Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1.
The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 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 in 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 multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.
Hemmerich
P
P
Institut fuer Molekulare Biotechnologie, Abteilung Molekularbiologie, Beutenbergstrasse 11, 07745 Jena, Germany. phemmer@imb-jena.de
Stoyan
T
T
Wieland
G
G
Koch
M
M
Lechner
J
J
Diekmann
S
S
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002503:Centromere / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D018386:Kinetochores / Q000378:metabolism*; D016350:Leucine Zippers; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; C081071:CBF2 protein, S cerevisiae; C092348:CEP3 protein, S cerevisiae; C081354:CTF13 protein, S cerevisiae; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D044843:SKP Cullin F-Box Protein Ligases; C120507:SKP1 protein, S cerevisiae