Fig. 1. Ask1p, Dad2p, Spc19p and Spc34p are components of the DDD complex. (A) Immunoprecipitation of DDD complex components with anti-HA antibodies. Immunoblots are shown. The asterisk shows the IgG light chain that is detected by the secondary antibody. (B) Dad2p–ProA was purified from SPC34 DAD2-ProA or spc34-3 DAD2-ProA cells grown at 23°C (lane 1) or incubated for 4 h at 37°C (lanes 2 and 3). The purified proteins were analysed by SDS–PAGE and Coomassie Blue staining. Protein bands were identified by MALDI.

Fig. 3. Determinates of kinetochore association of Dad2p and Spc34p. (A) Association of Dad2p and Spc34p with kinetochores but not MTs is dependent on Ndc80p. Wild-type NDC80 and ndc80-1 cells with DAD2-GFP and SPC34-GFP were incubated at 37°C for 3 h. Cells were analysed by fluorescence microscopy. Arrows indicate kinetochores. Bar: 5 µm. (B) Centromere binding of Dad2p-3HA requires MTs. DAD2-3HA cells were incubated with (lanes 4–6) and without 10 µg/ml nocodazole (lanes 1–3) for 3 h at 30°C. ChIPs with anti-HA (lanes 2 and 5) and anti-Mcm21p antibodies (lanes 3 and 6) were performed as in Figure 2A. Lanes 1 and 4 represent the input material.

Fig. 5. Spc34p is required to maintain bipolarity. (A) Cells of SPC34 and spc34-3 with GFP-CEN5 Gal1-CDC20 were grown in galactose medium at 23°C. Cells were then incubated for 2 h at 23°C in glucose medium to repress Gal1-CDC20 followed by a shift to 37°C for 1 h. Cells were analysed as in Figure 2D. Bar: 5 µm. (B) Sister CEN5 DNA biorientation gradually decreases in spc34-3 cells. Cells of SPC34 and spc34-3 with CFP-TUB1 YFP-CEN5 Gal1-CDC20 were incubated for 2 h at 23°C in glucose medium to arrest cells in metaphase. Cells were then shifted to 37°C. Separation of sister CEN5 of cells with a metaphase spindle was determined by fluorescence microscopy. (C) Cells of (B) were synchronized with α-factor at 23°C to mark the mother cell body with a mating projection. After washing the cells with glucose medium, they arrested in metaphase with a short spindle. The sample designated ‘23°C’ was taken and fixed. Cells were then incubated for 1 h at 37°C. Sister CEN5 localization was determined by fluorescent microscopy; n >100.

Fig. 7. Sister kinetochores of spc34-3 cells are functional. (A) Gal1-3HA-SCC1 spc34-3 cells carrying CFP-TUB1 YFP-CEN5 were grown in galactose medium at 23°C (‘Gal’). Cells were resuspended in glucose medium with α-factor and incubated for 4 h at 23°C to suppress SCC1 expression (‘Glc’). Shown is an immunoblot with anti-HA antibodies. Tub2p was detected as loading control. (B) Cells of (A) with depleted Scc1p were washed with glucose medium to remove α-factor and simultaneously shifted to 37°C. After 2 h at 37°C, spindle formation (CFP–Tub1p) and CEN5 localization (CEN5–YFP) were analysed by fluorescence microscopy. The asterisk indicates CFP–Tub1p, which is not associated with the spindle. Bar: 5 µm. (C) Quantification of (B). Shown is the average of two independent experiments. The CEN5 DNA localization of at least 100 large budded cells was determined. Cartoons are as in Figure 6A.

Fig. 2. Ask1p, Dad1p, Dad2p, Spc19p and Spc34p are kinetochore components and localize along nuclear MTs. (A) ChIP analysis. CEN3 DNA was co-immunoprecipitated with Dad1p-3HA (lane 4), Dad2p-3HA (lane 6), Spc19p-3HA (lane 8), Ask1p-6HA (lane 10) and Spc34p-3HA (lane 12) but not with Bim1p-3HA (lane 2). CEN3 DNA and control DNAs flanking CEN3 (ChIII-R and ChIII-L) were detected equally in the input material (lanes 1, 3, 5, 7, 9 and 11, load). (B) Spc34p co-localizes with the kinetochore protein Mcm21p. Fixed SPC34-GFP MCM21-9Myc cells were subjected to indirect immunofluorescence with anti-Myc antibodies. The GFP signal was analysed by fluorescence microscopy. The arrows indicate localization of Spc34p–GFP at the nuclear MTs. (C) Localization of Dad1p-3Myc by immunoelectron microscopy. Bar: 0.25 µm. (D) Dad2p, Duo1p and Spc34p do not localize with SPBs. Cells of DAD2-GFP, DUO1-GFP and SPC34-GFP were analysed as in (B) except using anti-Spc72p and anti-Tub2p antibodies. Cells in panels 1 and 2 are identical but, in (1), the tubulin signal is not shown. (E) Cells of cdc15-1 with the indicated gene fusions were incubated for 3 h at 37°C. DNA was stained with DAPI. Cells were analysed by fluorescence microscopy. Bars in (B), (D) and (E): 5 µm.

Fig. 4. Conditional lethal cells of SPC34 are defective in anaphase spindle formation and biorientation of sister centromeres. (A–C) Cells of SPC34 and spc34-3 with PDS1-6HA or GFP-TUB1 were synchronized with α-factor (t = 0). After release from the cell cycle block by washing, cells were shifted to 37°C. (A) Samples were analysed every 20 min for DNA content, nuclear Pds1p by indirect immunofluorescence, and budding index (n >100). (B) Spindle morphology was determined 100 min after release from α-factor by fluorescence microscopy. The smaller panels in 3–7 are 2-fold magnifications of the spindles below. (C) Quantification of the spindle phenotypes of spc34-3 cells of (B); n >100. The blue area in the cartoons to the bottom symbolizes the DAPI-staining regions. The lines inside the cells indicate MTs and the dotted lines defective MTs. (D) Cells of SPC34-GFP and spc34-3-GFP were incubated for 3 h at 37°C. Cells were inspected by fluorescence microscopy. Protein extracts of the indicated cells were analysed by immunoblotting with anti-GFP and anti-Tub2p antibodies. (E) Duplicated CEN5 of spc34-3 cells fails to attach to the spindle in a bipolar manner. α-Factor-synchronized SPC34 GFP-CEN5 and spc34-3 GFP-CEN5 cells were fixed after 3 h at 37°C and analysed as in Figure 2D. (F) Chromosomes of spc34-3 cells are segregated in the presence of nuclear Pds1p. SPC34 GFP-CEN5 PDS1-9Myc and spc34-3 GFP-CEN5 PDS1-9Myc cells were shifted for 2 h (panels 1 and 2) or 3 h (panel 3) to 37°C. Cells were subjected to indirect immunofluorescence to stain Pds1p-9Myc. GFP–CEN5 was visualized by fluorescence microscopy. Bars: 5 µm.

Fig. 6. Sister CEN5 DNAs of spc34-3 cells are associated preferentially with the old SPB. (A) SPC34 and spc34-3 cells with CFP-TUB1 YFP-CEN5 were arrested with α-factor to mark the mother cell. Washed cells were then shifted to 37°C for 3 h. CFP–Tub1p and YFP–CEN5 were analysed by fluorescence microscopy. The percentage of anaphase cells with YFP–CEN5 (red dot) at both or only one of the two spindle poles was determined in two independent experiments (n >100). The black lines in the drawn cells symbolize the MTs. The mating projection of the mother cell body is shown as an extension. (B) Cells of GFP-CEN5 RFP-SPC42 in which the old SPB is marked by the fluorescent RFP signal were grown as described in Materials and methods. The Spc42p–RFP and the GFP–CEN5 signals were detected by fluorescence microscopy. The red dot in the cartoons indicates the old SPB and the green dots the GFP–CEN5 signals. The mating projection marking the mother cell body is indicated as an extension. (C) Quantification of (B); n >100. Symbols are as in (B). (D) Preferential binding of sister chromatids to the old SPB is dependent on the time of centromere replication. Cells of spc34-3 with the late and early replicating plasmids pT334 and pT331 were analysed as in (A). Cartoon as in (A). (E) Gal1-3HA-SCC1 SPC34 cells carrying CFP-TUB1 YFP-CEN5 were grown and analysed as described in Figure 7A. (F) Cells of (E) were treated and analysed as in Figure 7B. (G) Quantification of (F). Shown is the average of two independent experiments; n >100. Cartoons are as in (A). Bars: 5 µm.

Fig. 8. Spc34p is required to maintain an anaphase spindle. (A) SPC34 (lanes 1–4) and spc34-3 cells (lanes 6–8) with Gal1-lacI-TEM1 GFP-TUB1 were grown in 2% galactose 3% raffinose medium at 23°C (lanes 1 and 5) followed by the addition of 2% glucose to repress Ga11-lacI-TEM1 expression (lanes 2 and 6). Anaphase cells were then incubated in glucose medium for up to 2 h at 37°C (lanes 3, 4, 7 and 8). Cells were tested by immunoblotting for Tem1p, Clb2p and Tub2p. Cells of Δclb2 in lane 9 were used as control for the anti-Clb2p antibodies. (B) Cells of (A), after incubation in glucose medium at 37°C, were fixed, stained with DAPI and analysed by fluorescence microscopy. Shown are cells after 1 h at 37°C. (C) Quantification of (B); n >100. Only the percentage of large-budded cells is indicated.