👤 Raymond K Cross

The Cln3 cyclin homolog of Saccharomyces cerevisiae functions to promote cell cycle START for only a short time following its synthesis. Cln3 protein is highly unstable and is stabilized by C-terminal truncation. Cln3 binds to Cdc28, a protein kinase catalytic subunit essential for cell cycle START, and Cln3 instability requires Cdc28 activity. The long functional lifetime and the hyperactivity of C-terminally truncated Cln3 (Cln3-2) relative to those of full-length Cln3 are affected by mutations in CDC28: the functional lifetime of Cln3-2 is drastically reduced by the cdc28-13 mutation at the permissive temperature, and the cdc28-4 mutation at the permissive temperature completely blocks the function of Cln3-2 while only partially reducing the function of full-length Cln3. Thus, sequences in the C-terminal third of Cln3 might help stabilize functional Cdc28-Cln3 association, as well as decreasing the lifetime of the Cln3 protein. These and other results strongly support the idea that Cln proteins function to activate Cdc28 at START. Show less

In budding yeast, a switch between the mutually exclusive pathways of cell cycle progression and conjugation is controlled at Start in late G1 phase. Mating pheromones promote conjugation by arresting cells in G1 phase before Start. Pheromone-induced cell cycle arrest requires a functional FAR1 gene. We have found that FAR1 transcription and protein accumulation are regulated independently during the cell cycle. FAR1 RNA and protein are highly expressed in early G1, but decline sharply at Start. Far1 is phosphorylated just before it disappears at Start, suggesting that modification may target Far1 for degradation. Although FAR1 mRNA levels rise again during late S or G2 phase, reaccumulation of Far1 protein to functional levels is restricted until after nuclear division. Show less

Budding yeast strains have three CLN genes, which have limited cyclin homology. At least one of the three is required for cell cycle START. Four B cyclins are known in yeast; two have been shown to function in mitosis. We have discovered a fifth B-cyclin gene, called CLB5, which when cloned on a CEN plasmid can rescue strains deleted for all three CLN genes. CLB5 transcript abundance peaks in G1, coincident with the CLN2 transcript but earlier than the CLB2 transcript. CLB5 deletion does not cause lethality, either alone or in combination with other CLN or CLB deletions. However, strains deleted for CLB5 require more time to complete S phase, suggesting that CLB5 promotes some step in DNA synthesis. CLB5 is the only yeast cyclin whose deletion lengthens S phase. CLB5 may also have some role in promoting the G1/S transition, because cln1 cln2 strains require both CLN3 and CLB5 for viability on glycerol media and cln1,2,3- strains require CLB5 for rescue by the Drosophila melanogaster cdc2 gene. In conjunction with cln1,2,3- rescue by CLB5 overexpression and the coincident transcriptional regulation of CLB5 and CLN2, these observations are suggestive of partial functional redundancy between CLB5 and CLN genes. Show less

The cell cycle in Saccharomyces cerevisiae is controlled by regulation of START in late G1. The CLN1, CLN2 and CLN3 family of cyclin homologues is required for cells to pass START. They probably act by activating the CDC28 protein kinase. Expression of CLN1 or CLN3 under the control of an inducible promoter shows that transcription of either gene is sufficient for cyclin-deficient strains arrested in G1 to traverse START. A model of START regulation involves activation of CDC28 kinase by any CLN protein, leading to activation of CLN1 and CLN2 transcription in a positive feedback loop and passage through START. The cell cycle-dependent transcriptional regulators SWI4 and SWI6 may be components of the feedback loop. Cell cycle commitment entails resistance to the inhibitory action of mating factor, which correlates with peak levels of CLN1 and CLN2 mRNAs. FAR1 encodes an alpha-factor-dependent inhibitor of CLN function whose expression is markedly reduced at the time of START. The interplay of all these factors may sharpen the START transition such that it is close to an all-or-nothing switch event. This may be important for several START-dependent events to be activated at the same time, leading to coordinated cell cycle progression. Show less

The CLN1, CLN2, and CLN3 genes of S. cerevisiae form a redundant family essential for the G1-to-S phase transition. CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not. The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene. An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene. mRNA from a nonfunctional cln1 gene was regulated similarly. Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression. Show less

Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest). In cln1 cln2 cln3 strains bearing plasmids containing the CLN3 (also called WHI1 or DAF1) coding sequence under the transcriptional control of a galactose-regulated promoter, shift from galactose to glucose medium (shutting off synthesis of CLN3 mRNA) allowed completion of cell cycles in progress but caused arrest in the ensuing unbudded G1 phase. Cell growth was not inhibited in arrested cells. Cell division occurred in glucose medium even if cells were arrested in S phase during the initial 2 h of glucose treatment, suggesting that CLN function may not be required in the cell cycle after S phase. However, when the coding sequence of the hyperactive C-terminal truncation allele CLN3-2 (formerly DAF1-1) was placed under GAL control, cells went through multiple cycles before arresting after a shift from galactose to glucose. These results suggest that the C terminus of the wild-type protein confers functional instability. cln-arrested cells are mating competent. However, cln arrest is distinct from constitutive activation of the mating-factor signalling pathway because cln-arrested cells were dependent on the addition of pheromone both for mating and for induction of an alpha-factor-induced transcript, FUS1, and because MATa/MAT alpha (pheromone-nonresponsive) strains were capable of cln arrest in G1 (although a residual capacity for cell division before arrest was observed in MATa/MAT alpha strains). These results are consistent with a specific CLN requirement for START transit. Show less

Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3. Show less