The cell cycle–regulated genes of schizosaccharomyces pombe

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S.pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

Genome-wide screen of genes required for caffeine tolerance in fission yeast

Background: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to atoxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.Methodology/Principal Findings: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeinecontainingplates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 aresensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. Conclusions/Significance: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we havedemonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

The peroxiredoxin Tpx1 is essential as a H202-scavenger during aerobic growth in fission yeast

Peroxiredoxins are known to interact with hydrogen peroxide (H2O2) and to participate in oxidant scavenging, redox signal transduction, and heat-shock responses. The two-cysteine peroxiredoxin Tpx1 of Schizosaccharomyces pombe has been characterized as the H2O2 sensor that transduces the redox signal to the transcription factor Pap1. Here, we show that Tpx1 is essential for aerobic, but not anaerobic, growth. We demonstrate that Tpx1 has an exquisite sensitivity for its substrate, which explains its participation in maintaining low steady-state levels of H2O2. We also show in vitro and in vivo that inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid is always preceded by a sulfinic acid form in a covalently linked dimer, which may be important for understanding the kinetics of Tpx1 inactivation. Furthermore, we provide evidence that a strain expressing Tpx1.C169S, lacking the resolving cysteine, can sustain aerobic growth, and we show that small reductants can modulate the activity of the mutant protein in vitro, probably by supplying a thiol group to substitute for cysteine 169.

Activation of Srk1 by the Mitogen-activated Protein Kinase Sty1/Spc1 Precedes Its Dissociation from the Kinase and Signals Its Degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. Methodology/Principal Findings: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. Conclusion/Significance: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.

Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calciumactivated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Fusion of the human gene for the polyubiquitination co-effector UEV-1 with Kua, a newly identified gene

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination.[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).