title: A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A.
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.
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Hornung
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Paulina
authors.initials: P
authors.lastname: Troc
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Francesca
authors.initials: F
authors.lastname: Malvezzi
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Maier
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Demianova
authors.affiliation: Department of Biochemistry, Gene Center, Ludwig-Maximilians Universität München, 81377 Munich, Germany.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Zimniak
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Litos
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Lampert
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Institute of Molecular Biotechnology GmbH, Austrian Academy of Sciences, 1030 Vienna, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Schleiffer
authors.affiliation: 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.
authors.forename: Matthias
authors.initials: M
authors.lastname: Brunner
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.affiliation: Department of Biochemistry, Gene Center, Ludwig-Maximilians Universität München, 81377 Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Herzog
authors.affiliation: 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.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Marlovits
authors.affiliation: Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria westermann@imp.ac.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Westermann
mesh_terms: D001324:Autoantigens / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002503:Centromere / Q000235:genetics; D000076247:Centromere Protein A; D002843:Chromatin / Q000235:genetics; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: C509718:Ame1 protein, S cerevisiae; D001324:Autoantigens; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C083768:MIF2 protein, S cerevisiae; C120362:MTW1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; C509719:Okp1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C060905:centromere protein C