Cdc123 and checkpoint forkhead associated with RING proteins control the cell cycle by controlling eIF2gamma abundance

Eukaryotic initiation factor 2 (eIF2) is a central regulator of translational initiation in times of growth and times of stress. Here we discovered three new conserved regulators of eIF2 in Saccharomyces cerevisiae. cdc123, homolog of mammalian D123, is a new cell division cycle mutant with a G2 delay at permissive temperature and a terminal, mating-proficient G1 arrest point. Cdc123 protein is regulated by nutrient availability. CHF1 and CHF2, homologs of mammalian checkpoint forkhead associated with RING genes, are required for G2 delay and G1 arrest of cdc123-4 and promote G1 delay when over-expressed. Cell cycle delaying activity and the natural instability of Chf1 and Chf2 depend on the integrity of both domains and association with Cdc123. Genetic analysis maps the Chf1 forkhead associated domain-binding site to the conserved Thr-274 of Cdc123, suggesting that mammalian D123 is a key target of Chfr. Gcd11, the gamma subunit of eIF2, is an additional Cdc123-interacting protein that is an essential target of the Cdc123 cell cycle promoting and Chf cell cycle arresting activity whose abundance is regulated by Cdc123, Chf1, and Chf2. Loss of cdc123 activity promotes Chf1 and Chf2 accumulation and Gcd11 depletion, accounting for the essentiality of Cdc123. The data establish the Cdc123-Chf-Gcd11 axis as an essential pathway for nutritional control of START that runs parallel to the Tor-Gcn2-Sui2 system of translational control.     

spo12 is a multicopy suppressor of mcs3 that is periodically expressed in fission yeast mitosis

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12. Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2. We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself. Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1. We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase. In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene. We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3. We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15. Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.     

Cdc4, a Protein Required for the Onset of S Phase, Serves an Essential Function during G2/M Transition in Saccharomyces cerevisiae

Saccharomyces cerevisiae proteins Cdc4 and Cdc20 contain WD40 repeats and participate in proteolytic processes. However, they are thought to act at two different stages of the cell cycle: Cdc4 is involved in the proteolysis of the Cdk inhibitor, Sic1, necessary for G(1)/S transition, while Cdc20 mediates anaphase-promoting complex-dependent degradation of anaphase inhibitor Pds1, a process necessary for the onset of chromosome segregation. We have isolated three mutant alleles of CDC4 (cdc4-10, cdc4-11, and cdc4-16) which suppress the nuclear division defect of cdc20-1 cells. However, the previously characterized mutation cdc4-1 and a new allele, cdc4-12, do not alleviate the defect of cdc20-1 cells. This genetic interaction suggests an additional role for Cdc4 in G(2)/M. Reexamination of the cdc4-1 mutant revealed that, in addition to being defective in the onset of S phase, it is also defective in G(2)/M transition when released from hydroxyurea-induced S-phase arrest. A second function for CDC4 in late S or G(2) phase was further confirmed by the observation that cells lacking the CDC4 gene are arrested both at G(1)/S and at G(2)/M. We subsequently isolated additional temperature-sensitive mutations in the CDC4 gene (such as cdc4-12) that render the mutant defective in both G(1)/S and G(2)/M transitions at the restrictive temperature. While the G(1)/S block in both cdc4-12 and cdc4Delta mutants is abolished by the deletion of the SIC1 gene (causing the mutants to be arrested predominantly in G(2)/M), the preanaphase arrest in the cdc4-12 mutant is relieved by the deletion of PDS1. Collectively, these observations suggest that, in addition to its involvement in the initiation of S phase, Cdc4 may also be required for the onset of anaphase.     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

The puc1 cyclin regulates the G1 phase of the fission yeast cell cycle in response to cell size

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.