title: Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex.
abstract: Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.
authors.affiliation: Molecular Biology, FLI, Jena, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Eskat
authors.forename: Wen
authors.initials: W
authors.lastname: Deng
authors.forename: Antje
authors.initials: A
authors.lastname: Hofmeister
authors.forename: Sven
authors.initials: S
authors.lastname: Rudolphi
authors.forename: Stephan
authors.initials: S
authors.lastname: Emmerth
authors.forename: Daniela
authors.initials: D
authors.lastname: Hellwig
authors.forename: Tobias
authors.initials: T
authors.lastname: Ulbricht
authors.forename: Volker
authors.initials: V
authors.lastname: Döring
authors.forename: James M
authors.initials: JM
authors.lastname: Bancroft
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McAinsh
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Cardoso
authors.forename: Patrick
authors.initials: P
authors.lastname: Meraldi
authors.forename: Christian
authors.initials: C
authors.lastname: Hoischen
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Stephan
authors.initials: S
authors.lastname: Diekmann
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006657:Histones; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C540903:CENPO protein, human; C559900:CENPQ protein, human; C485917:CENPU protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009687:Nuclear Proteins