Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

Subcellular localization and production kinetics of sucrase inSchizosaccharomyces pombe

The sucrase ofSchizosaccharomyces pombe is a repressible enzyme and its location and K_m values were found to be dependent on growth conditions. When cells were grown under repressive conditions, the enzyme activity was predominantly internal and the total sucrase activity exhibited two K_m values, indicating the presence of isoenzymes. When grown under derepressive conditions, the sucrase activity was predominantly external and the total activity exhibited only one K_m value which was smaller than those of the repressed sucrase. In resting cells, all of the activity was external. When RNA synthesis in cells growing under a repressive condition was inhibited, the sucrase activity became predominantly external after 7 h and its K_m value was similar to that of the derepressed sucrase. The results suggest a precursor-product relationship between the two isoenzymes.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

A selection system for diploid and against haploid cells inSchizosaccharomyces pombe

We have isolated a mutant ofSchizosaccharomyces pombe whose growth is temperature sensitive when it is haploid but not when it is diploid. This mutant may provide a useful system for selecting nonconditional mutants which are defective in diploid formation upon conjugation.     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

Division-site selection, cell separation, and formation of anucleate minicells inSchizosaccharomyces pombe mutants resistant to cell-wall lytic enzymes

Summary In most eukaryotic organisms that have cell walls, cell separation or cytokinesis is a degradative enzymatic process. In the fission yeastSchizosaccharomyces pombe, it is a post-M-phase event that includes the degradation of part of the cell wall and the primary septum. We describe the isolation of mutants partially defective in cytokinesis by enrichment of clones resistant to cell-wall lytic enzymes. The mutations confer mycelial morphology (chains of non-separated cells) and define four genes.Sep2-SA2 was subjected to detailed genetic and cytological analysis. Its cells frequently form complex septa composed of multiple layers, which appear as twin septa separated by anucleate minicells if the cell length is extended. This suggests that a polar signal-like mechanism may also operate inS. pombe during division-site selection andsep2 ⁺ takes part in it.Sep2 ⁺ seems to be involved in several cell cycle functions because its mutation can transiently block cell-cycle progression after nuclear division and provoke a transition from haploidy to diploidy in the double mutantsep2-SA2 cexl-SA2. Cexl-SA2 is another novel mutation which causes cell-length extension.Abbreviations DAPI 4,6-diamidino-2-phenylindole - YEA yeast extract agar - YEL yeast extract liquid - SMA synthetic minimal agar - MEA malt extract agar     

Interdependence of cell cycle events inSchizosaccharomyces pombe. Terminal phenotypes of cell division cycle mutants arrested during dna synthesis and nuclear division

Summary Temperature-sensitive cell division cycle (cdc) mutants of the fission yeastSchizosaccharomyces pombe, previously characterized as defective in nuclear division were examined by thin section electron microscopy. All of the mutants failed to enter mitosis, rather they accumulated at one of four distinct terminal phenotypes. Class one were arrested with a nucleus rectangular in cross-section and a laterally situated spindle pole body (SPB). The second group had spherical or rectangular nuclei with a single SPB. The sole member of the third group wascdc 27. K 3, which had a spherical crenated nucleus with a single SPB from which microtubules emerged and extended into the cytoplasm. Allelic variants ofcdc 25 comprised the fourth group all of which displayed aberrant nuclear morphologies. Utilizing this ultrastructural data together with a knowledge of the transition points of these mutants a model for the interdependence of certain cell cycle event is proposed in which the initiation of DNA synthesis is uncoupled from the replication and separation of the SPB. This paper also provides new information on SPB structure inS. pombe. This is discussed in connection with the transient assembly of both spindle and cytoplasmic microtubules.     

Complementation analysis in PtdInsP kinase-deficient yeast mutants demonstrates that Schizosaccharomyces pombe and murine Fab1p homologues are phosphatidylinositol 3-phosphate 5-kinases

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) is widespread in eukaryotic cells. In Saccharomyces cerevisiae, PtdIns(3,5)P(2) synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P(2) is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P(2) synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P(2) is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P(2) in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1 homologues both restored basal PtdIns(3,5)P(2) synthesis in Deltafab1 cells and made PtdIns(3,5)P(2) in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I PIPkin supported no PtdIns(3,5)P(2) synthesis. Thus, FAB1 and its homologues constitute a distinct class of Type III PIPkins dedicated to PtdIns(3,5)P(2) synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Deltafab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P(2) synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.     

Complementation of a glucose transporter mutant of Schizosaccharomyces pombe by a novel Trypanosoma brucei gene

The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells.     

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.     

Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants.

Avian retroviruses (with the notable exception of spleen necrosis virus) express their protease (PR) both in their gag and their gag-pol polyprotein precursors, in contrast to other retroviruses, notably, the mammalian retroviruses, in which PR is encoded in the gag-pol polyprotein or in a separate reading frame as a gag-pro product. The consequence is that the avian PR is expressed in stoichiometric rather than catalytic amounts. To investigate the significance of the particular genome organization of the avian retrovirus prototype Rous sarcoma virus, we developed an assay that measures complementation between the gag and the gag-pol polyproteins by expressing them from two different plasmids in transfected cells. By using this assay, we showed that the protease PR from the gag-pol polyprotein is capable of autocatalytic self-cleavage and -activation when coexpressed with a protease-deficient gag protein and that the PR domain has a role in viral particle assembly. Furthermore, this complementation assay can be used to investigate the role of the gag domain in the gag-pol polyprotein by determining whether it can rescue a defect in the gag polyprotein. We report here the results of such an experiment, which studied a mutation in the N terminus of the gag gene.     

Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.