Cyclins of the fission yeast Schizosaccharomyces pombe

Five cyclin-like genes, cig1, cig2/cyc17, mcs2, puc1 and cdc13, have been discovered in S. pombe to date. It is not yet clear what their functions are or even whether they are all involved with control of the cell cycle. Conflicting data for cig1 and cig2/cyc17 have obscured analysis of their function and cig1 remains largely uncharacterized, although clues to the role of cig2/cyc17 have emerged. There is genetic data available for the more distant cyclin homologue mcs2, which has an essential although as yet unspecified role. Puc1 may be involved in regulation of exit from the cell cycle. The first cyclin to be discovered, and the best understood, is cdc13 which with cdc2 promotes mitosis. Studies of the roles of cdc2 and cdc13 in the overall ordering of the cell cycle suggest that cdc13 and probably other cyclins are key regulators, maintaining the order of S phase and mitosis during the cell cycle.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Featured organism: Schizosaccharomyces pombe, the fission yeast

Schizosaccharomyces pombe, the fission yeast, has long been a crucial model for the study of the eukaryote cell cycle. We take a look at this important yeast, whose genome has recently been completed, featuring comments from Valerie Wood, Jürg Bähler, Ramsay McFarlane, Susan Forsburg, Iain Hagan and Paul Nurse on the implications of having the complete sequence and future prospects for pombe genomics.     

Acetate assimilation by the fission yeast, Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe utilizes acetate at subinhibitory concentrations in the presence of D-glucose. The nonionized form of acetate is preferentially utilized, oxidized to 14CO2, and assimilated into lipids and proteins. Acetyl CoA synthetase activity greatly increases in the yeast cells grown in media containing acetate. However, glyoxylate cycle enzymes are not detectable in Schizosaccharomyces pombe. [1-14C]Acetate is incorporated into stereols, sterol esters, neutral lipids, and phospholipids. Assimilation of [1-14C]acetate into the peptide structure of proteins was confirmed by a proteolytic digestion experiment.     

Pgt1, a glutathione transporter from the fission yeast Schizosaccharomyces pombe

The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter ( Pgt1 ) by a genetic strategy that exploited the requirement of the cys1a Δ strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1a Δ strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione ( K _m=63 μM) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu–cys–gly, an analogue of glutathione (γ-glu–cys–gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1⁺ expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast.     

Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.

ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.     

Pheromone communication in the fission yeast Schizosaccharomyces pombe

Conjugation between two haploid yeast cells is generally controlled by the reciprocal action of diffusible mating pheromones, cells of each mating type releasing pheromones that induce mating-specific changes in cells of the opposite type. Recent studies into pheromone signalling in the fission yeast Schizosaccharomyces pombe have revealed significant parallels with processes in higher eukaryotes and could provide the opportunity for investigating communication in an organism that is amenable to both biochemical and genetic manipulation.     

A galactosyltransferase from the fission yeast Schizosaccharomyces pombe

A membrane-associated galactosyltransferase has been purified to homogeneity from the fission yeast, Schizosaccharomyces pombe. The enzyme has a molecular weight of 61,000 and is capable of transfering galactose from UDP-galactose (UDP-Gal) to a variety of mannose-based acceptors to form an alpha-1,2 galactosyl mannoside linkage. Immunofluorescence localization of the protein is consistent with the presence of the enzyme in the Golgi apparatus of S. pombe. This, together with the presence of terminal, alpha-linked galactose on the N- linked oligosaccharides of S. pombe secretory proteins, suggests that the galactosyltransferase is an enzyme involved in the processing of glycoproteins transported through the Golgi apparatus in fission yeast.     

The proteolytic system of the fission yeast Schizosaccharomyces pombe

Proteinase and peptidase activities of the fission yeast Schizosaccharomyces pombe were investigated. Several intracellular proteolytic enzymes were found: two endoproteinases, one carboxypeptidase, one aminopeptidase and one dipeptidyl-aminopeptidase. In addition, proteinase inhibitors were detected. In fresh crude extracts an activation procedure is needed to measure maximal activities of endoproteinases and carboxypeptidase, whose level is markedly dependent on growth medium composition and on growth phase, while aminopeptidase and dipeptidyl-aminopeptidase activities are very little, if at all, regulated by the carbon source.     

Dikaryotic cell division of the fission yeast Schizosaccharomyces pombe

Dikaryons, cells with two haploid nuclei contributed by the members of a mating pair, are part of the life cycle of many filamentous fungi, but the molecular mechanisms underlying the division of dikaryons are largely unknown. We found that the fission yeast Schizosaccharomyces pombe has a latent ability to divide as a dikaryon. Cells capable of restarting the mitotic cycle with two nuclei were prepared by transient inactivation of the septation initiation network. Close pairing of the two nuclei before mitosis was dependent on minus-end-directed kinesin Klp2p and was essential for propagation as a dikaryon. The two spindles extended in opposite directions, keeping their old spindle pole bodies at the prospective site of cell division until the mid-anaphase. The spindles then overlapped, exchanging the inner nuclei. Finally, twin mitosis was followed by a single cytokinesis, producing two daughter dikaryons carrying copies of the original pair of nuclei.     

Transport of L-lysine in the fission yeast Schizosaccharomyces pombe

Systems of L-lysine transport in Schizosaccharomyces pombe are not constitutive, as at no phase of growth in a rich medium is lysine taken up. Transport activity appears only after preincubation of harvested cells with glucose or another suitable source of energy. If cycloheximide is added during this preincubation no transport systems are synthesized. After removal of glucose, the activity of the transport system decays with a half-time of 13 min. The transport of L-lysine into S. pombe cells from the stationary phase of growth preincubated for 60 min with 1% D-glucose is mediated by at least two systems, the high-affinity one with a Kt of 26 mumol/l and Jmax of 4.95 nmol/min per mg dry wt., the low-affinity one with a KT of 1.1 mmol/l and Jmax of 11.8 nmol/min per mg dry wt. The transport of lysine mediated by these two systems proceeds uphill. The high-affinity system has a pH optimum at 4.0-4.2, the accumulation ratio is highest at a cell density 2-5 mg dry wt. per ml and decreases with increasing lysine concentrations. Lysine accumulated by this system does not exit from cells. The only potent competitive inhibitors are L-arginine, L-histidine and D-lysine. The other amino acids tested do not behave as competitive inhibitors. Of the various metabolic inhibitors tested, the most potent were proton conductors and antimycin A.