The isolation of pGAL-CSE4 mutants that are lethal when overexpressed. (A) Fivefold serial dilutions of wild-type (SBY3), pGAL-CSE4 (SBY1812), pGAL-CSE4-350 (SBY1737), pGAL-CSE4-351 (SBY1738), pGAL-CSE4-352 (SBY1739), pGAL-CSE4-353 (SBY1740), pGAL-CSE4-354 (SBY1775), pGAL-CSE4-356 (SBY1741), pGAL-CSE4-358 (SBY1742), pGAL-CSE4-359 (SBY1743), pGAL-CSE4-360 (SBY1744), pGAL-CSE4-361 (SBY1745), and pGAL-CSE4-362 (SBY1746) cells were incubated on GLU or GAL media at 23°. Although the overexpression of wild-type pGAL-CSE4 has no growth defect, overexpression of the pGAL-CSE4 mutants is lethal. (B) Most Cse4 mutant proteins are expressed at 10- to 100-fold higher levels than the wild-type Cse4 protein. Strains in A were induced with GAL for 6 hr and harvested for immunoblot analysis with anti-Cse4p and anti-Tub1p antibodies (loading control). To normalize for loading, 0.1× was loaded for the indicated Cse4p immunoblots.

pGAL-CSE4 mutants can be divided into three classes on the basis of localization and spindle checkpoint activation. (A) The localization of wild-type Cse4p in cells overexpressing pGAL-CSE4-353. Cse4p-myc cells with (SBY1949) or without pGAL-CSE4-353 (SBY617) were induced with GAL for 6 hr and harvested for ChIP analysis with primers to CEN3 and PGK1. Threefold serial dilutions of input and IP are shown. The wild-type Cse4 protein still localizes to the centromere in cells overexpressing the Cse4-353 mutant protein. (B) Localization of the Cse4-352 and Cse4-353 mutant proteins. Cells containing pGAL-CSE4-myc (SBY1071), pGAL-CSE4-352-myc (SBY2640), or pGAL-CSE4-353-myc (SBY2567) were grown and processed for ChIP as in A. The Cse4-352 mutant protein localizes to CEN3, PGK1, HMR, and TEL6, while the Cse4-353 mutant protein does not localize to any of these loci. (C) FACS analysis of cells overexpressing pGAL-CSE4-350 and pGAL-CSE4-353. Wild-type (SBY3), pGAL-CSE4 (SBY1812), pGAL-CSE4-350 (SBY1737), and pGAL-CSE4-353 (SBY1740) cells were arrested in G₁, released into GAL media, and harvested at the indicated times (hours) for FACS analysis. Wild-type and pGAL-CSE4 cells progress normally through the cell cycle, while cells overexpressing pGAL-CSE4-353 arrest in G₂/M. Cells overexpressing pGAL-CSE4-350 show an increase in G₂/M cells. (D) Cells overexpressing pGAL-CSE4-353 arrest due to spindle checkpoint activation. pGAL-CSE4-353 (SBY1893) and pGAL-CSE4-353 mad2Δ (SBY1894) cells were grown as in A and FACS was performed. (E) Schematic of the classes of pGAL-CSE4 mutants obtained. Class I mutants are defined by the localization of mutant Cse4p to euchromatin and by the absence of spindle checkpoint activation. Class II mutants are defined by Cse4p mutant localization to the centromere and activation of the spindle checkpoint. In this class, the endogenous Cse4 protein may or may not localize to the centromere and the mutant protein may or may not localize to euchromatin. Only two possibilities are shown. Class III mutants are defined by the absence of mutant Cse4p throughout the genome and activation of the spindle checkpoint.

Kinetochore assembly and cohesin localization are normal in cells overexpressing pGAL-CSE4-353. (A–F) pGAL-CSE4-353 cells containing the indicated myc epitope-tagged kinetochore protein were grown in GLU or GAL media for 6 hr and harvested for ChIP analysis with primers to CEN3 and PGK1. Threefold serial dilutions of input and IP are shown. Mif2-myc13 (A, SBY4279), Ctf13-myc13 (B, SBY4280), Ctf19-myc13 (C, SBY4282), Okp1-myc13 (D, SBY4283), Mtw1-myc13 (E, SBY4278), and Ndc80-myc13 (F, SBY4281) all localize to kinetochores in the presence or absence of pGAL-CSE4-353 overexpression. (G and H) ChIP on microarray analysis of the cohesin Mcd1p in cells overexpressing pGAL-CSE4-353. Cells containing Mcd1-6HA that expressed pGAL-CSE4 (RC66) or pGAL-CSE4-353 (RC65) were grown in GAL and nocodazole and ChIP on chip was performed to localize Mcd1/Scc1 throughout the genome. The genomic region around CEN3 (*) is shown and indicates that there is no significant difference in the localization of Mcd1p when Cse4-353p is overexpressed.

Cells overexpressing pGAL-CSE4-353 have an increased distance between kinetochore clusters. Wild-type cells (SBY5437) and pGAL-CSE4-353 cells (SBY5446) expressing the kinetochore protein Mtw1-GFP3 and the SPB protein Spc29-CFP were arrested in G₁ with αF, released into the cell cycle in GAL media, and analyzed 100 min after release from G₁ by microscopy. (A) The Mtw1-GFP3 kinetochore clusters appear closer to the SPBs in pGAL-CSE4-353 cells compared to wild-type cells (compare panels 6 and 3). Bar, 5 μm. (B–D). Histograms of 200 cells of each strain indicate the following: (B) the distance between kinetochore clusters (KT-cluster distance), (C) the distance between SPBs (SPB–SPB distance), and (D) the ratio of the kinetochore cluster distance to SPB distance (KT/SPB normalized). The average of 200 cells is indicated. Cells overexpressing pGAL-CSE4-353 have a significantly greater distance between kinetochore clusters (P < 0.01).

Cells overexpressing pGAL-CSE4-353 are defective in kinetochore biorientation. (A) Analysis of biorientation in metaphase-arrested cells. pGAL-CDC20 (SBY5050) and pGAL-CSE4-353 cells (SBY5104) containing CEN8-GFP were grown in GLU or GAL media, respectively, for 6 hr. Cells with one CEN8 focus were scored as mono-oriented and cells with two distinct CEN8 foci were scored as bioriented. (B) Analysis of the segregation of a pair of sister chromatids in cells overexpressing pGAL-CSE4-353. Mad2Δ cells with (SBY5313) or without pGAL-CSE4-353 (SBY5314) that contain CEN8-GFP and Tub1-CFP were arrested in G₁, released into GAL media, and scored for CEN8 segregation after 150 min. Bar, 5 μm. Representative pictures of sister-chromatid segregation and spindle elongation are shown at t = 150 min as well as quantification of the classes of phenotypes scored. Although spindles elongate in mad2Δ cells overexpressing pGAL-CSE4-353, 53% of the cells segregate both sisters to the same pole. (C) Cells overexpressing pGAL-CSE4-353 require the Ipl1/aurora protein kinase to activate the spindle checkpoint. Wild-type (SBY818), pGAL-CSE4-353 (SBY5014), ipl1-as5 (SBY5456), and pGAL-CSE4-353 ipl1-as5 (SBY5455) cells that contain Pds1-myc18 were arrested in G₁ with αF and released into GAL media. The inhibitor 1NA-PP1 was added 80 min after release when small budded cells were first visible. Cells were harvested for immunoblot every 30 min and analyzed for Pds1p and Tub1p levels (data not shown).