Naomi Kamasawa

In situ localization of β-glucans in the cell wall of Schizosaccharomyces pombe

The chemical composition of the cell wall of Sz. pombe is known as β‐1,3‐glucan, β‐1,6‐glucan, α‐1,3‐glucan and α‐galactomannan; however, the three‐dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three‐layered structure: the outer layer is electron‐dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high‐resolution scanning electron microscope reveals a surface completely filled with α‐galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of β‐glucan by immuno‐electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly β‐1,6‐branched β‐1,3‐glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the α‐galactomannan layer, suggesting that some of the radial filaments contain β‐1,6‐branched β‐1,3‐glucan. β‐1,6‐glucan is preferentially located underneath the α‐galactomannan layer. Linear β‐1,3‐glucan is exclusively located in the primary septum of dividing cells. β‐1,6‐glucan only labels the secondary septum and does not co‐localize with linear β‐1,3‐glucan, while β‐1,6‐branched β‐1,3‐glucan is present in both septa. Linear β‐1,3‐glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear β‐1,3‐glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found β‐1,6‐glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that β‐1,6‐glucan is synthesized in the endoplasmic reticulum–Golgi system and exported to the cell membrane. Copyright

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme

Abstract. NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C4 species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C4 subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts.

Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses.

Abstract. The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as α-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 104–105 molecules of α-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

Abstract The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

Abstract We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form.

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

Abstract An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration.

Abstract. We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.

Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.

Pob1 ensures cylindrical cell shape by coupling two distinct rho signaling events during secretory vesicle targeting.

Proper cell morphogenesis requires the co‐ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1‐664 has a curious lemon‐like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin‐polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon‐like cell morphology similar to pob1‐664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin‐mediated vesicle transport and exocyst‐mediated vesicle tethering during secretory vesicle targeting.

Characterization of a dicarboxylic acid-producing mutant of the yeast Candida tropicalis

Abstract A comparative study was carried out on the biosynthesis of peroxisomal enzymes in the n -alkane-assimilating yeast Candida tropicalis 1098 and its dicarboxylic acid-producing mutant M2030. The mutant strain showed a drastic decrease in protein levels in acyl-CoA oxidase and 3-ketoacyl-CoA thiolase, constituting the yeast peroxisomal β-oxidation system, in comparison with those of the wild-type strain. Interestingly, the development of peroxisomes was scarcely observed in the mutant strain.