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Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex

Deng, Wen
Hofmeister, Antje
Rudolphi, Sven
Emmerth, Stephan
Hellwig, Daniela
Ulbricht, Tobias
Döring, Volker
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Published in PloS one. 2012, vol. 7, no. 9, e44717
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.
Keywords Blotting, WesternCell Line, TumorChromosomal Proteins, Non-Histone/genetics/metabolismFluorescence Resonance Energy TransferHeLa CellsHumansKinetochores/metabolismNuclear Proteins/genetics/metabolism
PMID: 23028590
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Research group Groupe Meraldi Patrick (physiologie cellulaire et métabolisme) (922)
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ESKAT, Anja et al. Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex. In: PloS one, 2012, vol. 7, n° 9, p. e44717. doi: 10.1371/journal.pone.0044717 https://archive-ouverte.unige.ch/unige:28855

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Deposited on : 2013-07-09

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