Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes

Abstract

Kinetochores are multiprotein complexes that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Over the past six years, the number of proteins known to localize to the Saccharomyces cerevisiae kinetochore has increased from around 10 to over 60. However, relatively little is known about the protein-protein interactions that mediate kinetochore assembly or about the overall structure of microtubule-attachment sites. Here we used biophysical techniques, affinity purification, mass spectrometry, and in vivo assays to examine the state of association of 31 centromere-binding proteins, including six proteins newly identified as kinetochore subunits. We found that yeast kinetochores resemble transcriptional enhancers in being composed of at least 17 discrete subcomplexes that assemble on DNA to form a very large structure with a mass in excess of 5 MD. Critical to kinetochore assembly are proteins that bridge subunits in direct contact with DNA and subunits bound to microtubules. We show that two newly identified kinetochore complexes, COMA (Ctf19p-Okp1p-Mcm21p-Ame1p) and MIND (Mtw1p including Nnf1p-Nsl1p-Dsn1p) function as bridges. COMA, MIND, and the previously described Ndc80 complex constitute three independent and essential platforms onto which outer kinetochore proteins assemble. In addition, we propose that the three complexes have different functions with respect to force generation and MT attachment.[Keywords: Kinetochore; chromosome segregation; mitosis; centromere; proteomics; hydrodynamics] Supplemental material is available at http://www.genesdev.org.

Reconstruction of the Kinetochore during Meiosis in Fission Yeast Schizosaccharomyces pombe

“…NMS group proteins reassemble to the kinetochore during prophase and toward metaphase in meiosis, and subsequently DASH group proteins localize at the centromere during chromosome segregation. These groupings are generally consistent with the complex structures revealed by genetic interactions and proteomic analyses (De Wulf et al, 2003;Cheeseman et al, 2004;Obuse et al, 2004;Liu et al, 2005).…”

“…Biochemical analyses have revealed that the kinetochore complex is comprised of subcomplexes of proteins. Many of these proteins are conserved in other eukaryotes, from yeasts to humans (De Wulf et al, 2003;Nekrasov et al, 2003;Cheeseman et al, 2004;Obuse et al, 2004;reviewed in Meraldi et al, 2006).…”

“…In spite of the variation in centromere DNA sequences, kinetochore proteins are well conserved among organisms. The S. cerevisiae kinetochore complex is composed of four subcomplexes: MIND, NDC80, COMA, and Ctf19 (De Wulf et al, 2003). In addition, the DASH complex is localized at the kinetochore and the spindle and is required for spindle attachment to the kinetochore (Cheeseman et al, 2001;Li et al, 2002).…”

Sgt1p and Skp1p Modulate the Assembly and Turnover of CBF3 Complexes Required for Proper Kinetochore Function

“…The protocol used for chromatin immunoprecipitation analysis was performed as described previously (De Wulf et al, 2003). Cells containing temperature-sensitive alleles were grown to log phase at 25°C, shifted to 37°C for 5 h, and fixed in 1% formaldehyde for 15 min.…”

The evolutionary conserved BER1 gene is involved in microtubule stability in yeast

“…In order to get further insights into the putative involvement of Ber1 in kinetochore function, the ber1 mutant was crossed with temperature-sensitive mutants of the following essential components of the kinetochore: the aurora kinase Ipl1 (Chan and Botstein 1993), which regulates microtubule-kinetochore attachment (Buvelot et al 2003); Nnf1, a component of the central MIND kinetochore complex that contributes to linking the inner and outer kinetochore layers (De Wulf et al 2003); Ndc10 (Goh and Kilmartin 1993), which is a key component of the inner kinetochore that is necessary for kinetochore assembly (Espelin et al 2003); and Cse4 (Stoler et al 1995), a centromere-speciWc histone variant involved in kinetochore assembly (Meluh et al 1998). Altogether, these proteins are quite representative of the diVerent components of the kinetochore.…”