Architecture of the budding yeast kinetochore reveals a conserved molecular core.
How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.
Department of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, Berkeley, CA 94720, USA. gbarnes@socrates.berkeley.edu
Stefan
S
Westermann
Iain M
IM
Cheeseman
Scott
S
Anderson
John R
JR
Yates
David G
DG
Drubin
Georjana
G
Barnes
D000595:Amino Acid Sequence; D064127:Aurora Kinases; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002503:Centromere / Q000378:metabolism; D002843:Chromatin / Q000302:isolation & purification / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D006657:Histones / Q000302:isolation & purification; D018386:Kinetochores / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009707:Nucleosomes / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000378:metabolism
C121933:CENPH protein, human; C092746:CSE4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006657:Histones; C083768:MIF2 protein, S cerevisiae; C120362:MTW1 protein, S cerevisiae; C108276:NNF1 protein, S cerevisiae; D009707:Nucleosomes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases