Roles for the conserved spc105p/kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint.
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.


RESULTS
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
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.
Pagliuca
Cinzia
C
Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.
Draviam
Viji M
VM
Marco
Eugenio
E
Sorger
Peter K
PK
De Wulf
Peter
P
D000595:Amino Acid Sequence; D000705:Anaphase; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D010641:Phenotype; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus
C081071:CBF2 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C546125:Kre28 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; C546124:Spc105 protein, S cerevisiae