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Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly.

Subramanian L, Toda NR, Rappsilber J, Allshire RC - Open Biol (2014)

Bottom Line: CENP-A chromatin forms the foundation for kinetochore assembly.Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors.The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

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Eic1 and Eic2 are Mis18-interacting proteins. (a) LC-MS/MS analysis of Myc-tagged Mis16RbAp46/48/Hat2 and Mis18 immunoprecipitates from S. pombe whole cell extracts identifies two previously uncharacterized proteins, Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16). Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Primary sequence alignments of Eic1 (top) and Eic2 (bottom) orthologues identified among Schizosaccharomyces species. (c) Eic1-GFP co-immunoprecipitates with both Mis16-Myc and Mis18-Myc, while Eic2-GFP only co-immunoprecipitates with Mis18-Myc. The asterisk (*) in the bottom panel denotes the IgG heavy chain. (d) Eic1 directly interacts with Mis18 and Mis16. 6xHis-Eic1 was co-expressed with GST alone, GST-Mis18 or GST-Mis16 in E. coli. Coomassie-stained SDS-PAGE gels showing reciprocal GST (lanes 1–3) and His (lanes 6–8) pulldowns from E. coli lysates are shown. Also shown are reciprocal pulldowns of 6xHis-Mis16 co-expressed with GST alone or GST-Mis18 (lanes 4–5 and 9–10).
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RSOB140043F1: Eic1 and Eic2 are Mis18-interacting proteins. (a) LC-MS/MS analysis of Myc-tagged Mis16RbAp46/48/Hat2 and Mis18 immunoprecipitates from S. pombe whole cell extracts identifies two previously uncharacterized proteins, Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16). Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Primary sequence alignments of Eic1 (top) and Eic2 (bottom) orthologues identified among Schizosaccharomyces species. (c) Eic1-GFP co-immunoprecipitates with both Mis16-Myc and Mis18-Myc, while Eic2-GFP only co-immunoprecipitates with Mis18-Myc. The asterisk (*) in the bottom panel denotes the IgG heavy chain. (d) Eic1 directly interacts with Mis18 and Mis16. 6xHis-Eic1 was co-expressed with GST alone, GST-Mis18 or GST-Mis16 in E. coli. Coomassie-stained SDS-PAGE gels showing reciprocal GST (lanes 1–3) and His (lanes 6–8) pulldowns from E. coli lysates are shown. Also shown are reciprocal pulldowns of 6xHis-Mis16 co-expressed with GST alone or GST-Mis18 (lanes 4–5 and 9–10).

Mentions: To explore the interactions of Mis16RbAp46/48/Hat2 and Mis18 with other proteins in fission yeast, Myc-tagged Mis16 and Mis18 were immunoprecipitated from extracts of cells expressing either Mis16-Myc or Mis18-Myc as fusion proteins from the endogenous genes and the captured proteins subjected to LC-MS/MS analyses. Mis18 immunoprecipitates were reproducibly found to contain two previously uncharacterized proteins, which we named Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16) for Eighteen Interacting Centromere protein 1 and 2, respectively. Mis16RbAp46/48/Hat2 immunoprecipitates also reproducibly contained Eic1, along with two other known interactors: Mis18 and the histone H4 acetyltransferase Hat1 (figure 1a) [11,35]. Proteins orthologous to Eic1 were identifiable in all the sequenced genomes of the three other fission yeast species (S. octosporus, S. cryophilus and S. japonicus) by homology and synteny searches (figure 1b). However, no orthologue of Eic2 was apparent in S. japonicus (figure 1b).Figure 1.


Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly.

Subramanian L, Toda NR, Rappsilber J, Allshire RC - Open Biol (2014)

Eic1 and Eic2 are Mis18-interacting proteins. (a) LC-MS/MS analysis of Myc-tagged Mis16RbAp46/48/Hat2 and Mis18 immunoprecipitates from S. pombe whole cell extracts identifies two previously uncharacterized proteins, Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16). Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Primary sequence alignments of Eic1 (top) and Eic2 (bottom) orthologues identified among Schizosaccharomyces species. (c) Eic1-GFP co-immunoprecipitates with both Mis16-Myc and Mis18-Myc, while Eic2-GFP only co-immunoprecipitates with Mis18-Myc. The asterisk (*) in the bottom panel denotes the IgG heavy chain. (d) Eic1 directly interacts with Mis18 and Mis16. 6xHis-Eic1 was co-expressed with GST alone, GST-Mis18 or GST-Mis16 in E. coli. Coomassie-stained SDS-PAGE gels showing reciprocal GST (lanes 1–3) and His (lanes 6–8) pulldowns from E. coli lysates are shown. Also shown are reciprocal pulldowns of 6xHis-Mis16 co-expressed with GST alone or GST-Mis18 (lanes 4–5 and 9–10).
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Related In: Results  -  Collection

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RSOB140043F1: Eic1 and Eic2 are Mis18-interacting proteins. (a) LC-MS/MS analysis of Myc-tagged Mis16RbAp46/48/Hat2 and Mis18 immunoprecipitates from S. pombe whole cell extracts identifies two previously uncharacterized proteins, Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16). Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Primary sequence alignments of Eic1 (top) and Eic2 (bottom) orthologues identified among Schizosaccharomyces species. (c) Eic1-GFP co-immunoprecipitates with both Mis16-Myc and Mis18-Myc, while Eic2-GFP only co-immunoprecipitates with Mis18-Myc. The asterisk (*) in the bottom panel denotes the IgG heavy chain. (d) Eic1 directly interacts with Mis18 and Mis16. 6xHis-Eic1 was co-expressed with GST alone, GST-Mis18 or GST-Mis16 in E. coli. Coomassie-stained SDS-PAGE gels showing reciprocal GST (lanes 1–3) and His (lanes 6–8) pulldowns from E. coli lysates are shown. Also shown are reciprocal pulldowns of 6xHis-Mis16 co-expressed with GST alone or GST-Mis18 (lanes 4–5 and 9–10).
Mentions: To explore the interactions of Mis16RbAp46/48/Hat2 and Mis18 with other proteins in fission yeast, Myc-tagged Mis16 and Mis18 were immunoprecipitated from extracts of cells expressing either Mis16-Myc or Mis18-Myc as fusion proteins from the endogenous genes and the captured proteins subjected to LC-MS/MS analyses. Mis18 immunoprecipitates were reproducibly found to contain two previously uncharacterized proteins, which we named Eic1 (SPBC27B12.02) and Eic2 (SPBC776.16) for Eighteen Interacting Centromere protein 1 and 2, respectively. Mis16RbAp46/48/Hat2 immunoprecipitates also reproducibly contained Eic1, along with two other known interactors: Mis18 and the histone H4 acetyltransferase Hat1 (figure 1a) [11,35]. Proteins orthologous to Eic1 were identifiable in all the sequenced genomes of the three other fission yeast species (S. octosporus, S. cryophilus and S. japonicus) by homology and synteny searches (figure 1b). However, no orthologue of Eic2 was apparent in S. japonicus (figure 1b).Figure 1.

Bottom Line: CENP-A chromatin forms the foundation for kinetochore assembly.Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors.The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, UK.

ABSTRACT
CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.

Show MeSH