title: CCAN Assembly Configures Composite Binding Interfaces to Promote Cross-Linking of Ndc80 Complexes at the Kinetochore.
abstract: Partitioning of the genome requires kinetochores, large protein complexes that mediate dynamic attachment of chromosomes to the spindle. Kinetochores contain two supramolecular protein assemblies. The ten-protein KMN network harbors key microtubule-binding sites in the Ndc80 complex and mediates assembly of checkpoint complexes via the KNL-1/Spc105 protein [1, 2]. As KMN does not contact DNA directly, it relies on different centromere-binding proteins for recruitment and cell-cycle-dependent assembly. These proteins are collectively referred to as the CCAN (constitutive centromere-associated network) [2-4]. The molecular mechanisms by which CCAN subunits associate, however, have remained incompletely defined. In particular, it is unclear how CCAN subunits facilitate the assembly of a microtubule-binding interface that contains multiple Ndc80 molecules bound to different receptors [5]. Here, we dissect molecular mechanisms that underlie targeting of the CCAN subunit Cnn1/CENP-T to the sequence-determined point centromeres of budding yeast. Systematic quantitative mass spectrometry experiments reveal association dependencies within the yeast CCAN network. We show that evolutionarily conserved residues in the histone-fold domain of Cnn1 are required for the formation of a stable five-subunit CCAN subassembly with the Ctf3 complex. Cnn1 localizes in a Ctf3-dependent manner to the core of the yeast point centromere, overlapping with the yeast CENP-A protein Cse4. By arranging the N-terminal domains of the CCAN subunits Mcm16, Mcm22, and Cnn1 into close proximity, the Ctf3c-Cnn1-Wip1 complex configures a composite interaction site for two molecules of the Ndc80 complex. Our experiments show how cooperative assembly mechanisms organize the microtubule-binding interface of the kinetochore.
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Gülsah
authors.initials: G
authors.lastname: Pekgöz Altunkaya
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Francesca
authors.initials: F
authors.lastname: Malvezzi
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Demianova
authors.affiliation: Gene Center and Department of Biochemistry, Ludwig-Maximilians Universität München, Fedor-Lynen Strasse 25, 81377 Munich, Germany.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Zimniak
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Litos
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Weissmann
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.affiliation: Gene Center and Department of Biochemistry, Ludwig-Maximilians Universität München, Fedor-Lynen Strasse 25, 81377 Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Herzog
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, 1030 Vienna, Austria; Department of Molecular Genetics, Faculty of Biology, Center for Medical Biotechnology, University of Duisburg-Essen, Universitätsstrasse 5, 45117 Essen, Germany. Electronic address: stefan.westermann@uni-due.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Westermann
mesh_terms: D002503:Centromere / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D029701:Saccharomyces cerevisiae Proteins
keywords: CCAN; centromere; histone-fold; kinetochore; nucleosome; spindle