The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex.
Kinetochores are smaller and more accessible experimentally in budding yeast than in any other eukaryote. Believing that simple and complex kinetochores have important structural and functional properties in common, we characterized the structure of CBF3, the essential centromere-binding complex that initiates kinetochore formation in Saccharomyces cerevisiae. We find that the four subunits of CBF3 are multimeric in solution: p23(Skp1) and p58(Ctf13) form a heterodimer, and p64(Cep3) and p110(Ndc10) form homodimers. Subcomplexes involving p58 and each of the other CBF3 subunits can assemble in the absence of centromeric DNA. In these subcomplexes, p58 appears to function as a structural core mediating stable interactions among other CBF3 proteins. p58 has a short half-life in yeast, being subject to ubiquitin-dependent proteolysis, but we find that it is much more stable following association with p64. We propose that p23(Skp1)-p58-p64 complexes constitute the primary pool of active p58 in yeast cells. These complexes can either dissociate, reexposing p58 to the degradation pathway, or can bind to p110 and centromeric DNA, forming a functional CBF3 complex in which p58 is fully protected from degradation. This pathway may constitute an editing mechanism preventing the formation of ectopic kinetochores and ensuring the fidelity of chromosome segregation.
Russell
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ID
Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts 02139, USA.
Grancell
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AS
Sorger
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PK
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
C092348:CEP3 protein, S cerevisiae; C081354:CTF13 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins