We have developed methods to reconstitute the centromere DNA (CEN)-bound Saccharomyces cerevisiae kinetochore complex, CBF3, from isolated CBF3 components in vitro. This revealed that cooperation of at least three CBF3 components is imperatively required to form an activity that specifically binds to the centromere DNA in vitro. Two of the CBF3 proteins, Cbf3a and Cbf3b, that were used in the reconstitution were obtained from heterologous systems. In contrast, Cbf3c, the third CBF3 component known, had to be purified from S. cerevisiae to obtain a Cbf3c preparation that was competent to reconstitute the CBF3-CEN complex in combination with Cbf3a and Cbf3b. This led to the identification of a 29 kDa protein that co-purified with Cbf3c. The 29 kDa protein was shown to be a fourth component of CBF3 and therefore was named Cbf3d. Analysing the Cbf3d gene revealed that Cbf3d exhibits strong homology to p19SKP1, a human protein that is part of active cyclin A-CDK2 complexes. Therefore, Cbf3d is the only CBF3 protein that has a known homologue in higher eukaryotes and may provide the anchor that directs cell cycle-regulated proteins to the kinetochore.
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