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.     

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.     

Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation.

Cyclin-dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras-associated GTPase-activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation.     

The F-box protein Grr1 regulates the stability of Ccn1, Cln3 and Hof1 and cell morphogenesis in Candida albicans

Both G1 and mitotic cyclins have been implicated in regulating Candida albicans filamentous growth. We have investigated the functions of Grr1 whose orthologue in Saccharomyces cerevisiae is known to mediate ubiquitin-dependent degradation of the G1 cyclins Cln1 and Cln2. Here, we report that deleting C. albicans GRR1 causes significant stabilization of two G1 cyclins Ccn1 and Cln3 and pseudohyphal growth. grr1Delta cells are highly heterogeneous in length and many of them fail to separate after cytokinesis. Interestingly, some isolated rod-like G1 cells of similar sizes are present in the grr1Delta culture. Time-lapse microscopy revealed that the rod-shaped G1 cells first grew exclusively in width before budding and then the bud grew exclusively by apical extension until after cytokinesis, yielding rod-like daughter cells. Consistently, actin patches persistently localize to the bud tip until around the time of cytokinesis. Despite the pseudohyphal phenotype, grr1Delta cells respond normally to hyphal induction. Hyperphosphorylated Cln3 isoforms accumulate in grr1Delta cells, indicating that Grr1 selectively mediates their degradation in wild-type cells. grr1Delta pseudohyphal growth requires neither Hgc1 nor Swel, two important regulators of cell morphogenesis. Furthermore, the cellular level of Hof1, a protein having a role in cytokinesis, is also significantly increased in grr1Delta cells.     

C1n3缺失对酿酒酵母生长的影响

Cln3是酿酒酵母G1期周期蛋白中的一种,为了研究Cln3在细胞周期与形态发生中的作用,我们构建了酿酒酵母CLN3基因的缺失株,并对其表型进行了分析。结果显示,cln3缺失株对α信息素的敏感性增强,α信息素诱导的细胞周期停滞现象明显大于野生型菌株,这种增强作用不受Sgvl因子的影响。同时,与野生菌相比cln3缺失株的细胞形态也有明显变化,双倍体cln3缺失株细胞的顶端生长能力增强而单倍体细胞的侵入生长能力则受到抑制。结果表明,与酿酒酵母的另外两个G1期周期蛋白Clnl、Cln2不同,Cln3在形态发生中有其独特的功能与作用方式。     

The yeast kinase Swe1 is required for proper entry into cell cycle after arrest due to ribosome biogenesis and protein synthesis defects

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6Delta), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6Delta cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.     

The Yeast Kinase Swe1 is Required for Proper Entry into Cell Cycle After Arrest Due to Ribosome Biogenesis and Protein Synthesis Defects

Sda1 is an essential protein required for cell cycle progression in Saccharomyces cerevisiae. Here, we show that the sda1-1 mutation causes a defect in the formation and nuclear export of 60S ribosomal subunits. Moreover, the sda1-1, but also other mutants defective in ribosome biogenesis (e.g., rix1-1 and tif6D), exhibit a G1 arrest, which could be the consequence of impaired ribosome biogenesis. Interestingly, additional deletion of the non-essential Swe1 kinase, the homolog of S. pombe Wee1, causes a pronounced delay in entering a new cell cycle in sda1-1, rix1-1 and tif6D cells, when shifted back from restrictive to permissive conditions. However, such a prolonged delay is independent of the Tyr19 phosphorylation in Cdc28. Moreover, the lack of Swe1 causes delay in budding and DNA replication in cells released from the G1 arrest due to the block of protein synthesis. Our data suggest that Swe1 is required for timely entry into cell cycle after a G1 arrest caused by impairment in pre-60S biogenesis and in protein synthesis. Therefore we propose that Swe1, which is required for coordination of cell growth and cell division in G2/M, also has a role in the beginning of the cell cycle.     

Counteractive control of polarized morphogenesis during mating by mitogen-activated protein kinase Fus3 and G1 cyclin-dependent kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis.     

G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover.

The 545-residue Cln2 protein, like the other G1 cyclins of Saccharomyces cerevisiae, is a very unstable protein. This instability is thought to play a critical role in regulating cell cycle progression. The carboxyl-terminal domains of Cln2 and the other G1 cyclins contain sequences rich in Pro, Glu (and Asp), Ser, and Thr (so-called PEST motifs) that have been postulated to make up the signals that are responsible for the rapid degradation of these and other unstable proteins. To test this hypothesis, the carboxyl-terminal 178 residues of Cln2 were fused to the C terminus of a reporter enzyme, a truncated form of human thymidine kinase (hTK delta 40). The resulting chimeric protein (hTK delta 40-Cln2) retained thymidine kinase activity but was markedly less stable than hTK, hTK delta 40, or an hTK-beta-galactosidase fusion protein, as judged by enzyme assay, immunoblotting with anti-hTK antibodies, pulse-chase analysis of the radiolabeled polypeptides, and ability to support the growth of a thymidylate auxotroph (cdc21 mutant) on thymidine-containing medium. Thus, the presence of the Cln2 PEST domain was sufficient to destabilize a heterologous protein. Furthermore, the half-life of hTK delta 40-Cln2 was similar to that of authentic Cln2, and the rate of degradation of neither protein was detectably enhanced by treatments known to cause G1 arrest, including exposure of MATa haploids to alpha-factor mating pheromone and shifting cdc28ts and cdc34ts mutants to the restrictive temperature. These results suggest that the major signals responsible for Cln2 instability are confined to its C-terminal third. Because hTK delta 40-Cln2 and Cln2 were expressed from heterologous promoters yet their half-lives both in asynchronous cultures and when arrested at various cell cycle stages were always similar, the Cln2 PEST domain contains a signal for rapid protein turnover that is constitutively active and operative throughout the cell cycle. Removal of the 37 codons that encode the most prominent PEST-like segment from either hTK delta 40-Cln2 or Cln2 decreased the turnover rate of the resulting proteins, as expected; however, an hTK delta 40 chimera containing only this 37-residue segment was not detectably destabilized, suggesting that this PEST sequence, when removed from its normal context, is not a self-contained determinant of protein instability.     

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.     

Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway.

Recombinant G1 cyclin Cln2p can bind to and stimulate the protein kinase activity of p34CDC28 (Cdc28p) in an extract derived from cyclin-depleted and G1-arrested Saccharomyces cerevisiae cells. Upon activating Cdc28p, Cln2p is extensively phosphorylated and conjugated with multiubiquitin chains. Ubiquitination of Cln2p in vitro requires the Cdc34p ubiquitin-conjugating enzyme, Cdc28p, protein phosphorylation and unidentified factors in yeast extract. Ubiquitination of Cln2p by Cdc34p contributes to the instability of Cln2p in vivo, as the rate of Cln2p degradation is reduced in cdc34ts cells. These results provide a molecular framework for G1 cyclin instability and suggest that a multicomponent, regulated pathway specifies the selective ubiquitination of G1 cyclins.     

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.     

p34Cdc28-mediated control of Cln3 cyclin degradation.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.