Eskat, Anja, Deng, Wen, Hofmeister, Antje, Rudolphi, Sven, Emmerth, Stephan, Hellwig, Daniela, Ulbricht, Tobias, Döring, Volker, Bancroft, James M., McAinsh, Andrew D., Cardoso, M. Cristina, Meraldi, Patrick, Hoischen, Christian, Leonhardt, Heinrich and Diekmann, Stephan (2012) Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex. PLoS One, Vol.7 (No.9). e44717. doi:10.1371/journal.pone.0044717 ISSN 1932-6203.

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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.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School
Library of Congress Subject Headings (LCSH):	Mitosis
Journal or Publication Title:	PLoS One
Publisher:	Public Library of Science
ISSN:	1932-6203
Official Date:	September 2012
Dates:	Date Event September 2012 Published
Volume:	Vol.7
Number:	No.9
Page Range:	e44717
DOI:	10.1371/journal.pone.0044717
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access (Creative Commons)
Date of first compliant deposit:	22 December 2015
Date of first compliant Open Access:	22 December 2015
Funder:	Deutsche Forschungsgemeinschaft (DFG), Thüringer Aufbaubank
Grant number:	DI 258/17-1 (DFG), SPP1128 (DFG), SPP1395 (DFG), 2007 FE 9011 (TA)

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