Figure 2.

The genetic interactions of BFA1-DR2 mutants with various mitotic exit–defective cells. (A and B) The effects of BFA1-DR2 mutants on Δlte1Δbfa1 cells. (A) Δlte1BFA1 (YSK2052), Δlte1Δbfa1 (YSK2051), and Δlte1BFA1-DR2 mutants (YSK2058, 2059, 2060, and 2061) were serially diluted on YPAD and incubated at either 25°C for 2 d or 13°C for 10 d. (B) Cells (YSK2051, 2052, 2058, 2059, 2060, and 2061) were synchronized with α-factor at room temperature and released to fresh medium at 13°C. Cells were fixed and stained with DAPI. Anaphase cells were determined per indicated time point (n = 200). (C and D) The effects of BFA1-DR2 mutants on cdc5-2Δbfa1 cells. (C) Top, cdc5-2BFA1 (YSK2030), cdc5-2Δbfa1 (YSK823), and cdc5-2BFA1-DR2 mutants (YSK2036, 2037, 2038, and 2039) were serially diluted on YPAD and incubated at either 25 or 37°C. Bottom, cdc5-2BFA1-TAP cells (YSK1122) synchronized with α-factor at 25°C were released at 37°C, harvested at each time point, and their protein extracts were subjected to Western blotting with PAP. cdc15-2BFA1-TAP cells (YSK1153) incubated for 3 h at 37°C were included as a positive control for slowly migrating forms of Bfa1. Arrow indicates the slowest migrating form of Bfa1. (D) Cells (YSK823, 2030, 2036, 2037, 2038, and 2039) were synchronized with α-factor at 25°C and allowed to progress the cell cycle in YPAD at 37°C. At each time point, cells were collected to analyze DNA content by FACS (top, n = 50,000), and the cells with indicated phenotypes were counted (bottom, n = 200).