title: Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore.
abstract: Kinetochores orchestrate mitotic chromosome segregation. Here, we use quantitative mass spectrometry of mitotic chromosomes isolated from a comprehensive set of chicken DT40 mutants to examine the dependencies of 93 confirmed and putative kinetochore proteins for stable association with chromosomes. Clustering and network analysis reveal both known and unexpected aspects of coordinated behavior for members of kinetochore protein complexes. Surprisingly, CENP-T depends on CENP-N for chromosome localization. The Ndc80 complex exhibits robust correlations with all other complexes in a "core" kinetochore network. Ndc80 associated with CENP-T interacts with a cohort of Rod, zw10, and zwilch (RZZ)-interacting proteins that includes Spindly, Mad1, and CENP-E. This complex may coordinate microtubule binding with checkpoint signaling. Ndc80 associated with CENP-C forms the KMN (Knl1, Mis12, Ndc80) network and may be the microtubule-binding "workhorse" of the kinetochore. Our data also suggest that CENP-O and CENP-R may regulate the size of the inner kinetochore without influencing the assembly of the outer kinetochore.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.
authors.forename: Itaru
authors.initials: I
authors.lastname: Samejima
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.
authors.forename: Christos
authors.initials: C
authors.lastname: Spanos
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.
authors.forename: Flavia de Lima
authors.initials: Fde L
authors.lastname: Alves
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hori
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Marinela
authors.initials: M
authors.lastname: Perpelescu
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.
authors.forename: Juan
authors.initials: J
authors.lastname: Zou
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Fukagawa
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK bill.earnshaw@ed.ac.uk.
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002645:Chickens; D002875:Chromosomes / Q000235:genetics; D018386:Kinetochores / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D020543:Proteome / Q000235:genetics* / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D020543:Proteome