Limits...
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.

Show MeSH

Related in: MedlinePlus

Eic1 interacts with three essential subunits of the CCAN/Mis6/Ctf19 complex. (a) LC-MS/MS analysis of GFP-tagged Eic1 or Eic2 immunoprecipitates from S. pombe whole cell extracts identifies Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 as Eic1-interacting proteins. Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Eic1-GFP co-immunoprecipitates with Fta7-Myc and Cnl2-Myc. In the top panel, the asterisk (*) denotes the IgG heavy chain, and the hash tag (#) denotes a non-specific band.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4043117&req=5

RSOB140043F7: Eic1 interacts with three essential subunits of the CCAN/Mis6/Ctf19 complex. (a) LC-MS/MS analysis of GFP-tagged Eic1 or Eic2 immunoprecipitates from S. pombe whole cell extracts identifies Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 as Eic1-interacting proteins. Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Eic1-GFP co-immunoprecipitates with Fta7-Myc and Cnl2-Myc. In the top panel, the asterisk (*) denotes the IgG heavy chain, and the hash tag (#) denotes a non-specific band.

Mentions: To further investigate the functional niche of Eic1 and Eic2, immunoprecipitates of both GFP-tagged proteins were subjected to LC-MS/MS analyses to identify associated factors. Only Eic1 and Mis18 were detected in Eic2-GFP immunoprecipitates (figure 7a). However, three CCAN/Mis6/Ctf19 complex components were found to associate with Eic1-GFP: Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 (figure 7a). Co-immunoprecipitation of Myc-tagged Fta7CENP-Q/Okp1 or Cnl2Nkp2 with Eic1-GFP verified that these CCAN/Mis6/Ctf19 complex components associate with Eic1 (figure 7b). ChIP analyses revealed that the association of Myc-tagged Fta7CENP-Q/Okp1, Myc-tagged Cnl2Nkp2 and GFP-tagged Mal2CENP-O/Mcm21 with centromeres is only partially dependent on functional Eic1 (figure 8a–c), as these proteins are detectable to a significant degree at centromeres in eic1-1 cells even at non-permissive temperature. This is consistent with the finding that centromeric association of the CCAN component Mis6CENP-I/Ctf3 also only partly depends on Eic1 function (figure 4d).Figure 7.


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 interacts with three essential subunits of the CCAN/Mis6/Ctf19 complex. (a) LC-MS/MS analysis of GFP-tagged Eic1 or Eic2 immunoprecipitates from S. pombe whole cell extracts identifies Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 as Eic1-interacting proteins. Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Eic1-GFP co-immunoprecipitates with Fta7-Myc and Cnl2-Myc. In the top panel, the asterisk (*) denotes the IgG heavy chain, and the hash tag (#) denotes a non-specific band.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043117&req=5

RSOB140043F7: Eic1 interacts with three essential subunits of the CCAN/Mis6/Ctf19 complex. (a) LC-MS/MS analysis of GFP-tagged Eic1 or Eic2 immunoprecipitates from S. pombe whole cell extracts identifies Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 as Eic1-interacting proteins. Average number of unique peptides reproducibly identified from three independent experiments is shown. (b) Eic1-GFP co-immunoprecipitates with Fta7-Myc and Cnl2-Myc. In the top panel, the asterisk (*) denotes the IgG heavy chain, and the hash tag (#) denotes a non-specific band.
Mentions: To further investigate the functional niche of Eic1 and Eic2, immunoprecipitates of both GFP-tagged proteins were subjected to LC-MS/MS analyses to identify associated factors. Only Eic1 and Mis18 were detected in Eic2-GFP immunoprecipitates (figure 7a). However, three CCAN/Mis6/Ctf19 complex components were found to associate with Eic1-GFP: Fta7CENP-Q/Okp1, Cnl2Nkp2 and Mal2CENP-O/Mcm21 (figure 7a). Co-immunoprecipitation of Myc-tagged Fta7CENP-Q/Okp1 or Cnl2Nkp2 with Eic1-GFP verified that these CCAN/Mis6/Ctf19 complex components associate with Eic1 (figure 7b). ChIP analyses revealed that the association of Myc-tagged Fta7CENP-Q/Okp1, Myc-tagged Cnl2Nkp2 and GFP-tagged Mal2CENP-O/Mcm21 with centromeres is only partially dependent on functional Eic1 (figure 8a–c), as these proteins are detectable to a significant degree at centromeres in eic1-1 cells even at non-permissive temperature. This is consistent with the finding that centromeric association of the CCAN component Mis6CENP-I/Ctf3 also only partly depends on Eic1 function (figure 4d).Figure 7.

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
Related in: MedlinePlus