Exo84 phosphorylation is required for secretion block at metaphase. (A) Glycoprotein secretion defects in cells during metaphase. The wild-type and exo84-A cells were synchronized with α-factor and then released for 30 min (approximately interphase stage) and 90 min (approximately metaphase stage), respectively. These are the two time points at which electrophoretic mobility (i.e., phosphorylation state) of Exo84p is significantly distinct from each other according to Fig. 3 B. The media glycoproteins were collected and analyzed. Asterisks indicate the glycoproteins that were changed comparing the wild-type and exo84-A strains. Protein molecular weights are indicated to the left (kD). (B) Quantification of the glycoprotein band indicated by the arrow in A. Error bars represent standard deviation. *, P < 0.01; n = 3. (C) The phospho-deficient exo84-A mutant rescued the Bgl2 secretion defect of the cdc20-1 cells. The cdc20-1 and cdc20-1 exo84-A mutants were grown at 25°C and then shifted to 37°C for 1.5 h. Internal (In) and external (Ex) pools of Bgl2 were examined by Western blot analysis. ADH was probed as a protein loading control. (D) Quantification of Bgl2 accumulation in the cdc20-1 and cdc20-1 exo84-A mutants at 37°C. Error bars represent standard deviation. *, P < 0.01; n = 3. (E) Cells were fixed with permanganate and processed for thin-section EM. The cdc20-1 cells accumulated post-Golgi secretory vesicles at the restrictive temperature, whereas cdc20-1 exo84-A double mutant did not. “SV” and the arrow indicate one of the vesicles. Bars, 1 µm. (F) Quantification of accumulated vesicles in the cdc20-1 and cdc20-1 exo84-A mutants at 37°C. Error bars represent standard error of the mean. *, P < 0.01; n = 35.