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KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans.

ABSTRACT: Segregation of the replicated genome during cell division requires kinetochores, mechanochemical organelles that assemble on mitotic chromosomes to connect them to spindle microtubules. CENP-A, a histone H3 variant, and CENP-C, a conserved structural protein, form the DNA-proximal foundation for kinetochore assembly. Using RNA interference-based genomics in Caenorhabditis elegans, we identified KNL-1, a novel kinetochore protein whose depletion, like that of CeCENP-A or CeCENP-C, leads to a "kinetochore-null" phenotype. KNL-1 is downstream of CeCENP-A and CeCENP-C in a linear assembly hierarchy. In embryonic extracts, KNL-1 exhibits substoichiometric interactions with CeCENP-C and forms a near-stoichiometric complex with CeNDC-80 and HIM-10, the C. elegans homologs of Ndc80p/HEC1p and Nuf2p-two widely conserved outer kinetochore components. However, CeNDC-80 and HIM-10 are not functionally equivalent to KNL-1 because their inhibition, although preventing formation of a mechanically stable kinetochore-microtubule interface and causing chromosome missegregation, does not result in a kinetochore-null phenotype. The greater functional importance of KNL-1 may be due to its requirement for targeting multiple components of the outer kinetochore, including CeNDC-80 and HIM-10. Thus, KNL-1 plays a central role in translating the initiation of kinetochore assembly by CeCENP-A and CeCENP-C into the formation of a functional microtubule-binding interface.

SUBMITTER: Desai A

PROVIDER: S-EPMC218079 | biostudies-other | 2003 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain approximately 70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores. PRINCIPAL FINDINGS:We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells. CONCLUSIONS/SIGNIFICANCE:Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.

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KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans.

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