We describe the identification and characterization of a transcription factor encoded by the atf1+ gene of the fission yeast Schizosaccharomyces pombe. The factor Atf1, contains a bZIP domain at its C‐terminus with strong homology to members of the ATF/CREB family of mammalian factors and in vitro binds specifically to ATF/CRE recognition sites. Furthermore the ATF‐like binding activity detected in extracts from fission yeast cells is entirely lost upon deletion of the atf1+ gene. Upon growth to saturation, fission yeast cells exit the mitotic cycle and enter a G0‐like stationary phase. However, on rich medium, entry of atf1‐ cells into stationary phase is restricted and they rapidly lose viability; this does not occur on minimal medium unless cAMP levels are raised. Thus stationary phase entry appears to be regulated negatively by cAMP and positively by Atf1. atf1‐ cells are also sterile and this sterility appears to be due to a combination of two defects: first, upon nitrogen starvation the majority of atf1‐ cells fail to arrest in the G1 phase of the cell cycle and second, the induction of ste11+ expression is lost. Thus expression of ste11+ represents a second example of an event that is negatively regulated by the cAMP pathway and positively regulated by Atf1. Despite their close association however, these two regulatory pathways function independently and Atf1 activity is not directly modulated by cAMP levels or mutations that alter the activity of components of the cAMP signalling pathway. Thus Atf1 is a transcription factor that plays an important role in the response of cells to adverse environmental conditions, which is to exit the mitotic cell cycle and either sexually differentiate or enter a resting state.

In fission yeast, maintenance of genome ploidy is controlled by at least two mechanisms. One operates through the Cdc2/Cdcl3 kinase, which also involves the CDK inhibitor Ruml, and the other through the S-phase regulator Cdcl8. By screening for sterile mutants that show increased ploidy, we have identified a new gene, popl ÷, in mutants that become polyploid. The popl mutation shows a synthetic lethal interaction with the temperature-sensitive cdc2 or cdcl3 mutation. In a popl mutant Ruml and Cdcl8 proteins become accumulated to high levels. The high ploidy phenotype in the popl mutant is dependent on the presence of the rural ÷ gene, whereas the accumulation of Cdcl8 is independent of Ruml. The predicted sequence of the Popl protein indicates that it belongs to a WD-repeat family with highest homology to budding yeast Cdc4, which participates in the ubiquitin-dependent pathway. Consistent with this notion, in a mutant of the 26S proteasome, higher molecular weight forms of Ruml and Cdcl8 are accumulated corresponding to polyubiquitination of these proteins. In the popl mutant, however, no ubiquitinated forms of these proteins are detected. Finally we show that Popl binds Cdcl8 in vivo. We propose that Popl functions as a recognition factor for Ruml and Cdcl8, which are subsequently ubiquitinated and targeted to the 26S proteasome for degradation.

SME1 was cloned due to its high copy number effect: it enabled MATa/MAT alpha diploid cells to undergo meiosis and sporulation in a vegetative medium. Disruption of SME1 resulted in a recessive Spo- phenotype. These results suggest that SME1 is a positive regulator for meiosis. DNA sequencing analysis revealed an open reading frame of 645 amino acids. An amino terminal peptide of ca 400 amino acids in the deduced protein was similar to known protein kinases. Transcription of SME1 was regulated negatively by nitrogen and glucose and positively by MATa/MAT alpha and IME1, another positive regulator gene of meiosis. By complementation analysis, SME1 was found to be identical to IME2, which had been shown to be important in meiosis. These results suggest that IME1 product stimulates meiosis by activating transcription of SME1 (IME2) and that protein phosphorylation is required for initiation of meiosis.

Background: In the ubiquitin-dependent proteolysis pathway, a ubiquitin ligase (E3) is responsible for substrate selectivity and timing of degradation. A novel E3, SCF (Skp1-Cullin-1/Cdc53-F-box) plays a pivotal role in cell cycle progression. In fission yeast, F-box/WD-repeat protein Pop1 regulates the level of the CDK (cyclin-dependent kinase) inhibitor Rum1 and the S phase regulator Cdc18.

Many eukaryotic cells arrest the cell cycle at G 1 phase upon nutrient deprivation. In fission yeast, during nitrogen starvation, cells divide twice and arrest at G 1 . We have isolated a novel type of sterile mutant, which undergoes one additional S phase upon starvation and, as a result, arrests at G 2 . Three loci (apc10, ste9/ srw1 and rum1) were identified. The apc10 mutants, previously unidentified, show, in addition to sterility, temperature-sensitive growth with defects in chromosome segregation. apc10 ⍣ is essential for viability, encodes a conserved protein (a homologue of budding yeast Apc10/Doc1) and is required for ubiquitination and degradation of mitotic B-type cyclins. Apc10 does not co-sediment with the 20S APC-cyclosome, a ubiquitin ligase for B-type cyclins, and in the apc10 mutant the 20S complex is intact, suggesting that it is a novel regulator for this complex. A subpopulation of Apc10 does co-immunoprecipitate with the anaphase-promoting complex (APC). A second gene, ste9 ⍣ /srw1 ⍣ , encodes a member of the fizzy-related family, also regulators of the APC. Finally, Rum1 is a cyclindependent kinase (CDK) inhibitor which exists only in G 1 . The results suggest that dual downregulation of CDK, one via the APC and the other via the CDK inhibitor, is a universal mechanism that is used to arrest cell cycle progression at G 1 .

The nin1‐1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1‐1 strains fail to activate histone H1 kinase after release from alpha factor‐imposed G1 block and after release from hydroxyurea‐imposed S block. The nin1‐1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28‐IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP‐Mg2+, and (ii) nin1‐1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.

Ninlp, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the ninl-l mutant, we have screened for genes encoding proteins with related functions to Ninlp and have cloned and characterized two new multicopy suppressors, SUNI and SlUN2, of the ninl-l mutation. SUNI can suppress a null ninl mutation, whereas SlUN2, an essential gene, does not. Sunlp is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sunlp binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sunlp and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sunlp and Sun2p are components of the regulatory module of the yeast 26S proteasome.

We describe a novel set of oscillation mechanisms for the fission yeast S phase cyclin Cig2, which contains an authentic destruction box and is destroyed at anaphase via the APC/cyclosome (APC/C). Unlike the mitotic cyclin Cdc13, however, Cig2 mRNA and protein peak at the G1/S boundary and decline to low levels in G2 and M phases. We show here that SCF(Pop1, Pop2) plays a role in transcriptional periodicity, as pop mutations result in constitutive cig2(+) transcripts. The instability of Cig2 during G2 and M is independent of either the APC/C or Pop1/Pop2, but requires Skp1, a core component of SCF. These data indicate that the APC/C and SCF control Cig2 levels differentially at different stages of the cell cycle.