Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and oligomycin-sensitivity during the cell cycle of catabolite-repressed and -de-repressed cells.

1. Changes in activity of ATPase (adenosine triphosphatase) during the cell cycle of Schizosaccharomyces pombe were analysed in cell-free extracts of cells harvested from different stages of growth of synchronous cultures and also after cell-cycle fractionation. 2. Oligomycin-sensitive ATPase oscillates in both glucose-repressed synchronous cultures and shows four maxima of activity approximately equally spaced through the cell cycle. The amplitude of the oscillations accounts for between 13 and 80% of the total activity at different times in the cell cycle. 3. Oligomycin sensitivity varies over a fourfold range at different stages of the cell cycle. 4. The periodicity of maximum oligomycin sensitivity is one-quarter of a cell cycle. 5. These results were confirmed for the first three-quarters of the cell cycle by cell-cycle fractionation. 6. In cells growing synchronously with glycerol, ATPase activity increases in a stepwise pattern, with two steps per cell cycle; the first of these occurs at 0.54 of the cell cycle and the second at 0.95. 7. These results are discussed in relation to previously obtained data on the development of mitochondrial activities during the cell cycle.     

Mitochondrial adenosine triphosphatase of the fission yeast Schizosaccharomyces pombe 972h-. Changes in inhibitor sensitivities during the cell cycle indicate similarities and differences in binding sites.

1. We used 11 different inhibitors of energy conservation as inhibitors of ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe obtained from cells at different stages of the cell cycle. 2. All the inhibitors showed cell-cycle-dependent variations in their I50 values (microng of inhibitor/mg of protein giving 50% inhibition of inhibitor-sensitive ATPase at pH 8.6). 3. From the sensitivity profiles through the cell cycle it was concluded that: (a) oligomycin, venturicidin, triethyltin sulphate and dibutylchloromethyltin chloride all act at closely associated site(s); (b) NN'-dicyclohexylcarbodi-imide and leucinostatin both act at a similar site, which is, however, distinct from that at which other inhibitors of the membrane factor (Fo) act. 4. The variations in I50 values for efrapeptin closely followed changes in specific activity of ATPase, as would be expected for an inhibitor acting at catalytic sites; these fluctuations were different from those for aurovertin, Dio-9, 4-chloro-7-nitrobenzofurazan, quercetin and spegazzinine, all of which show different sensitivity profiles from one another. 5. Anomalous stepwise inhibitor-titration curves were obtained for spegazzinine, NN'-dicyclohexylcarbodiimide, dibutylchloromethyltin chloride and leucinostatin. 6. Possible explanations are proposed for the discontinuous expression of inhibitor-binding sites during the cell cycle.     

Ammonia assimilation in the fission yeast Schizosaccharomyces pombe 972

Glutamine synthetase (GS) activity of Schizosaccharomyces pombe 972 was high in ammonia-limited cultures, low in phosphate-and sulphate-limited cultures and not detected in glucose-limited cultures. When ammonia was pulsed into an ammonia-limited culture then GS activity decreased at a rate faster than that calculated if enzyme synthesis ceased and enzyme was diluted out by growth. Enzyme activity increased in ammonia-starved, phosphate-limited cultures and in the ammonia pulse system when the added ammonia had been utilised. These increases in enzyme activity were prevented by the presence of 100 g/ml cycloheximide. GS activity was inversely related to the intracellular concentration of glutamate.Abbreviations Gs Glutamine synthetase, EC 6.3.1.2 - GOGAT Glutamine: 2-oxo-glutarate amino transferase, EC 2.6.1.53 - GDH Glutamate dehydrogenase, EC 1.4.1.3     

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.     

Respiratory metabolism of a "petite negative"yeast Schizosaccharomyces pombe 972h-

The respiratory metabolism of Schizosaccharomyces pombe 972h(-), a fission, haplontic, "petite negative" yeast, was studied. Glucose and glycerol are good growth substrates and are oxidized under appropriate conditions. l-Lactate, ethanol, malate, and succinate are oxidized but are poor substrates for growth. d-Lactate and pyruvate are neither oxidized nor used for growth. Limited growth was observed under anaerobic conditions. The addition of 0.3% KNO(3) to a rich medium relieves the oxygen requirement. A continuous increase of cell respiration during growth on repressive concentration of glucose was observed, suggesting the presence of glucose repression of respiration. Reduced nicotinamide adenine dinucleotide (NADH), succinate, alpha-glycerophosphate, and ascorbate plus tetramethyl-p-phenylenediamine are oxidized by a mitochondrial fraction. NADH and succinate oxidations are inhibited by antimycin A and NaCN but not by rotenone, suggesting the absence of the phosphorylation site I and the presence of sites II and III. The effects of several mitochondrial inhibitors on growth and respiration indicate that the requirement of an oxidant for growth is related neither to the functioning of the respiratory electron transport chain nor to the formation of respiratory energy. The previously suggested correlations between the nonviability of vegetative "petites" mutants, the absence of repression of respiration by glucose, and the incapacity to grow under anaerobic conditions are thus not strictly valid for S. pombe.     

Genes involved in glucose repression and oxidative stress response in the fission yeast Schizosaccharomyces pombe

We looked for changes in gene expression and novel genes that could be involved in the interaction between glucose repression and oxidative stress response in the fission yeast, Schizosaccharomyces pombe, using a constitutive invertase mutant, ird11, which is resistant to glucose. BLAST analysis was made of the S. pombe genome database of cDNAs whose expression ratios differentially decreased or increased upon exposure to mild oxidative stress in this mutant compared to the wild type. Genes with this type of activity were identified as rpl302, encoding 60S ribosomal protein L3, and mpg1, encoding mannose-1-phosphate guanyltransferase; their expression patterns were measured using quantitative real-time PCR. We found that the expression levels of rpl302 and mpg1 genes in ird11 under unstressed conditions were increased compared to those of the wild type. Under stress conditions, the expression levels of the rpl302 gene were decreased in both strains, while mpg1 expression levels remained unchanged. These results suggest that these genes play a role in the response to oxidative stress in this mutant strain.     

The reaction of cytochrome oxidase with oxygen in the fission yeast Schizosaccharomyces pombe 972h-. Studies at subzero temperatures and measurement of apparent oxygen affinity.

1. Cytochrome alpha 3 in whole-cell suspensions of the fission yeast Schizosaccharomyces pombe reacted in the reduced form with CO to give a photodissociable CO complex with absorption maxima at 429, 543 and 591 nm in CO-liganded reduced-minus-reduced difference spectra. 2. Other CO-bound haemoproteins, cytochromes P-420 and P-450, were not photodissociated under the conditions employed. 3. Measurements of the rates of reassociation of CO with cytochrome alpha 3 after flash photolysis over the temperature range from -101 to -109 degrees C gave a value for Eact. of 28.6 kJ/mol. 4. Between -94 and -106 degrees C, O2 reacted with cytochrome oxidase in intact cells to give an oxygenated intermediate (compound A). 5. At -70 degrees C compound A was converted into a second spectrally distinct intermediate (compound B). 6. Electron transport, indicated by the oxidation of cytochromes alpha + alpha 3 and cytochrome c, did not occur until the temperature was raised to -50 degrees C. 7. At room temperature cytochfome oxidase was oxidized to 50% of its steady-state concentration by 0.35 microM-O2.     

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.     

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.     

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.     

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.     

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.     

Apoptosis and lipoapoptosis in the fission yeast Schizosaccharomyces pombe

Yeasts being simple eukaryotes are established genetic systems that are often employed to solve important biological questions. Recently, it has become evident that certain cell death programs exist in these unicellular organisms. For example, it has been shown recently that strains of the fission yeast Schizosaccharomyces pombe deficient in triacylglycerol synthesis undergo cell death with prominent apoptotic markers. This minireview is intended to discuss key developments that have rendered fission yeast useful both as a tool and as a model for apoptosis and lipoapoptosis research. It is attempted to delineate a putative signaling pathway leading to the execution of lipoapoptosis in the fission yeast. Although in its infancy, apoptosis research in the fission yeast promises exciting breakthroughs in the near future.