Transcriptional regulation of glutathione synthetase in the fission yeast Schizosaccharomyces pombe

Glutathione (GSH), an important antioxidant involved in the stress response, is synthesized in two sequential reactions involving glutamylcysteine synthetase (GCS), followed by glutathione synthetase (GS). Expression of the unique GS gene in the fission yeast Schizosaccharomyces pombe was previously found to be regulated by nitric oxide and by L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GCS. In this work, expression of S. pombe GS gene is shown to be induced by menadione (MD), which generates superoxide. The responsible DNA sequence between -365 and -234 bp from the translation start site, was convinced using five GS-lacZ fusion plasmids. Expression of GS gene is also induced by low glucose, fructose and disaccharides, apparently dependent on Pap1 protein; GS mRNA increases in low concentrations of glucose in wild type S. pombe but not in Pap1-negative cells. Although nonfermentable carbon sources such as acetate and ethanol stimulate expression of GS gene, they also arrest the growth of the yeast cells. These results indicate that the biosynthesis of glutathione is regulated by superoxide radicals and carbon source limitation.     

Characterization and regulation of the gamma-glutamyl transpeptidase gene from the fission yeast Schizosaccharomyces pombe

The structural gene for the putative gamma-glutamyl transpeptidase (GGT) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined sequence contained 3324 bp and encoded the predicted 630 amino acid sequence of GGT, which resembles counterparts in Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, and Escherichia coli. The S. pombe cells harboring the cloned GGT gene showed about twofold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGT gene was functional. To monitor the expression of the S. pombe GGT gene, we fused the fragment 1085 bp upstream of the cloned GGT gene into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98. The synthesis of beta-galactosidase from the fusion plasmid in S. pombe cells was enhanced by treatments with NO-generating sodium nitroprusside (SN), L-buthionine-(S,R)-sulfoximine (BSO), and glycerol. The GGT mRNA level in the S. pombe cells was increased by SN and BSO. Involvement of Pap1 in the induction of the GGT gene by SN and BSO was observed.     

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.     

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.     

Regulation of the gene encoding glutathione synthetase from the fission yeast

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.     

The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe

We have cloned and sequenced the alcohol dehydrogenase gene of the fission yeast Schizosaccharomyces pombe. The gene was isolated by transformation and complementation of a Saccharomyces cerevisiae strain which lacked functional alcohol dehydrogenase with an S. pombe gene bank constructed in the autonomously replicating yeast plasmid YEp13. Southern hybridization analysis indicates that S. pombe contains only one alcohol dehydrogenase gene. The structural region of the gene is 50% homologous to the alcohol dehydrogenase encoding genes of the budding yeast S. cerevisiae. The gene exhibits a very strong codon usage bias; with the set of predominantly used codons generally resembling that which S. cerevisiae employs preferentially. All of the differences in codon usage bias between S. pombe and S. cerevisiae are in the direction of greater G + C content in S. pombe codons. It is argued that this observation supports the hypothesis that selection toward uniform codon-anticodon binding energies contributes to codon usage bias and that the optimum binding energy is, on the average, higher in S. pombe than S. cerevisiae.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

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.     

Properties of actin from the fission yeast Schizosaccharomyces pombe and interaction with fission yeast profilin

The fission yeast Schizosaccharomyces pombe serves as a model system for studying role of actin cytoskeleton, since it has simple actin cytoskeletons and is genetically tractable. In contrast, biochemical approaches using this organism are still developing; fission yeast actin has so far not been isolated in its native form and characterized, and therefore, biochemical assays of fission yeast actin-binding proteins (ABPs) or myosin have been performed using rabbit skeletal muscle actin that may interact with the fission yeast ABPs in a manner different from fission yeast actin. Here, we report a novel method for isolating functionally active actin from fission yeast cells. The highly purified fission yeast actin polymerized with kinetics somewhat different from those of muscle actin and forms filaments that are structurally indistinguishable from skeletal muscle actin filaments. The fission yeast actin was a significantly weaker activator of Mg(2+)-ATPase of HMM of skeletal muscle myosin than muscle actin. The fission yeast profilin Cdc3 suppressed polymerization of fission yeast actin more effectively than that of muscle actin and showed an affinity for fission yeast actin higher than for muscle actin. The establishment of purification of fission yeast actin will enable reconstruction of physiologically relevant interactions between the actin and fission yeast ABPs or myosins and contribute to clarification of function of actin cytoskeleton in various cellular activities.     

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.     

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.     

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.