The assembly of the exocyst complex is inhibited during M phase. (A) Exo84 was tagged by the myc epitope by chromosomal integration. Cells expressing Exo84-myc were synchronized in G1, S, and M phase with α-factor (α), hydroxyurea (HU), and nocodazole (Noc), respectively. Exo84-myc was immunoprecipitated using the anti-myc antibody, and Western blots were performed to detect the amounts of coprecipitating Sec5-GFP, Sec10, Sec15, and Exo70. (left) The expression of the exocyst subunits cell extracts (“10% input”). (right) The amounts of the exocyst subunits coimmunoprecipitated with Exo84-myc. (B) Quantification of the amounts of the exocyst coimmuniprecipitated with Exo84-myc. The levels of binding in different groups with respect to that of the nontreated (NT) cells (“relative binding”) were shown. The levels of binding for Sec10, Sec15, and Exo70 decrease in the nocodazole-treated cells. Sec5 binding remains unchanged. Error bars represent standard deviation. *, P < 0.01; n = 3. (C) Inhibition of Cdk1 promotes exocyst assembly. The wild-type and cdk1-as cells were treated with DMSO (mock) or 1NM-PP1 before Exo84-myc coimmunoprecipitation. The levels of binding of Sec10, Sec15, and Exo70 increased in the cdk1-as cells treated with 1NM-PP1, whereas their binding with Sec5 remained unchanged. (D) Quantification of binding for C was performed as described in B. Error bars represent standard deviation. *, P < 0.01; n = 3. (E) The cdc20-1 mutant exhibits exocyst assembly defects at the restrictive temperature. The wild-type and different cell cycle mutant cells were grown at 25°C and shifted to 37°C for 2 h. Exo84-myc immunoprecipitation was performed using these cells. The levels of binding of Sec10, Sec15, and Exo70 decreased in the cdc20-1 mutant. (F) Quantification of the amounts of the exocyst coimmuniprecipitated with Exo84-myc. The levels of binding for Sec10, Sec15, and Exo70 decreased in the cdc20-1 mutant. Sec5 binding remains unchanged. Error bars represent standard deviation. *, P < 0.01; n = 3.