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

Ohkuni K, Abdulle R, Tong AH, Boone C, Kitagawa K - PLoS ONE (2008)

Bottom Line: 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.Chromatin-immunoprecipitation analyses revealed that Ybp2 associates specifically with CEN DNA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

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.

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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).
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pone-0001617-g005: 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).

Mentions: Given the interaction of Ybp2 with the central kinetochore subcomplexes (COMA, MIND, and Ndc80), we expected that Ybp2 might be important for the interaction between the Ndc80 and the COMA complexes (Figure 4C). As per this hypothesis, the Ndc80–COMA association would be disrupted in the ybp2Δ background but the Ndc80–MIND and the COMA–MIND associations would not (Figure 5A). To test this hypothesis, we performed coimmunoprecipitation assays using ybp2Δ mutant cells. The Spc25–Mtw1 interaction for the Ndc80–MIND association, the Mtw1–Ctf19 interaction for the MIND–COMA association, and the Ndc80–Ctf19 interaction for the Ndc80–COMA association (Figure 5A) have been previously reported [37], [38]. First, we tested the interaction between Spc25 in the Ndc80 complex and Mtw1 in the MIND complex. We constructed an Spc25-myc Mtw1-HA double-tagged strain in the wild-type or ybp2Δ strain, performed an anti-myc immunoprecipitation assay, and identified the Mtw1-HA tagged protein by Western blot. The interaction of Spc25 with Mtw1 was slightly increased or unaffected in the absence of Ybp2 (Figure 5B). Next, we checked whether the lack of Ybp2 disrupted the interaction between Ctf19 in the COMA complex and Mtw1 in the MIND complex. The Ctf19–Mtw1 interaction was slightly increased in the absence of Ybp2 (Figure 5C). Finally, we checked whether the lack of Ybp2 disrupted the interaction between Ctf19 in the COMA complex and Ndc80 in the Ndc80 complex. Unexpectedly, the Ctf19–Ndc80 interaction was increased in the absence of Ybp2 (Figure 5D). Together, these results indicate that the lack of Ybp2 enhances the interactions among the central kinetochore subcomplexes (COMA, MIND, and Ndc80).


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

Ohkuni K, Abdulle R, Tong AH, Boone C, Kitagawa K - PLoS ONE (2008)

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).
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pone-0001617-g005: 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).
Mentions: Given the interaction of Ybp2 with the central kinetochore subcomplexes (COMA, MIND, and Ndc80), we expected that Ybp2 might be important for the interaction between the Ndc80 and the COMA complexes (Figure 4C). As per this hypothesis, the Ndc80–COMA association would be disrupted in the ybp2Δ background but the Ndc80–MIND and the COMA–MIND associations would not (Figure 5A). To test this hypothesis, we performed coimmunoprecipitation assays using ybp2Δ mutant cells. The Spc25–Mtw1 interaction for the Ndc80–MIND association, the Mtw1–Ctf19 interaction for the MIND–COMA association, and the Ndc80–Ctf19 interaction for the Ndc80–COMA association (Figure 5A) have been previously reported [37], [38]. First, we tested the interaction between Spc25 in the Ndc80 complex and Mtw1 in the MIND complex. We constructed an Spc25-myc Mtw1-HA double-tagged strain in the wild-type or ybp2Δ strain, performed an anti-myc immunoprecipitation assay, and identified the Mtw1-HA tagged protein by Western blot. The interaction of Spc25 with Mtw1 was slightly increased or unaffected in the absence of Ybp2 (Figure 5B). Next, we checked whether the lack of Ybp2 disrupted the interaction between Ctf19 in the COMA complex and Mtw1 in the MIND complex. The Ctf19–Mtw1 interaction was slightly increased in the absence of Ybp2 (Figure 5C). Finally, we checked whether the lack of Ybp2 disrupted the interaction between Ctf19 in the COMA complex and Ndc80 in the Ndc80 complex. Unexpectedly, the Ctf19–Ndc80 interaction was increased in the absence of Ybp2 (Figure 5D). Together, these results indicate that the lack of Ybp2 enhances the interactions among the central kinetochore subcomplexes (COMA, MIND, and Ndc80).

Bottom Line: 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.Chromatin-immunoprecipitation analyses revealed that Ybp2 associates specifically with CEN DNA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

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.

Show MeSH
Related in: MedlinePlus