Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.     

A new pair of B-type cyclins from Saccharomyces cerevisiae that function early in the cell cycle.

Two new B-type cyclin genes from Saccharomyces cerevisiae, called CLB5 and CLB6, are located in a tail to tail arrangement adjacent to the G2/M phase promoting cyclins CLB2 and CLB1, respectively. These genomic cyclin arrays are flanked by tRNAs and repeated sequences of Ty elements suggesting an intrachromosomal gene duplication followed by an interchromosomal gene duplication. Based on their deduced protein sequence the CLB5 and CLB6 genes form a new pair of B-type cyclins. They are most related to each other and then to the deduced protein sequence of their adjacent genes CLB1 and CLB2. Both genes are periodically expressed, peaking early in the cell cycle. Loss of function mutants are viable, but clb5- mutants exhibit a delay in S phase whereas clb6- mutants show a delay in late G1 and/or S phase. The clb5 mutant phenotype is somewhat more pronounced in a double null mutant. Both cyclins have the potential to interact with the p34CDC28 kinase in vivo.     

Functions of the mitotic B-type cyclins CLB1, CLB2, and CLB3 at mitotic exit antagonized by the CDC14 phosphatase

In the budding yeast Saccharomyces cerevisiae, cell cycle progression and cytokinesis at mitotic exit are proposed to be linked by CDC14 phosphatase antagonizing the function of mitotic B-type cyclin (CLBs). We have isolated a temperature-sensitive mutant, cdc14(A280V), with a mutation in the conserved phosphatase domain. Prolonged arrest in the cdc14(A280V) mutant partially uncoupled cell cycle progression from the completion of cytokinesis as measured by bud re-emergence, in the form of elongated apical projections, and DNA re-replication. In contrast to previous mitotic exit mutants, cdc14(A280V) mutants displayed a strong bias for the first apical projection to form in the mother cell body. Using cdc14(A280V) mutant phenotypes, the functions of the B-type cyclins at mitotic exit were investigated. The preference in mother-daughter apical projection formation was observed to be independent of any individual CLB function. However, cdc14(A280V)clb1Δ cells displayed a pronounced increase in apical projections, while cdc14(A280V)clb3Δ cells were observed to form round cellular chains. While cdc14(A280V) cells arrested at mitotic exit, both cdc14(A280V)clb1Δ and cdc14(A280V)clb3Δ cells completed cytokinesis, but failed cell separation. cdc14(A280V)clb2Δ cells displayed a defect in actin ring assembly. These observations differentiate the functions of CLB1, CLB2, and CLB3 at mitotic exit, and are consistent with the hypothesis that CLB activities are antagonized by the CDC14 phosphatase in order to couple cell cycle progression with cytokinesis at mitotic exit.     

Specialization of B-Type Cyclins for Mitosis or Meiosis in S. Cerevisiae

The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be much more important for meiosis than for mitosis and may play some special role in meiosis II. In contrast, CLB2 is important for mitosis but not meiosis. The level of Cdc28-Clb activity may be important in determining whether meiosis II will occur.     

Specialization and targeting of B-type cyclins.

The B-type cyclins of S. cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function. We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus. CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p. Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity. Thus, Clb5p may have targeting domains required for CLB5-specific biological activity.     

Distinct mechanisms control the stability of the related S-phase cyclins Clb5 and Clb6

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 clb5Delta mutants but not in clb6Delta 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 clb5Delta 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.     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999