catalytic subunit, and Tax immunoblots as for panel A. Times after release and induction (0, 30, 60, 90, 120, and 150 min) are indicated. Flow cytometry was carried out as for Fig. 2A. (C) Tax causes a reduction in Pds1p level. W303-1a cells carrying a gene encoding Pds1-HA integrated at the PDS1 locus were transformed with Gal10-Tax. Cells were grown at 30°C and synchronized with hydroxyurea as for panel B. Two hours into the hydroxyurea treatment, galactose or glucose was added to the culture to 2% to induce Tax expression or not for 2 h. Cells were then released from the cell cycle block in galactose-containing (rows +) or glucose-containing (row −) medium. Cell lysates were prepared and blotted for Pds1p-HA, protein phosphatase 2A catalytic subunit, and Tax as before. Times (0, 30, 60, 90, and 120 min) after release from G₁/S arrest are indicated.