CBF3 complex

Abstract: 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: p23Skp1 and p58Ctf13 form a heterodimer, and p64Cep3 and p110Ndc10 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 p23Skp1-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.

Abstract: During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.

Abstract: Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G2/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.

Abstract: The budding yeast PLC1 gene encodes a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p associates with the kinetochore complex CBF3. This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo. Deletion of PLC1, as well as plc1 mutations which abrogate the interaction of Plc1p with the CBF3 complex, results in a higher frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay. Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1 mutants, implying that the association of Plc1p with CBF3 is important for optimal kinetochore function. Chromatin extracts from plc1Δ cells exhibit reduced microtubule binding to minichromosomes. These results suggest that Plc1p associates with kinetochores and regulates some aspect of kinetochore function and demonstrate an intranuclear function of phospholipase C in eukaryotic cells.