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.     

Raf-1 is a binding partner of DSCR1

Down syndrome critical region 1 (DSCR1) is recognized as an endogenous calcineurin inhibitor. DSCR1 is induced in endothelial cells and may play an important role in inflammation and angiogenesis. To address a novel function of DSCR1, we searched interacting partners of DSCR1. We performed pull-down analysis using DSCR1 as a bait and identified Raf-1 as a binding partner. The association of Raf-1 was confirmed by co-immunoprecipitation in GM7373 cells expressing green fluorescence protein tagged DSCR1. We determined two Raf-1 binding regions in DSCR1; one in the N-terminus and the other in the C-terminus regions. We further demonstrated that calpain cleaved DSCR1 and generated fragments with different binding affinity to Raf-1 or calcineurin. These results constitute the first demonstration of Raf-1 as a binding partner of DSCR1, and suggest a novel role of DSCR1.     

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.     

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.     

Pseudo-exponential growth in length of the fission yeast, Schizosaccharomyces pombe

The growth patterns of individual cells of the fission yeast (Schizosaccharomyces pombe wild-type cells, strain 972 h-; cells exposed to hydroxyurea; and cdc mutants, 11-123, 2-33) were investigated by time-lapse photomicrography. Wild-type cells showed one, two, or three linear-growth segments followed by a constant-length stage. Cells with two segments were most frequent. Hydroxyurea cells that divided as oversized cells (about three times the birth length) had three linear-growth segments in a cycle. Mutant cdc11-123 cells did not divide but had a constant-length stage separating the cycles; both the first and second cycles consisted of two linear-growth segments, and cells were oversized at the second constant-length stage (about 3.5 times the birth length). Elongating cdc2-33 cells that did not divide and were oversized (about five times the birth length) while under observation, showed four linear-growth segments. Cells of all strains showed 30 to 40% increase in growth rate at the rate-change point and maintained approximate exponential (pseudo-exponential) growth. We conclude that the normal growth pattern of individual fission-yeast cells is the pseudo-exponential pattern.     

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     

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.     

Distribution, levels and phosphorylation of Raf-1 in Alzheimer's disease

Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase pathway, has been increasingly implicated in the pathogenesis of Alzheimer's disease due to its critical role in brain function. While we previously demonstrated that ERK is activated in Alzheimer's disease, the upstream cascade leading to its activation had not been fully examined. In this study, we focused on Raf-1, one of the physiological activators of the ERK pathway. Raf-1 is activated by phosphorylation at Ser338 and Tyr340/341 and inhibited by phosphorylation at Ser259. Interestingly, phosphorylation at all three sites on Raf-1 was increased as evidenced by both immunocytochemistry and immunoblot analysis in Alzheimer's disease brains compared to age-matched controls. Both phospho-Raf-1 (Ser259) and phospho-Raf-1 (Ser338) were localized to intracytoplasmic granular structures, whereas phospho-Raf-1 (Tyr340/341) was localized to neurofibrillary tangles and granules in pyramidal neurons in Alzheimer's disease hippocampus. There is extensive overlap between phospho-Raf-1 (Ser338) and phospho-Mek1/2, the downstream effector of Raf-1, suggestive of a mechanistic link. Additionally, increased levels of Raf-1 are associated with Ras and MEK1 in Alzheimer's disease as evidenced by its coimmunoprecipitation with Ras and Mek1, respectively. Based on these findings, we speculate that Raf-1 is activated to effectively mediate Ras-dependent signals in Alzheimer's disease.     

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.     

The Cdc42p GTPase is targeted to the site of cell division in the fission yeast Schizosaccharomyces pombe

The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.     

A review of mitosis in the fission yeast Schizosaccharomyces pombe

Mitosis and cell division are the final events of the cell cycle, resulting in the precise segregation of chromosomes into two daughter cells. A highly controlled and accurate segregation of the chromosomes is required to ensure that each daughter cell receives a complete genome and remains viable. The fission yeast, Schizosaccharomyces pombe, is a unicellular eukaryotic organism which is particularly convenient for investigating these problems. It is very amenable to genetic analysis and its predominantly haploid life cycle has allowed the isolation of recessive temperature-sensitive mutants unable to complete the cell cycle. Classical genetic analysis of these mutants has been used to identify over 40 gene functions that are required for cell cycle progress in S. pombe. Many of these genes have now been cloned and sequenced and in some cases the encoded gene product has been identified. This approach, coupling classical and molecular genetics, allows identification of the molecules important in the mitotic processes and provides a means for establishing what functional roles they may play.     

Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe

We have succeeded to produce a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we solved the problem by identifying plg7, encoding phospholipase A2, as a multicopy suppressor. Characterization of the RA-tolerant strains suggested that the removal of RA moieties from phospholipids would be the suppression mechanism by plg7. In this study, we extended our analysis and report our new discovery that the overexpression of plg7 enabled cells to secrete free RA into culture media. When the FAH12 integrant in the absence of the overexpressed plg7 was grown at 20 °C for 11 days, the amount of intracellular RA reached 200.1 μg/ml of culture and only 69.3 μg/ml of RA was detected in culture media. On the other hand, the FAH12 integrant harboring the plg7 multicopy plasmid secreted RA in the media (184.5 μg/ml) without decreasing the amount in the cells, i.e., a significantly higher total secretion and a lead to making RA by its secretory production in S. pombe.     

Degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe

Although the overall structures of flagellar and cytoplasmic microtubules are understood, many details have remained a matter of debate. In particular, studies of the arrangement of tubulin subunits have been hampered by the low contrast of the tubulin subunits. This problem can now be addressed by the kinesin decoration technique. We have shown previously that the recombinant kinesin head domain binds to beta-tubulin, thus enhancing the contrast between alpha- and beta- tubulin in the electron microscope; this allows one to study the arrangement of tubulin dimers. Here we describe the lattices of the four different types of microtubules in eukaryotic flagellar axonemes (outer doublet A and B, central pair C1 and C2). They could all be labeled with kinesin head with an 8-nm axial periodicity (the tubulin dimer repeat), and all of them showed the B-surface lattice. This lattice is characterized by a 0.92-nm stagger between adjacent protofilaments. The B-lattice was observed on the axonemal microtubules as well as on extensions made by polymerizing porcine brain tubulin onto axonemal microtubules in the proximal and distal directions. This emphasizes that axonemal microtubules serve as high fidelity templates for seeding microtubules. The presence of a B-lattice implies that there must be a helical discontinuity ("seam") in the wall. This discontinuity is now placed near protofilaments A1 and A2 of the A- tubule, close to the inner junction between A- and B-microtubules. The two junctions differ in structure: the protofilaments of the inner junction (A1-B10) are staggered roughly by half a dimer, those of the outer junction (A10-B1) are roughly in register. Of the two junctions the inner one appears to have the stronger bonds, whereas the outer one is more labile and opens up easily, generating "composite sheets" with chevron patterns from which the polarity can be deduced (arrow in the plus direction). Decorated microtubules have a clear polarity. We find that all flagellar microtubules have the same polarities. The orientation of the dimers is such that the plus end terminates with a crown of alpha subunits, the minus end terminates with beta subunits which thus could be in contact with gamma-tubulin at the nucleation centers.     

Repair of UV damage in the fission yeast Schizosaccharomyces pombe

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.     

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast

The fission yeast plc1 ⁺ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 ⁺ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 ⁺ and rad24 ⁺ belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins. Received: 10 July 1997 / Accepted: 15 December 1997