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CENP-T proteins are conserved centromere receptors of the Ndc80 complex

Nature Cell Biology volume 14, pages 604–613 (2012)Cite this article

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Abstract

Centromeres direct the assembly of kinetochores, microtubule-attachment sites that allow chromosome segregation on the mitotic spindle. Fundamental differences in size and organization between evolutionarily distant eukaryotic centromeres have in many cases obscured general principles of their function. Here we demonstrate that centromere-binding proteins are highly conserved between budding yeast and humans. We identify the histone-fold protein Cnn1CENP-T as a direct centromere receptor of the microtubule-binding Ndc80 complex. The amino terminus of Cnn1 contains a conserved peptide motif that mediates stoichiometric binding to the Spc24–25 domain of the Ndc80 complex. Consistent with the critical role of this interaction, artificial tethering of the Ndc80 complex through Cnn1 allows mini-chromosomes to segregate in the absence of a natural centromere. Our results reveal the molecular function of CENP-T proteins and demonstrate how the Ndc80 complex is anchored to centromeres in a manner that couples chromosome movement to spindle dynamics.

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Figure 1: Identification of Cnn1 as a budding yeast CENP-T homologue.
Figure 2: Proteomic analysis of the yeast CCAN and identification of the subunit Wip1.
Figure 3: Localization dependencies of yeast CCAN histone-fold proteins.
Figure 4: Cnn1CENP-T is an exclusive and direct interaction partner of the Ndc80 complex.
Figure 5: Cell-cycle-dependent interaction between Ndc80 and Cnn1CENP-T.
Figure 6: The N terminus of Cnn1CENP-T contains a conserved binding motif for the Spc24/25 domain of the Ndc80 complex.
Figure 7: Artificial tethering of the Ndc80 complex through Cnn1CENP-T allows mini-chromosomes to segregate in the absence of a centromere.

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Acknowledgements

The authors thank all members of the Westermann laboratory for discussions and J. M. Peters for critical reading of the manuscript. We thank T. Burkard for suggestions, W. Lugmayr for support with high-performance computing, O. Hudecz for help with presentation of the mass spectrometry results and M. Madalinski for peptide synthesis. We thank P. De Wulf (European Institute of Oncology, Milan, Italy) for the Cnn1–3GFP strain and communicating results before publication. Research in the Westermann laboratory receives funding from the European Research Council under the European Community’s Seventh Framework Programme (S.W. FP7/2007-2013)/ERC grant agreement no. 203499, and from the Austrian Science Fund FWF (S.W., SFB F34-B03).

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Authors and Affiliations

  1. IMP/IMBA Bioinformatics Core Facility, Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, 1030 Vienna, Austria

    Alexander Schleiffer

  2. Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, 1030 Vienna, Austria

    Michael Maier, Gabriele Litos, Fabienne Lampert, Peter Hornung, Karl Mechtler & Stefan Westermann

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Contributions

A.S. performed bioinformatic sequence analysis. M.M. purified CCAN proteins and performed characterization of proteins. P.H. performed interaction studies. F.L. conducted ChIP and biochemical experiments. K.M. guided the mass spectrometry analysis. S.W. guided the study and performed biochemical and genetic experiments supported by G.L. All authors discussed results and analysed data. S.W. and A.S. wrote the manuscript.

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Correspondence to Stefan Westermann.

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Schleiffer, A., Maier, M., Litos, G. et al. CENP-T proteins are conserved centromere receptors of the Ndc80 complex. Nat Cell Biol 14, 604–613 (2012). https://doi.org/10.1038/ncb2493

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