Genetic analysis of Cln/Cdc28 regulation of cell morphogenesis in budding yeast.

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity.     

Regulation of B-type cyclin proteolysis by Cdc28-associated kinases in budding yeast.

In budding yeast, stability of the mitotic B-type cyclin Clb2 is tightly cell cycle-regulated. B-type cyclin proteolysis is initiated during anaphase and persists throughout the G1 phase. Cln-Cdc28 kinase activity at START is required to repress B-type cyclin-specific proteolysis. Here, we show that Clb-dependent kinases, when expressed during G1, are also capable of repressing the B-type cyclin proteolysis machinery. Furthermore, we find that inactivation of Cln- and Clb-Cdc28 kinases is sufficient to trigger Clb2 proteolysis and sister-chromatid separation in G2/M phase-arrested cells, where the B-type cyclin-specific proteolysis machinery is normally inactive. Our results suggest that Cln- and Clb-dependent kinases are both capable of repressing B-type cyclin-specific proteolysis and that they are required to maintain the proteolysis machinery in an inactive state in S and G2/M phase-arrested cells. We propose that in yeast, as cells pass through START, Cln-Cdc28-dependent kinases inactivate B-type cyclin proteolysis. As Cln-Cdc28-dependent kinases decline during G2, Clb-Cdc28-dependent kinases take over this role, ensuring that B-type cyclin proteolysis is not activated during S phase and early mitosis.     

Coherence and timing of cell cycle start examined at single-cell resolution

Cell cycle "Start" in budding yeast involves induction of a large battery of G1/S-regulated genes, coordinated with bud morphogenesis. It is unknown how intra-Start coherence of these events and inter-Start timing regularity are achieved. We developed quantitative time-lapse fluorescence microscopy on a multicell-cycle timescale, for following expression of unstable GFP under control of the G1 cyclin CLN2 promoter. Swi4, a major activator of the G1/S regulon, was required for a robustly coherent Start, as swi4 cells exhibited highly variable loss of cooccurrence of regular levels of CLN2pr-GFP expression with budding. In contrast, other known Start regulators Mbp1 and Cln3 are not needed for coherence but ensure regular timing of Start onset. The interval of nuclear retention of Whi5, a Swi4 repressor, largely accounts for wild-type mother-daughter asymmetry and for variable Start timing in cln3 mbp1 cells. Thus, multiple pathways may independently suppress qualitatively different kinds of noise at Start.     

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.