Fig. 4. Cell-cycle regulation of Ace2p. (A) cdc25-22 ace2-myc₁₃ (KGY4472) cells were shifted to 36°C for 4 hours to block cells in G2. Cell pellets were collected every 15 minutes after shift down to 25°C and were fixed in ethanol or frozen. The septation index (SI) was determined to monitor progression through the cell cycle. The ethanol-fixed samples were stained with DAPI to visualize the nuclei and allow microscopic calculation of the percentage of binucleates (BN). Protein lysates were prepared and resolved by SDS-PAGE. Ace2p-Myc₁₃ levels were determined by immunoblotting with 9E10 antibody. In these and other experiments with multi-septated cells, the fluctuation of Cdc13p abundance (determined as in Fig. 1C) was used to assess mitotic progression and Cdc2p abundance (determined by blotting with anti-PSTAIRE) served as a loading control. (B) Denatured protein lysates prepared from wild-type (KGY246), ace2-myc₁₃ (KGY1124) and ace2-myc₁₃ sep1 (KGY1141) strains were resolved on a 4-12% Bis-Tris gel and immunoblotted with antibodies to myc (9E10) (top panel) or to Cdc2p (PSTAIRE) (lower panel). (C) The nmt1sep1-HA ace2-myc₁₃ (KGY4735) strain was grown to mid-log phase in the absence or presence of thiamine (–T/+T) at 32°C for 20 hours. Protein lysates were prepared, resolved on a 4-12% Bis-Tris gel, and immunoblotted as in (B) or with the 12CA5 antibody. (D) The nmt1sep1-HA₃ (KGY4221) strain was grown as in (C). Cells were fixed in ethanol, stained with Methyl Blue and DAPI and imaged. (E) The ace2-myc₁₃ mts3-1 (KGY4536) and ace2-HA₃ mts3-1 (KGY5314) strains were grown at 25°C and then shifted to 36°C for 4 hours to block proteasome function and arrest cells in mitosis. Ace2p-Myc₁₃ was immunoprecipitated with the 9E10 antibody and Ace2p-HA₃ was immunoprecipitated with the 12CA5 antibody, followed by incubation in the presence or absence of -phosphatase. (F) The mid2-myc₁₃ mts3-1 (KGY1976; lane 1), mts3-1 (KGY1135; lane 2), ace2-myc₁₃ mts3-1 (KGY5231) strains containing the pREP1-His6-Ubiquitin plasmid (pKG1284; lane 4) and the ace2-myc₁₃ mts3-1 (KGY4536; lane 3) strain without vector were grown at 25°C for 22 hours in the absence of thiamine in order to induce His-Ub production. This was followed by a 4-hour shift to 36°C. Substrates modified with ubiquitin were isolated and detected by immunoblotting with anti-Myc antibodies.