Distinct Mechanisms Control the Stability of the Related S-Phase Cyclins Clb5 and Clb6

Abstract

The yeast S-phase cyclins Clb5 and Clb6 are closely related proteins that are synthesized late in G₁. Although often grouped together with respect to function, Clb5 and Clb6 exhibit differences in their ability to promote S-phase progression. DNA replication is significantly slowed in clb5Δ mutants but not in clb6Δ mutants. We have examined the basis for the differential functions of Clb5 and Clb6 and determined that unlike Clb5, which is stable until mitosis, Clb6 is degraded rapidly at the G₁/S border. N-terminal deletions of CLB6 were hyperstabilized, suggesting that the sequences responsible for directing the destruction of Clb6 reside in the N terminus. Clb6 lacks the destruction box motif responsible for the anaphase promoting complex-mediated destruction of Clb5 but contains putative Cdc4 degron motifs in the N terminus. Clb6 was hyperstabilized in cdc34-3 and cdc4-3 mutants at restrictive temperatures and when S/T-P phosphorylation sites in the N terminus were mutated to nonphosphorylatable residues. Efficient degradation of Clb6 requires the activities of both Cdc28 and Pho85. Finally, hyperstabilized Clb6 expressed from the CLB6 promoter rescued the slow S-phase defect exhibited by clb5Δ cells. Taken together, these findings suggest that the SCF^Cdc4 ubiquitin ligase complex regulates Clb6 turnover and that the functional differences exhibited by Clb5 and Clb6 arise from the distinct mechanisms controlling their stability.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Danny Lew, Peter Kaiser, and Dave Stuart for yeast strains and plasmids, Kevan Shokat for a generous supply of 1-NM-PP1, and David Orlando and Todd Wasson for computational assistance. We also thank Duncan Clarke, Peter Kaiser, Danny Lew, and members of the Haase lab for helpful discussions and Danny Lew and Mark Chee for critical reading of the manuscript.