FIG.4.

APC^Cdc20p mediates Tax-induced Pds1p and Clb2p degradation. (A) The PDS1 gene in the W303-1a-derived strain 785-cdc23^ts was replaced with a gene encoding HA-Pds1. The resultant strain was further transformed with the Gal10-Tax plasmid. Cdc23^ts/Gal10-Tax cells grown to mid-log phase at 23°C were treated with hydroxyurea for 2 h and divided into four portions, each grown at 23°C or 37°C in hydroxyurea- and galactose- or glucose-containing (Tax + and Tax −, respectively) medium for 2 h more (to maintain cell cycle arrest and induce Tax expression or not at the same time). The cells were then released from the G₁/S arrest in medium without hydroxyurea but with galactose or glucose (to induce Tax expression or not) for 1 h and blotted for Pds1p and Clb2p (left and middle panels). W303-1a/Gal10-Tax grown at 37°C under the same conditions was included as a control (WT, right panel). Cell lysates were prepared at 1 h after release. Immunoblots (WB) for Clb2p, Pds1p-HA, protein phosphatase 2A catalytic subunit (PP2Ac), and Tax were carried out as before. Clb2p antibody was from Santa Cruz. (B) The PDS1 genes in a cdc20^ts, a mad1-null (mad1Δ), and a cdh1-null (cdh1Δ) derivative of W303-1a were replaced with the gene encoding HA-Pds1 as above. The resultant strains were transformed with the Gal10-Tax plasmid. The conditions for cell cycle arrest, Tax induction, temperature shift, release from G₁/S block, and immunoblotting were as for panel A. The experiments for cdc20^ts were carried out at 23°C and 37°C, and those for the cdh1-null and mad1-null mutants were done at 30°C. (C) The Tax phenotype is exaggerated in the cdh1-null background. Cell cycle arrest, Tax induction, release from G₁/S block, and immunoblotting were as for panel A. Times after release from G₁/S arrest (0, 30, 60, 90, and 120 min) are indicated.