Abstract
Kinetochores are chromatin‐bound multi‐protein complexes that allow high‐fidelity chromosome segregation during mitosis and meiosis. Kinetochore assembly is exclusively initiated at chromatin containing Cse4/CENP‐A nucleosomes. The molecular mechanisms ensuring that subcomplexes assemble efficiently into kinetochores only at centromeres, but not anywhere else, are incompletely understood. Here, we combine biochemical and genetic experiments to demonstrate that auto‐inhibition of the conserved kinetochore subunit Mif2/CENP‐C contributes to preventing unscheduled kinetochore assembly in budding yeast cells. We show that wild‐type Mif2 is attenuated in its ability to bind a key downstream component in the assembly pathway, the Mtw1 complex, and that addition of Cse4 nucleosomes overcomes this inhibition. By exchanging the N‐terminus of Mif2 with its functional counterpart from Ame1/CENP‐U, we have created a Mif2 mutant which bypasses the Cse4 requirement for Mtw1 binding in vitro, thereby shortcutting kinetochore assembly. Expression of this Mif2 mutant in cells leads to mis‐localization of the Mtw1 complex and causes pronounced chromosome segregation defects. We propose that auto‐inhibition of Mif2/CENP‐C constitutes a key concept underlying the molecular logic of kinetochore assembly.
Highlights
To properly distribute their genomes during cell division, eukaryotic cells have to assemble kinetochores from hundreds of individual proteins
The KMN network is recruited to the kinetochore through two axes: (i) recruitment of Mis12c/Mtw1c to CENP-C in human or Mif2 and Ame1 in S. cerevisiae (Przewloka et al, 2011; Screpanti et al, 2011; Hornung et al, 2014), which in turn leads to recruitment of Ndc80c, or (ii) direct recruitment of Ndc80c via CENP-T/Cnn1 in human and yeast cells (Gascoigne et al, 2011; Schleiffer et al, 2012; Nishino et al, 2013)
To distinguish between these possibilities, we tested a potential competition between Ame1 and Mif2 in their binding to Mtw1c in a solid-phase binding assay in which GST or GST-Mif2-N1-41 was incubated with the Mtw1-Nnf1 heterodimer (MN) in the presence of increasing concentrations of soluble Ame1-N peptide
Summary
To properly distribute their genomes during cell division, eukaryotic cells have to assemble kinetochores from hundreds of individual proteins. The CCAN in turn is responsible for recruitment of the microtubule binding outer kinetochore, including the KMN network (Cheeseman et al, 2006; DeLuca et al, 2006; Ciferri et al, 2008) (Fig 1). The KMN network is recruited to the kinetochore through two axes: (i) recruitment of Mis12c/Mtw1c to CENP-C in human or Mif and Ame in S. cerevisiae (Przewloka et al, 2011; Screpanti et al, 2011; Hornung et al, 2014), which in turn leads to recruitment of Ndc80c, or (ii) direct recruitment of Ndc80c via CENP-T/Cnn in human and yeast cells (Gascoigne et al, 2011; Schleiffer et al, 2012; Nishino et al, 2013). Two CCAN subunits, CENP-C and CENP-N, recognize structural features that distinguish CENP-A- from H3 nucleosomes and contribute to downstream kinetochore assembly at centromeres (Carroll et al, 2009, 2010; Kato et al, 2013; Xiao et al, 2017). Along the CENP-C polypeptide chain, an Mtw1c binding domain at the extreme N-terminus
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