Curt Wittenberg

An essential G1 function for cyclin-like proteins in yeast

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.

G1-specific cyclins of S. cerevisiae: Cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase

The S. cerevisiae CLN genes encode cyclin homologs essential for progression from G1 to S phase. The CLN2 gene encodes a 62 kd polypeptide that accumulates periodically, peaking during G1 and decreasing rapidly thereafter, and is rapidly lost following exposure of cells to mating pheromone. Cln2 abundance can be explained by the G1-specific accumulation of the CLN2 transcript coupled with instability of the Cln2 protein. The abundance of the CLN1 and CLN2 transcripts increases greater than 5-fold during the G1 interval, decreasing dramatically as cells enter S phase. Both transcripts decrease in cells responding to mating pheromone. Finally, we demonstrate that the Cln2 polypeptide interacts with p34CDC28 to form an active protein kinase complex. This physical interaction is consistent with the genetic interaction between the CLN genes and CDC28 and suggests that Cln proteins are an essential component of the active protein kinase complex required for the G1 to S transition.

Cdc53 Targets Phosphorylated G1 Cyclins for Degradation by the Ubiquitin Proteolytic Pathway

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.

Regulation of Transcription by Ubiquitination without Proteolysis: Cdc34/SCFMet30-Mediated Inactivation of the Transcription Factor Met4

Polyubiquitination of proteins by Cdc34/SCF complexes targets them for degradation by the 26S proteasome. The essential F-box protein Met30 is the substrate recognition subunit of the ubiquitin ligase SCF(Met30). The critical target of SCF(Met30) is the transcription factor Met4, as deletion of MET4 suppresses the lethality of met30 mutants. Surprisingly, Met4 is a relatively stable protein and its abundance is not influenced by Met30. However, transcriptional repression of Met4 target genes correlates with Cdc34/SCF(Met30)-dependent ubiquitination of Met4. Functionally, ubiquitinated Met4 associates with target promoters but fails to form functional transcription complexes. Our data reveal a novel proteolysis-independent function for Cdc34/SCF and indicate that ubiquitination of transcription factors can be utilized to directly regulate their activities.

Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5.

G1-specific transcriptional activation by Cln3/CDK initiates the budding yeast cell cycle. To identify targets of Cln3/CDK, we analyzed the SBF and MBF transcription factor complexes by multidimensional protein interaction technology (MudPIT). Whi5 was identified as a stably bound component of SBF but not MBF. Inactivation of Whi5 leads to premature expression of G1-specific genes and budding, whereas overexpression retards those processes. Whi5 inactivation bypasses the requirement for Cln3 both for transcriptional activation and cell cycle initiation. Whi5 associates with G1-specific promoters via SBF during early G1 phase, then dissociates coincident with transcriptional activation. Dissociation of Whi5 is promoted by Cln3 in vivo. Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes. Mutation of putative CDK phosphorylation sites, at least five of which are phosphorylated in vivo, strongly reduces SBF-dependent transcription and delays cell cycle initiation. Like mammalian Rb, Whi5 is a G1-specific transcriptional repressor antagonized by CDK.

DNA Polymerase ε Catalytic Domains Are Dispensable for DNA Replication, DNA Repair, and Cell Viability

DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.

Rapid Degradation of the G1 Cyclin Cln2 Induced by CDK-Dependent Phosphorylation

Cyclins regulate the major cell cycle transitions in eukaryotes through association with cyclin-dependent protein kinases (CDKs). In yeast, G1 cyclins are essential, rate-limiting activators of cell cycle initiation. G1-specific accumulation of one G1 cyclin, Cln2, results from periodic gene expression coupled with rapid protein turnover. Site-directed mutagenesis of CLN2 revealed that its phosphorylation provides a signal that promotes rapid degradation. Cln2 phosphorylation is dependent on the Cdc28 protein kinase, the CDK that it activates. These findings suggest that Cln2 is rendered self-limiting by virtue of its ability to activate its cognate CDK subunit.

A cyclin B homolog in S. cerevisiae: Chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.

Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes

In the budding yeast, Saccharomyces cerevisiae, a significant fraction of genes (>10%) are transcribed with cell cycle periodicity. These genes encode critical cell cycle regulators as well as proteins with no direct connection to cell cycle functions. Cell cycle-regulated genes can be organized into ‘clusters’ exhibiting similar patterns of regulation. In most cases periodic transcription is achieved via both repressive and activating mechanisms. Fine-tuning appears to have evolved by the juxtaposition of regulatory motifs characteristic of more than one cluster within the same promoter. Recent reports have provided significant new insight into the role of the cyclin-dependent kinase Cdk1 (Cdc28) in coordination of transcription with cell cycle events. In early G1, the transcription factor complex known as SBF is maintained in a repressed state by association of the Whi5 protein. Phosphorylation of Whi5 by Cdk1 in late G1 leads to dissociation from SBF and transcriptional derepression. G2/M-specific transcription is achieved by converting the repressor Fkh2 into an activator. Fkh2 serves as a repressor during most of the cell cycle. However, phosphorylation of a cofactor, Ndd1, by Cdk1 late in the cell cycle promotes binding to Fkh2 and conversion into a transcriptional activator. Such insights derived from analysis of specific genes when combined with genome-wide analysis provide a more detailed and integrated view of cell cycle-dependent transcription.