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Vol. 16, Issue 10, 4882-4892, October 2005

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Measuring the Stoichiometry and Physical Interactions between Components Elucidates the Architecture of the Vertebrate Kinetochore

Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, VA 22908

Submitted March 22, 2005; Revised July 18, 2005; Accepted July 25, 2005
Monitoring Editor: Kerry Bloom

Vertebrate kinetochores contain over 50 different proteins organized into three distinct regions: the inner plate, outer plate, and fibrous corona. The present study characterizes numerous precursors of kinetochore assembly in a system free of centromeric chromatin, Xenopus extracts. Hydrodynamic analysis suggests there are a minimum of two monomeric proteins and six preassembled complexes that accumulate on centromeres to form the kinetochore. The inner and outer kinetochore assemble from at least two distinct kinetochore complexes containing the proteins Mis12, Zwint, and Ndc80, all of which interact by immunoprecipitation. There is also a network of interactions between the fibrous corona proteins that is dissociated by microtubules. We quantify the number of molecules of specific proteins assembled into a single kinetochore. There are between 800 and 1200 molecules of the measured inner and outer kinetochore proteins, demonstrating that the components in these regions are in similar stoichiometry. In contrast, the measured fibrous corona proteins are present at 250-300 molecules per kinetochore. Zwint, but not Mis12, requires the Ndc80 complex for assembly into the kinetochore. Further, Ndc80 requires Zwint for assembly, indicating a codependency for these two proteins. Our data provide a model for the structural architecture and assembly pathway of the vertebrate kinetochore.

^* These authors contributed equally to this work.

Address correspondence to: P. Todd Stukenberg (pts7h{at}virginia.edu).

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