Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro.
Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
P K
PK
Sorger
F F
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Severin
A A
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Hyman
D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002503:Centromere / Q000378:metabolism; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D018386:Kinetochores / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; C092348:CEP3 protein, S cerevisiae; D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins