REGULATING THE YEAST KINETOCHORE BY UBIQUITIN-DEPENDENT DEGRADATION AND SKP1P-MEDIATED PHOSPHORYLATION

In S. cerevisiae, the four-protein Cbf3 complex binds to the essential CDEIII region of centromeric DNA to initiate kinetochore assembly. We report the reconstitution of Cbf3p from recombinant proteins and an a nalysis of its p58(Ctf13) and p23(Skp1) subunits. p23(Skp1) has both G 1- and G2-specific functions in yeast and binds to p58(Ctf13) and to t he essential Cdc4p component of the ubiquitin conjugating complex Scul (Cdc4). We show that the function of p23(Skp1) in Cbf3p is to activate p58(Ctf13) by phosphorylation. p58(Ctf13) is an unstable protein that is targeted to the proteosome, probably by Scul(Cdc4)-mediated ubiqui tination. Thus, p58 appears to be activated by phosphorylation in a p2 3(Skp1)-dependent step and degraded by the proteosome in a ubiquitin-d ependent step. We propose that coupled activation and destruction link the assembly of Cbf3p to the duplication of centromeres in S phase.