Abstract
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 analysis of its p58Ctf13 and p23Skp1 subunits. p23Skp1 has both G1- and G2-specific functions in yeast and binds to p58Ctf13 and to the essential Cdc4p component of the ubiquitin conjugating complex Scul(Cdc4). We show that the function of p23Skp1 in Cbf3p is to activate p58Ctf13 by phosphorylation. p58Ctf13 is an unstable protein that is targeted to the proteosome, probably by Scul(Cdc4)-mediated ubiquitination. Thus, p58 appears to be activated by phosphorylation in a p23Skp1-dependent step and degraded by the proteosome in a ubiquitin-dependent step. We propose that coupled activation and destruction link the assembly of Cbf3p to the duplication of centromeres in S phase.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Anaphase-Promoting Complex-Cyclosome
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Animals
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Cell Cycle Proteins / metabolism
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Cell Line
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DNA, Fungal / metabolism
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DNA-Binding Proteins / metabolism*
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F-Box Proteins*
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Fungal Proteins / metabolism*
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G1 Phase / physiology
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G2 Phase / physiology
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Insecta
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Kinetochores / metabolism*
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Ligases / genetics
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Ligases / physiology
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Nuclear Proteins / metabolism
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Phosphorylation
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Protein Binding
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins*
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Ubiquitin-Protein Ligase Complexes*
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Ubiquitin-Protein Ligases*
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Ubiquitins / metabolism*
Substances
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CDC4 protein, S cerevisiae
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CEP3 protein, S cerevisiae
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CTF13 protein, S cerevisiae
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Cell Cycle Proteins
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DNA, Fungal
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DNA-Binding Proteins
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F-Box Proteins
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Fungal Proteins
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Nuclear Proteins
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Ubiquitins
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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Ubiquitin-Protein Ligases
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Ligases