Figure 2.

Overexpression of BFA1 under GAL1 promoter control induces a drastic mitotic arrest. (A) Wild-type and various mutant cells bearing either GAL1-T7-BFA1–6His or GAL1-T7-BUB2–6His were streaked onto either YEP-glucose or YEP-galactose plate as indicated and incubated for 3 days. Overexpression of BFA1 completely inhibited the cell growth, whereas overexpression of BUB2 inhibited it modestly only in the presence of Bfa1. 1, KLY2170 (TEM1-myc); 2, KLY2243 (TEM1-myc GAL1-BFA1); 3, KLY3032 (TEM1-myc GAL1-BFA1 bub2Δ); 4, KLY2244 (TEM1-myc GAL1-BUB2); 5, KLY3044 (TEM1-myc GAL1-BUB2 bfa1Δ). (B) Reversal of the BFA1-induced mitotic arrest by overexpression of TEM1 and CDC15, but not by CDC5. Strain KLY2243 (GAL1-BFA1) was transformed with various constructs. The obtained transformants were streaked onto YEP-galactose to examine their cell growth. Wild-type cells were also cultured as a comparison for cell growth under these conditions. 1, Wild-type KLY1546; 2, KLY2243 (TEM1-myc GAL1-BFA1) transformed with pRS424-TEM1; 3, KLY2243 transformed with pRS424-CDC15; 4, KLY2243 transformed with pRS424-CDC14^TAB6–1 (25); 5, KLY2243 transformed with YEp351-CDC5; 6, KLY2243 transformed with pRS424 vector. (C) To closely monitor the arresting phenotype upon induction of either GAL1-BFA1 or GAL1-BUB2, samples were taken at the indicated time points after shifting the cultures to YEP-galactose medium. The fixed cells were subjected to DNA staining with 4′,6′-diamidino-2-phenylindole and then observed under a fluorescent microscope to determine the fraction of cells with separated sister chromatids. (Inset) The levels of either Bfa1 or Bub2 protein expressed under the induction conditions were determined by immunoblotting with an anti-T7 antibody. 1 and ▴, KLY2170 (TEM1-myc); 2 and ■, KLY2243 (TEM1-myc GAL1-BFA1); 3 and □, KLY3032 (TEM1-myc GAL1-BFA1 bub2Δ); 4 and ●, KLY2244 (TEM1-myc GAL1-BUB2); 5 and ○, KLY3044 (TEM1-myc GAL1-BUB2 bfa1Δ).