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Phosphorylation Controls Timing of Cdc6p Destruction: A Biochemical Analysis

Elsasser, Suzanne and Chi, Yong and Yang, Ping and Campbell, Judith L. (1999) Phosphorylation Controls Timing of Cdc6p Destruction: A Biochemical Analysis. Molecular Biology of the Cell, 10 (10). pp. 3263-3277. ISSN 1059-1524. http://resolver.caltech.edu/CaltechAUTHORS:20111206-092106846

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Abstract

The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell cycle. Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed. First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase. However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p. SCF-Cdc4p– and N-terminal phosphorylation site–dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle. The stable cdc6 mutants show no apparent replication defects in wild-type strains. However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex.


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Additional Information:© 1999 by The American Society for Cell Biology. Submitted March 5, 1999; Accepted August 2, 1999. Monitoring Editor: Mark J. Solomon. We thank Ray Deshaies and members of his laboratory for strains, discussions, and critical reading of the manuscript. The experiment in Figure 5 was carried out in the Deshaies laboratory.
Record Number:CaltechAUTHORS:20111206-092106846
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20111206-092106846
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:28310
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:11 Jan 2012 23:51
Last Modified:26 Dec 2012 14:34

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