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

PLoS One. 2012;7(9):e44717. doi: 10.1371/journal.pone.0044717. Epub 2012 Sep 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Cycle Proteins
  • Cell Line, Tumor
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Fluorescence Resonance Energy Transfer
  • HeLa Cells
  • Histones
  • Humans
  • Kinetochores / metabolism*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*

Substances

  • CENPO protein, human
  • CENPQ protein, human
  • CENPU protein, human
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • Histones
  • Nuclear Proteins

Grant support

This work was supported by Deutsche Forschungsgemeinschaft (DFG DI 258/17-1, SPP1128, SPP1395), and Thüringer Aufbaubank (2007 FE 9011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.