News and Publications

Control of Initiation of the Cell Cycle in Budding Yeast

C. Wittenberg, K. Flick, M. Guaderrama, Y. Hsiung, T. Kesti, S. Lanker, D. Stuart

In budding yeasts, as in most eukaryotic cells, proliferation is regulated primarily during the G₁ phase of the cell cycle. Progression through the cycle is governed by the cyclin-dependent protein kinases (CDKs). CDKs are heterodimeric enzymes that consist of a catalytic subunit and a positive regulatory subunit called a cyclin. They drive the major transitions in the cell cycle and are the targets of the negative regulatory signals that mediate checkpoints in the cycle.

Budding yeasts have a single CDK catalytic subunit, encoded by the gene CDC28, that collaborates with no fewer than 9 different cyclins to regulate events in the cell cycle. The cyclins can be categorized into 2 major classes, G₁ and B type, and these classes can be divided into subclasses on the basis of both primary sequence and pattern of expression. Each class appears to be optimized for a specific function in the cell cycle. This specificity is derived from the differences between the cyclins themselves and from the differences between the CDK complexes in which the cyclins participate. First, the patterns of cyclin accumulation are differentially regulated as a consequence of the combined effects of cell cycle--dependent transcription and regulated proteolytic degradation. Next, specific cyclin-CDK complexes differ in their substrate specificity. Finally, complexes composed of CDKs and G₁ or B-type cyclins can be inhibited by distinct CDK inhibitors.

We have focused our attention on regulation of initiation of the cell cycle during G₁ in the budding yeast Saccharomyces cerevisiae. Initiation involves a complex choreography of events driven by the sequential activation of CDKs that is just beginning to be fully elucidated. The pattern of CDK activity is a consequence of the interplay of cell cycle--dependent transcription and regulated proteolysis.

Cln3, which accumulates during the early part of G₁, activates transcription of the genes CLN1, CLN2, CLB5, and CLB6. The Cln1 and Cln2 proteins then accumulate, activating the Cdc28 CDK for several critical functions. First, these cyclin-dependent complexes promote formation of a bud, the presumptive daughter cell, and duplication of spindle poles. Next, they inactivate the machinery for B-type cyclin proteolysis. Finally, they drive degradation of several key proteins via phosphorylation-dependent ubiquitination. These key proteins include Sic1, a specific inhibitor of CDK activity associated with B-type cyclins, and the G₁ cyclins themselves. These events lead to activation of the Clb5 and Clb6 forms of CDK, which promote entry into S phase and set the stage for the accumulation of subsequent waves of CDK activity that drive cells into mitosis. Efforts to understand the mechanisms that govern G₁-specific transcriptional activation and phosphorylation-dependent ubiquitination form the core of our current research.

In addition to their role during mitotic growth, we are interested in the regulation of cyclin-CDK function in response to environmental signals and during cell differentiation. Cells growing mitotically monitor the availability and quality of nutrients and modulate the rate of progression of the cell cycle and cell size accordingly. We have shown that this modulation is due, in part, to alterations in the expression of the cyclin genes. If diploid cells are limited for nutrients, they can undergo meiosis and sporulation, an interesting and important modification of the mitotic cell cycle. We have shown that G₁ cyclins are not required for meiosis and sporulation but that the B-type cyclins Clb5 and Clb6 are absolutely essential. CLB5 and CLB6 are required both for premeiotic DNA replication and for activation of the checkpoint that restricts meiotic M phase in the absence of fully replicated chromosomes. Thus, cyclin-CDK complexes are critical not only for driving progression of the cell cycle but also for maintaining organization of the cycle. We plan to use biochemical, genetic, and cell biological approaches to better understand these processes and analogous processes in other systems.

PUBLICATIONS

Flick, K., Chapman-Shimshoni, D., Stuart, D., Guaderrama, M., Wittenberg, C. Regulation of cell size by glucose is exerted via repression of the CLN1 promoter. Mol. Cell. Biol. 18:2492, 1998.

Schneider, B.L., Patton, E., Lanker, S., Mendenhall, M., Wittenberg, C., Futcher, B., Tyers, M. Yeast G₁ cyclins are unstable in G₁ phase. Nature, in press.

Stuart, D., Wittenberg, C. S phase cyclins are essential for premeiotic DNA replication and activation of a meiotic S/M checkpoint. Genes Dev., in press.