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Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

Pidoux AL, Choi ES, Abbott JK, Liu X, Kagansky A, Castillo AG, Hamilton GL, Richardson W, Rappsilber J, He X, Allshire RC - Mol. Cell (2009)

Bottom Line: Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase.Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin.While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, 6.34 Swann Building, Edinburgh EH93JR, Scotland, UK. alison.pidoux@ed.ac.uk

ABSTRACT
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

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Scm3Sp Associates with Itself, CENP-ACnp1, and Mis18(A) Coaffinity purification of Scm3Sp and CENP-ACnp1. Strains expressing the following tagged protein(s) were analyzed: Scm3-13myc only, both Scm3-13myc and CENP-ACnp1-CTAP, CENP-ACnp1-GFP only, or both CENP-ACnp1-GFP and Scm3-CTAP. Tagged genes were expressed with the native promoters at their endogenous loci, except that CENP-ACnp1-GFP was overexpressed (labeled with “oe”). IgG beads were used for affinity purification from cell extracts. Input samples (I) and bead-bound samples (B) were subject to western blotting using the indicated antibodies.(B) In vitro binding assay. (Left) 35S-labeled Scm3Sp was produced by in vitro transcription-translation in reticulocyte lysate (see [C], right panel) and incubated with GST, GST-Cnp1, or GST-H3. Complexes were pulled down with glutathione agarose, washed, and analyzed by SDS-PAGE and fluorography.(C) 35S-labeled Sim3, Scm3Sp, Mis16, and Mis18 were produced (right panel) and used in vitro binding assay with GST or GST- Scm3Sp (left panel).(D) Model for Scm3Sp function. The Mis6/Sim4 complex and Mis16/Mis18 are required to recruit Scm3Sp to centromeres. Scm3Sp acts as a receptor at the centromere for incoming CENP-ACnp1 from the Sim3 escort-chaperone. In conjunction with Mis16/Mis18 and other factors, Scm3Sp mediates the incorporation of CENP-ACnp1 (C) in subkinetochore chromatin in place of H3.
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fig7: Scm3Sp Associates with Itself, CENP-ACnp1, and Mis18(A) Coaffinity purification of Scm3Sp and CENP-ACnp1. Strains expressing the following tagged protein(s) were analyzed: Scm3-13myc only, both Scm3-13myc and CENP-ACnp1-CTAP, CENP-ACnp1-GFP only, or both CENP-ACnp1-GFP and Scm3-CTAP. Tagged genes were expressed with the native promoters at their endogenous loci, except that CENP-ACnp1-GFP was overexpressed (labeled with “oe”). IgG beads were used for affinity purification from cell extracts. Input samples (I) and bead-bound samples (B) were subject to western blotting using the indicated antibodies.(B) In vitro binding assay. (Left) 35S-labeled Scm3Sp was produced by in vitro transcription-translation in reticulocyte lysate (see [C], right panel) and incubated with GST, GST-Cnp1, or GST-H3. Complexes were pulled down with glutathione agarose, washed, and analyzed by SDS-PAGE and fluorography.(C) 35S-labeled Sim3, Scm3Sp, Mis16, and Mis18 were produced (right panel) and used in vitro binding assay with GST or GST- Scm3Sp (left panel).(D) Model for Scm3Sp function. The Mis6/Sim4 complex and Mis16/Mis18 are required to recruit Scm3Sp to centromeres. Scm3Sp acts as a receptor at the centromere for incoming CENP-ACnp1 from the Sim3 escort-chaperone. In conjunction with Mis16/Mis18 and other factors, Scm3Sp mediates the incorporation of CENP-ACnp1 (C) in subkinetochore chromatin in place of H3.

Mentions: Mis16 and Mis18 have been reported to coimmunoprecipitate, indicating that they associate in a complex (Hayashi et al., 2004). However, although Mis16 and Mis18 affect CENP-ACnp1 recruitment to centromeres, neither has been found in association with CENP-ACnp1. A possible role for Scm3Sp might be to act as the receptor for CENP-ACnp1 at the central domain of centromeres after delivery by the Sim3 escort (Dunleavy et al., 2007). This scenario predicts that Scm3Sp would interact with CENP-ACnp1. In strains expressing CENP-ACnp1-TAP and Scm3Sp-myc, from their own promoter at the endogenous locus, Scm3Sp-myc is pulled down with CENP-ACnp1-TAP. Reciprocally, in Scm3Sp-TAP strains overexpressing GFP-tagged CENP-ACnp1, CENP-ACnp1-GFP is pulled down with Scm3Sp-TAP (Figure 7A). Scm3Sp-myc is also pulled down with sheep anti-Cnp1 serum (Figure S10). Interestingly, in cells lacking the CENP-ACnp1 escort Sim3 (sim3Δ), the interaction between Scm3-TAP and overexpressed CENP-ACnp1-GFP is disrupted, suggesting that Sim3 is required to facilitate interactions between CENP-ACnp1 and Scm3Sp (Figure S10). This may be consistent with the Sim3 handing over CENP-ACnp1 to Scm3Sp.


Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.

Pidoux AL, Choi ES, Abbott JK, Liu X, Kagansky A, Castillo AG, Hamilton GL, Richardson W, Rappsilber J, He X, Allshire RC - Mol. Cell (2009)

Scm3Sp Associates with Itself, CENP-ACnp1, and Mis18(A) Coaffinity purification of Scm3Sp and CENP-ACnp1. Strains expressing the following tagged protein(s) were analyzed: Scm3-13myc only, both Scm3-13myc and CENP-ACnp1-CTAP, CENP-ACnp1-GFP only, or both CENP-ACnp1-GFP and Scm3-CTAP. Tagged genes were expressed with the native promoters at their endogenous loci, except that CENP-ACnp1-GFP was overexpressed (labeled with “oe”). IgG beads were used for affinity purification from cell extracts. Input samples (I) and bead-bound samples (B) were subject to western blotting using the indicated antibodies.(B) In vitro binding assay. (Left) 35S-labeled Scm3Sp was produced by in vitro transcription-translation in reticulocyte lysate (see [C], right panel) and incubated with GST, GST-Cnp1, or GST-H3. Complexes were pulled down with glutathione agarose, washed, and analyzed by SDS-PAGE and fluorography.(C) 35S-labeled Sim3, Scm3Sp, Mis16, and Mis18 were produced (right panel) and used in vitro binding assay with GST or GST- Scm3Sp (left panel).(D) Model for Scm3Sp function. The Mis6/Sim4 complex and Mis16/Mis18 are required to recruit Scm3Sp to centromeres. Scm3Sp acts as a receptor at the centromere for incoming CENP-ACnp1 from the Sim3 escort-chaperone. In conjunction with Mis16/Mis18 and other factors, Scm3Sp mediates the incorporation of CENP-ACnp1 (C) in subkinetochore chromatin in place of H3.
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fig7: Scm3Sp Associates with Itself, CENP-ACnp1, and Mis18(A) Coaffinity purification of Scm3Sp and CENP-ACnp1. Strains expressing the following tagged protein(s) were analyzed: Scm3-13myc only, both Scm3-13myc and CENP-ACnp1-CTAP, CENP-ACnp1-GFP only, or both CENP-ACnp1-GFP and Scm3-CTAP. Tagged genes were expressed with the native promoters at their endogenous loci, except that CENP-ACnp1-GFP was overexpressed (labeled with “oe”). IgG beads were used for affinity purification from cell extracts. Input samples (I) and bead-bound samples (B) were subject to western blotting using the indicated antibodies.(B) In vitro binding assay. (Left) 35S-labeled Scm3Sp was produced by in vitro transcription-translation in reticulocyte lysate (see [C], right panel) and incubated with GST, GST-Cnp1, or GST-H3. Complexes were pulled down with glutathione agarose, washed, and analyzed by SDS-PAGE and fluorography.(C) 35S-labeled Sim3, Scm3Sp, Mis16, and Mis18 were produced (right panel) and used in vitro binding assay with GST or GST- Scm3Sp (left panel).(D) Model for Scm3Sp function. The Mis6/Sim4 complex and Mis16/Mis18 are required to recruit Scm3Sp to centromeres. Scm3Sp acts as a receptor at the centromere for incoming CENP-ACnp1 from the Sim3 escort-chaperone. In conjunction with Mis16/Mis18 and other factors, Scm3Sp mediates the incorporation of CENP-ACnp1 (C) in subkinetochore chromatin in place of H3.
Mentions: Mis16 and Mis18 have been reported to coimmunoprecipitate, indicating that they associate in a complex (Hayashi et al., 2004). However, although Mis16 and Mis18 affect CENP-ACnp1 recruitment to centromeres, neither has been found in association with CENP-ACnp1. A possible role for Scm3Sp might be to act as the receptor for CENP-ACnp1 at the central domain of centromeres after delivery by the Sim3 escort (Dunleavy et al., 2007). This scenario predicts that Scm3Sp would interact with CENP-ACnp1. In strains expressing CENP-ACnp1-TAP and Scm3Sp-myc, from their own promoter at the endogenous locus, Scm3Sp-myc is pulled down with CENP-ACnp1-TAP. Reciprocally, in Scm3Sp-TAP strains overexpressing GFP-tagged CENP-ACnp1, CENP-ACnp1-GFP is pulled down with Scm3Sp-TAP (Figure 7A). Scm3Sp-myc is also pulled down with sheep anti-Cnp1 serum (Figure S10). Interestingly, in cells lacking the CENP-ACnp1 escort Sim3 (sim3Δ), the interaction between Scm3-TAP and overexpressed CENP-ACnp1-GFP is disrupted, suggesting that Sim3 is required to facilitate interactions between CENP-ACnp1 and Scm3Sp (Figure S10). This may be consistent with the Sim3 handing over CENP-ACnp1 to Scm3Sp.

Bottom Line: Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase.Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin.While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, 6.34 Swann Building, Edinburgh EH93JR, Scotland, UK. alison.pidoux@ed.ac.uk

ABSTRACT
The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

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