Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1

The centromere-kinetochore complex of Saccharomyces cerevisiae is a special ized chromosomal substructure that mediates attachment of duplicated chromo somes to the mitotic spindle by a regulated network of protein-DNA and prot ein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromere-kinetocho re complex. Glutathione S-transferase pull-down experiments showed the dire ct interaction of in vitro translated p110, p64, and p58 of the essential C BF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the i n vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multime rization activity of Cbf1p in the presence of centromere DNA in vitro. Thes e results show the direct physical interaction of Cbf1p and CBF3 subunits a nd provide evidence that CBF3 components can promote the binding of Cbf1p t o its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 pr omoters reveals the strong analogy between centromeres and the MET16 promot er.