Effects of symmetrically localized myc-tagged Bub2 on Bfa1 and Tem1 localization and cdc5-2 cell viability. (A) Exponentially growing cells expressing Bub2-HA3 (ySP3866), Bfa1-HA6 (ySP2035), or Tem1-HA3 (ySP3641) were stained by indirect immunofluorescence with anti-HA antibodies and mounted with DAPI to stain DNA. (B) Cells expressing Bub2-myc9 alone (ySP710) or in combination with Bfa1-HA6 (ySP5087) or Tem1-HA3 (ySP4625) were treated as in A, using anti-myc antibodies to detect Bub2-myc9. Only anaphase cells were photographed. The arrows point to the bud. (C) Serial dilutions of logarithmically growing cultures of wild-type (ySP41), BUB2-myc9 (ySP710), MET3-CDC5 cdc5-2 (ySP3426), and MET3-CDC5 cdc5-2 BUB2-myc9 (ySP3442) cells, either untransformed or carrying one extra copy of BUB2 integrated at the URA3 locus (MET3-CDC5 cdc5-2 BUB2-myc9 BUB2 a and b, ySP3743, and ySP3744), were spotted on –Met (MET3 promoter on) or +Met (MET3 promoter off) plates and incubated for 2 d at 25°C.

Expression of Bub2-myc9 delays mitotic exit in cdc5-2 cells at permissive temperature. Cultures of wild-type (ySP41), MET3-CDC5 cdc5-2 (ySP3426), and MET3-CDC5 cdc5-2 BUB2-myc9 cells (ySP3480), exponentially growing in –Met medium, were arrested in G1 with α factor and released at 25°C in YEPD supplemented with 2 mM Met (MET3-CDC5 expression off). After 120 min, 10 μg/ml α factor was readded to prevent cells from entering a second cell cycle. Cells were collected at the indicated times for FACS analysis of DNA contents (A); kinetics of budding, nuclear division, and spindle elongation/breakdown after in situ immunostaining of tubulin (B); and Western analysis of Clb2 and Sic1, as well as Clb2/Cdk1 immunoprecipitations followed by kinase assays using histone H1 as substrate (C). Swi6 was used as loading control.

The mitotic exit delay of cdc5-2 BUB2-myc9 cells depends on functional Bfa1. Strains with the indicated genotypes (ySP3426, ySP3480, and ySP3481) were grown in –Met medium, arrested in G1 with α factor, and released at 25°C in the presence of Met. Cells were collected at the indicated time points for FACS analysis of DNA contents (top) and to measure kinetics of nuclear division and spindle elongation/breakdown (bottom) as described in Fig. 2 B.

Hyperactivation of the MEN restores viability of BUB2-myc9 cdc5-2 cells. Serial dilutions of wild-type (ySP41), MET3-CDC5 cdc5-2 (ySP3426), MET3-CDC5 cdc5-2 BUB2-myc9 (ySP3442 and ySP3480), MET3-CDC5 cdc5-2 BUB2-myc9 GAL1-LTE1 (ySP3482 and ySP3495), MET3-CDC5 cdc5-2 BUB2-myc9 GAL1-TEM1 (ySP3646), MET3-CDC5 cdc5-2 BUB2-myc9 GALs-TEM1 (ySP3904), MET3-CDC5 cdc5-2 BUB2-myc9 carrying a TEM1-bearing centromeric plasmid (YCp-TEM1), and MET3-CDC5 cdc5-2 BUB2-myc9 CDC14^TAB6-1 (ySP3891) strains, grown in –Met medium (MET3-CDC5 on), were spotted on –Met, YEPD +Met (MET3 and GAL1/GALs promoters off), and YEPRG +Met (MET3-CDC5 off, GAL1/GALs promoter on) plates, which were incubated at 25°C for 2 d.

Lack of Bub2 GAP activity leads to persistent symmetric localization of Bub2 on SPBs. (A, B, and D) Bacterially expressed GST-Bub2, MBP-Bfa1, and 6×His-Tem1 were used in in vitro GAP assays as previously described (Geymonat et al., 2002). In brief, 240 nM of 6×His-Tem1 was loaded with γ-[³²P]GTP in either the absence or presence of 150 nM MBP-Bfa1 and incubated at 30°C for 10 min. The mixture was then added to 15 μM of GST-Bub2 or buffer alone, and kinetics of GTP hydrolysis and dissociation was followed by filter binding assays (see Materials and methods). (C) Increasing amounts of the indicated Bub2 variants (0:1, 1:1, 2:1, 2.6:1, 6.6:1, 13:1, 26:1, 53:1, and 80:1 molar ratio Bub2/Bfa1) were added to 605 nM of 6×His-Tem1– γ-[³²P]GTP in the presence of 378 nM MBP-Bfa1 (final concentrations), and the fraction of filter-bound radioactivity was measured after 8 min. (E) Localization of Bub2R85A-HA3 at anaphase SPBs was monitored by in situ immunostaining with anti-HA antibodies. (F) Localization of Bfa1-HA6 in asynchronous BUB2R85A-myc9 cells was monitored as in E. (G, top) Protein extracts from BUB2-myc9 (ySP710), bub2R85A-myc9 (ySP4696), BUB2-myc9 BFA1-HA6 (ySP5087), and two different bub2R85A-myc9 BFA1-HA6 strains (ySP5118 and ySP5119) were used for Western blot analysis of either the total levels of Bub2-myc9 and Bfa1-HA6 (total) or the amount of Bub2-myc9 coimmunoprecipitating with Bfa1-HA6 (anti-HA IP). (bottom) Protein extracts from BFA1-HA6 (ySP2035), BUB2-myc9 BFA1-HA6 (ySP5087), and two different bub2R85A-myc9 BFA1-HA6 strains (ySP5118 and ySP5119) were used for Western blot analysis of either the total levels of Bub2-myc9 and Bfa1-HA6 (total) or the amount of Bfa1-HA6 coimmunoprecipitating with Bub2-myc9 (anti-myc IP).

Disappearance of Bub2 from the mother-bound SPB depends on proteins localized at the bud neck. (A) Localization of Bub2-HA3 was analyzed on formaldehyde-fixed cells undergoing properly oriented anaphase (as assessed by DAPI staining) from logarithmically growing cultures at 25°C, with the exception of BUB2-HA3 4× GAL1-CLA4t swe1Δ cells, which were grown in raffinose-containing medium, arrested in G1 by α factor, and released in the presence of galactose for 3 h to induce GAL1-CLA4t expression. At least 150 anaphase cells were scored for each strain. (B) Wild-type (ySP41), 4× GAL1-CLA4t swe1Δ (ySP2711), and 4× GAL1-CLA4t swe1Δ bub2Δ (ySP2728) cells were grown in YEPR at 25°C, arrested in G1 with α factor, and released in YEPRG at 25°C. Galactose was added 30 min before the release. 120 min after the release, 10 μg/ml α factor was readded to prevent cells from entering a second cell cycle. Cells were collected at the indicated times for kinetics of budding, nuclear division, and bipolar spindle formation/breakdown after in situ immunostaining of tubulin. Bipolar spindles include both metaphase and anaphase spindles. (C) Serial dilutions of wild-type (ySP41), cdc5-2 (ySP324), GAL1-CLA4t (ySP2622), GAL1-CLA4t cdc5-2 (ySP3565), GAL1-CLA4t cdc5-2 bub2Δ (ySP4802), and GAL1-CLA4t cdc5-2 bfa1Δ (ySP4804) cell cultures were spotted on either YPD (GAL1 promoter off) or YPRG (GAL1 promoter on) plates and incubated at 25°C for 3 d. (D) Serial dilutions of wild type (ySP41), cdc5-2 (ySP324), cdc12-1 (ySP293), cdc5-2 bfa1Δ (ySP3671), cdc12-1 cdc5-2 (ySP4473 and ySP4474), and cdc12-1 cdc5-2 bfa1Δ (ySP4518 and ySP4519) were spotted on YPD plates, which were incubated at 25 and 30°C for 2 d.

A schematic model for mitotic exit. In the presence of spindle positioning defects (A), Bub2-dependent Bfa1 retention at both SPBs prevents mitotic exit by inhibiting Tem1. When anaphase takes place correctly and one SPB crosses the bud neck (B), a signal is sent to the mother-bound SPB, where the Bub2–Bfa1 complex is removed through the action of Bub2 GAP activity, thus leading to Tem1 activation at this pole. This helps trigger mitotic exit, together with Bfa1 inhibition by Cdc5, presumably at the bud-directed SPB, and with increased Tem1 loading on the same SPB.