Recruitment of Cdc28 by Whi3 restricts nuclear accumulation of the G1 cyclin-Cdk complex to late G1

The G1 cyclin Cln3 is a key activator of cell-cycle entry in budding yeast. Here we show that Whi3, a negative G1 regulator of Cln3, interacts in vivo with the cyclin-dependent kinase Cdc28 and regulates its localization in the cell. Efficient interaction with Cdc28 depends on an N-terminal domain of Whi3 that is also required for cytoplasmic localization of Cdc28, and for proper regulation of G1 length and filamentous growth. On the other hand, nuclear accumulation of Cdc28 requires the nuclear localization signal of Cln3, which is also found in Whi3 complexes. Both Cln3 and Cdc28 are mainly cytoplasmic during early G1, and become nuclear in late G1. However, Whi3-deficient cells show a distinct nuclear accumulation of Cln3 and Cdc28 already in early G1. We propose that Whi3 constitutes a cytoplasmic retention device for Cln3-Cdc28 complexes, thus defining a key G1 event in yeast cells.     

Cks1 is required for G(1) cyclin-cyclin-dependent kinase activity in budding yeast

p13(suc1) (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G(1) cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G(1) cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G(1) cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G(1) cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G(1) phase in budding yeast.     

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.     

Enhanced Cell Polarity in Mutants of the Budding Yeast Cyclin-dependent Kinase Cdc28p

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.     

Cyclin Cln3 is retained at the ER and released by the J chaperone Ydj1 in late G1 to trigger cell cycle entry

G1 cyclin Cln3 plays a key role in linking cell growth and proliferation in budding yeast. It is generally assumed that Cln3, which is present throughout G1, accumulates passively in the nucleus until a threshold is reached to trigger cell cycle entry. We show here that Cln3 is retained bound to the ER in early G1 cells. ER retention requires binding of Cln3 to the cyclin-dependent kinase Cdc28, a fraction of which also associates to the ER. Cln3 contains a chaperone-regulatory Ji domain that counteracts Ydj1, a J chaperone essential for ER release and nuclear accumulation of Cln3 in late G1. Finally, Ydj1 is limiting for release of Cln3 and timely entry into the cell cycle. As protein synthesis and ribosome assembly rates compromise chaperone availability, we hypothesize that Ydj1 transmits growth capacity information to the cell cycle for setting efficient size/ploidy ratios.     

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.     

Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes.

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase.     

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.