Mitotic Cdc6 stabilizes anaphase-promoting complex substrates by a partially Cdc28-independent mechanism, and this stabilization is suppressed by deletion of Cdc55.
Ectopic expression of Cdc6p results in mitotic delay, and this has been attributed to Cdc6p-mediated inhibition of Cdc28 protein kinase and failure to activate the anaphase-promoting complex (APC). Here we show that endogenous Cdc6p delays a specific subset of mitotic events and that Cdc28 inhibition is not sufficient to account for it. The depletion of Cdc6p in G(2)/M cells reveals that Cdc6p is rate limiting for the degradation of the APC/Cdc20 substrates Pds1p and Clb2p. Conversely, the premature expression of Cdc6p delays the degradation of APC/Cdc20 substrates. Abolishing Cdc6p/Cdc28p interaction does not eliminate the Cdc6-dependent delay of these anaphase events. To identify additional Cdc6-mediated, APC-inhibitory mechanisms, we looked for mutants that reversed the mitotic delay. The deletion of SWE1, RAD24, MAD2, or BUB2 had no effect. However, disrupting CDC55, a PP2A regulatory subunit, suppressed the Cdc6p-dependent delay of Pds1 and Clb2 destruction. A specific role for CDC55 was supported by demonstrating that the lethality of Cdc6 ectopic expression in a cdc16-264 mutant is suppressed by the deletion of CDC55, that endogenous Cdc6p coimmunoprecipitates with the Cdc55 and Tpd3 subunits of PP2A, that Cdc6p/Cdc55p/Tpd3 interaction occurs only during mitosis, and that Cdc6 affects PP2A-Cdc55 activity during anaphase. This demonstrates that the levels and timing of accumulation of Cdc6p in mitosis are appropriate for mediating the modulation of APC/Cdc20.
Boronat
Susanna
S
Braun Laboratories 147-75, California Institute of Technology, Pasadena, CA 91125, USA.
Campbell
Judith L
JL
D064173:Anaphase-Promoting Complex-Cyclosome; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054648:Protein Phosphatase 2; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
C071246:CDC55 protein, S cerevisiae; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D017489:CDC28 Protein Kinase, S cerevisiae; D054648:Protein Phosphatase 2