Ybp2 associates with the central kinetochore of Saccharomyces cerevisiae and mediates proper mitotic progression

Abstract

The spindle checkpoint ensures the accurate segregation of chromosomes by monitoring the status of kinetochore attachment to microtubules. Simultaneous mutations in one of several kinetochore and cohesion genes and a spindle checkpoint gene cause a synthetic-lethal or synthetic-sick phenotype. A synthetic genetic array (SGA) analysis using a mad2Delta query mutant strain of yeast identified YBP2, a gene whose product shares sequence similarity with the product of YBP1, which is required for H(2)O(2)-induced oxidation of the transcription factor Yap1. ybp2Delta was sensitive to benomyl and accumulated at the mitotic stage of the cell cycle. Ybp2 physically associates with proteins of the COMA complex (Ctf19, Okp1, Mcm21, and Ame1) and 3 components of the Ndc80 complex (Ndc80, Nuf2, and Spc25 but not Spc24) in the central kinetochore and with Cse4 (the centromeric histone and CENP-A homolog). Chromatin-immunoprecipitation analyses revealed that Ybp2 associates specifically with CEN DNA. Furthermore, ybp2Delta showed synthetic-sick interactions with mutants of the genes that encode the COMA complex components. Ybp2 seems to be part of a macromolecular kinetochore complex and appears to contribute to the proper associations among the central kinetochore subcomplexes and the kinetochore-specific nucleosome.

Figure 1. Ybp2 has a mitotic function.

(A) The ybp2Δ mutant is sensitive to benomyl. Wild-type (BY4741), ybp2Δ (Y1028), and mad2Δ (Y516) strains were spotted in 5-fold dilutions from 5×10⁴ cells per spot on YPD plates containing 20 µg/mL benomyl and incubated at 25°C for 3 days. (B) The ybp2Δ cells accumulate at G2/M. Logarithmically growing cells, wild type (Y14) and ybp2Δ (Y1831), were cultured at 25°C and processed for flow cytometry. (C) Double mutants of ybp2Δ and the spindle checkpoint genes show temperature-dependent synthetic lethality. Yeast strains were spotted in 5-fold dilutions from 5×10⁴ cells per spot on YPD plates. The plates were incubated at the indicated temperatures for 2 days. Isogenic yeast strains were wild type (YPH500), ybp2Δ (Y1342), mad1Δ (Y550), mad2Δ (Y1323), mad3Δ (Y554), bub1Δ (Y530), bub3Δ (Y548), ybp2Δmad1Δ (Y1398), ybp2Δmad2Δ (Y1419), ybp2Δmad3Δ (Y1402), ybp2Δbub1Δ (Y1415), and ybp2Δbub3Δ (Y1396). (D) Immunofluorescence analysis of ybp2Δ cells. Logarithmically growing cells cultured at 25°C were fixed and stained with antitubulin antibodies and DAPI. The strains shown are wild type (Y14) and ybp2Δ (Y1831). For each sample, 300 cells were counted.

Figure 2. The mitotic defect of the ybp2Δ mutant is enhanced by mad2Δ but not ybp1Δ.

(A) The G2/M delay of ybp2Δ cells depends on MAD2. Logarithmically growing cells cultured at 25°C were processed for flow cytometry. (B) The ybp2Δ strain does not display a substantial chromosome-missegregation phenotype. Chromosome fragment segregation was analyzed by a colony color assay (see Materials and Methods). Loss of nonessential chromosome fragments results in a red sector in a white colony. Isogenic yeast strains used in (A) and (B) were wild type (Y14), ybp2Δ (Y1831), mad2Δ (Y1833), and ybp2Δmad2Δ (Y1834). (C) The phenotype of the ybp1Δ mutant was not similar to that of the ybp2Δ mutant. Yeast strains were spotted in 5-fold dilutions from 5×10⁴ cells per spot on YPD plates containing the indicated concentrations of benomyl and incubated at 30°C for 2–3 days. (D) The ybp2Δ cells showed a subtle chromosome-missegregation phenotype, but ybp1Δ cells did not. The ybp2Δybp1Δ double mutant exhibited the same subtle chromosome-missegregation phenotype, indicating that Ybp1 does not participate in the mitotic function of Ybp2. Isogenic yeast strains used in (C) and (D) were wild type (Y14), ybp2Δ (Y1831), ybp1Δ (Y1835), and ybp2Δybp1Δ (Y1836).

Figure 3. Ybp2 physically associates with the COMA and Ndc80 complexes and Cse4.

(A–E) The indicated strains were grown to log phase at 30°C, lysed, and anti-myc immunoprecipitations were performed. Total lysate (T), supernatant (S), and the immunoprecipitated fraction (IP) were subjected to SDS-PAGE, and Western blots were used to detect Ybp2, Ctf19, Cse4, or myc-tagged proteins with the respective antibodies. For the quantification of Ybp2, aliquots of the total lysate (1/500 of the total), supernatant (1/500 of the total), and IP fraction (2/5 of the total lysate) were loaded. Arbitrary number is defined as the ratio of the amount of coprecipitated protein to the amount of input protein. Intensity of the total lysate is defined as 1 in each strain. Error bars indicate standard deviations of 2 or 3 experiments. The yeast strains used were (A) untagged (YPH499), Ybp2-myc (Y1689), Okp1-myc (Y1707), Mcm21-myc (Y1708), and Ame1-myc (Y1706); (B) untagged (YPH499), Ndc80-myc (Y1713), Nuf2-myc (Y1714), Spc24-myc (Y1715), and Spc25-myc (Y1716); (C) untagged (YPH499), Mtw1-myc (Y1709), Nsl1-myc (Y1710), Nnf1-myc (Y1711), and Dsn1-myc (Y1712); (D) Mcm16-myc (YVM325), Mcm22-myc (YVM290), Ctf3-myc (YVM219), Spc105-myc (Y1717), and Chl4-myc (YPH1542); and (E) untagged (YPH499) and Ybp2-myc (Y1689). Asterisks indicate IgG bands in immunoprecipitates. (F) Summary of coimmunoprecipitation assay results. Several kinetochore proteins were detected (red) in the Ybp2 immune complexes and others were not (brown).

Figure 4. Ybp2 associates with CEN DNA in an Ndc10-dependent manner.

(A and B) Anti-myc chromatin immunoprecipitation (ChIP) assays were performed from log-phase cells at 30°C (A) or 25°C (B). Total lysate (T, 1/800 of the total) and coimmunoprecipitated DNA (IP, 1/10 of the total) were analyzed by PCR with primers specific to centromeric regions of chromosome I and XVI and to a noncentromeric region (PGK1) as a control for binding specificity. The myc-tagged Okp1 strain was used as positive control. For the quantification of CEN1 and CEN16, aliquots of the total lysate (2/25 of the total) and IP fraction (2/5 of the total) were loaded on an 8% acrylamide gel. Arbitrary number is defined as the ratio of the amount of coprecipitated DNA to the amount of input DNA. Intensity of the total lysate is defined as 1 in each strain. The yeast strains used were (A) untagged (YPH499), Ybp2-myc (Y1689), and Okp1-myc (Y1707); and (B) untagged (YPH499), Ybp2-myc (Y1689), Ybp2-myc ndc10-1 (Y1838), and Okp1-myc (Y1707). (C) A model of the role of Ybp2 in the central kinetochore. Ybp2 interacts strongly with proteins in the COMA and Ndc80 complexes (shown in red) but not as well with those in the MIND complex and other kinetochore proteins (shown in brown). We propose that Ybp2 is localized to bridge the COMA and Ndc80 complexes onto the centromeric nucleosome.

Figure 5. Interactions among 3 central kinetochore subcomplexes in the ybp2Δ mutant.

(A) The model indicates the positions of the 3 central kinetochore subcomplexes Ndc80, MIND, and COMA. Orange arrows show protein–protein interactions that link the subcomplexes. (B–D) The indicated strains were grown to log phase at 30°C, lysed, and anti-myc immunoprecipitations were performed. Total lysate (T), supernatant (S), and the immunoprecipitated fraction (IP) were subjected to SDS-PAGE, and Western blots were used to detect Ctf19, HA-tagged, or myc-tagged proteins with the respective antibodies. For the quantification of (B) Mtw1-HA, aliquots of the total lysate (1/500 of the total), supernatant (1/500 of the total), and IP fraction (2/25 of the total lysate) were loaded. ND, not detectable. For the quantification of (C) Ctf19, aliquots of the total lysate (1/500 of the total), supernatant (1/500 of the total), and IP fraction (2/5 of the total lysate) were loaded. For the quantification of (D) Ctf19, aliquots of the total lysate (1/1000 of the total), supernatant (1/1000 of the total), and IP fraction (1/5 of the total lysate) were loaded. Arbitrary number is defined as described in Figure 3. The yeast strains used were (B) untagged (YPH499), Spc25-myc Mtw1-HA (Y1837), and Spc25-myc Mtw1-HA ybp2Δ (Y1839); (C) untagged (YPH499), Mtw1-myc (Y1709), and Mtw1-myc ybp2Δ (Y1840); and (D) untagged (YPH499), Ndc80-myc (Y1713), and Ndc80-myc ybp2Δ (Y1841). (E and F) Ndc80, Ctf19, and Mcm21 bind more tightly to CEN DNA in the absence of Ybp2. ChIP assays were performed with nocodazole-arrested cells (treated with 20 µg/mL nocodazole for 3 h at 25°C). Total lysate (T, 1/800 of the total) and coimmunoprecipitated DNA (IP, 1/10 of the total) were analyzed by PCR with primers specific to the centromeric regions of chromosome I and to a noncentromeric region (PGK1) as a control for binding specificity. For the quantification of CEN1, aliquots of the total lysate (2/25 of the total) and IP fraction (2/5 of the total) were loaded in 8% acrylamide gel. Arbitrary number is defined as described in Figure 4. ND, not done. Isogenic yeast strains were (E) untagged (YPH499), Ndc80-myc (Y1713), Ndc80-myc ybp2Δ (Y1841), Ctf19-myc (IPY313), Ctf19-myc ybp2Δ (Y1842), Mtw1-myc (Y1709), and Mtw1-myc ybp2Δ (Y1840); and (F) untagged (YPH499), Mcm21-myc (Y1708), Mcm21-myc ybp2Δ (Y1866), Nuf2-myc (Y1714), Nuf2-myc ybp2Δ (Y1867), Dsn1-myc (Y1712), and Dsn1-myc ybp2Δ (Y1868). (G) Synthetic-sick interaction between ybp2Δ and mutation of COMA complex genes. Yeast strains were spotted in 5-fold dilutions from 5×10⁴ cells per spot on YPD plates. The plates were incubated at the indicated temperatures for 2 days. Isogenic yeast strains used were wild type (YPH499), ybp2Δ (Y1337), ctf19Δ (YPH1315), mcm21Δ (Y1824), ame1-4 (YPH1676), okp1-5 (YPH1678), ybp2Δctf19Δ (Y1826), ybp2Δmcm21Δ (Y1827), ctf19Δmcm21Δ (Y1828), ybp2Δame1-4 (Y1864), ybp2Δokp1-5 (Y1865), and ybp2Δctf19Δmcm21Δ (Y1829).