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J Cell Biol. 2014 Aug 18;206(4):509-24. doi: 10.1083/jcb.201403081.

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A.

Author information

1
Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
2
Department of Biochemistry, Gene Center, Ludwig-Maximilians Universität München, 81377 Munich, Germany.
3
Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Institute of Molecular Biotechnology GmbH, Austrian Academy of Sciences, 1030 Vienna, Austria.
4
Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Institute of Molecular Biotechnology GmbH, Austrian Academy of Sciences, 1030 Vienna, Austria Center for Structural Systems Biology, University Medical Center Eppendorf-Hamburg, 20246 Hamburg, Germany Deutsches Elektronen-Synchrotron, 22607 Hamburg, Germany.
5
Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria westermann@imp.ac.at.

Abstract

Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.

Comment in

PMID:
25135934
PMCID:
PMC4137059
DOI:
10.1083/jcb.201403081
[Indexed for MEDLINE]
Free PMC Article

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