A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

作者：Peter Hornung, Paulina Troć, Francesca Malvezzi, Michael Maier, Zuzana Demianová, Tomasz Zimniak, Gabriele Litos, Fabienne Lampert, Alexander Schleiffer, Matthias Brünner, Karl Mechtler, Franz Herzog, Thomas C. Marlovits, Stefan Westermann · 发表于：The Journal of Cell Biology · 年份：2014 · DOI：10.1083/jcb.201403081 · 被引用次数：122 · 研究领域：Microtubule and mitosis dynamics、Genomics and Chromatin Dynamics、Chromosomal and Genetic Variations

Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.