Kaoru Takegawa

We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) had high levels of transglycosylation activity. The enzyme activity of Endo-A was markedly increased by adding 4-L-aspartyl-glycosylamine (GlcNAc-Asn) to the reaction mixture. Digesting (Man)6(GlcNAc)2 with the enzyme in the presence of GlcNAc-Asn gave a mixture of hydrolytic ((Man)6GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Asn) products. By me

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enla

We describe a simple and efficient procedure for transformation of Schizosaccharomyces pombe. Sz. pombe colonies grown on minimal (SD) plates were directly removed and suspended in a 100 microl reaction mixture containing 70 microl PLATE solution (50% polyethylene glycol-4000, 100 mM lithium acetate, 10 mM Tris-HCl, pH 4.9, and 1 mM EDTA), 10 microl plasmid DNA (1 microg), 10 microl carrier DNA (100 microg) and 10 microl sterile distilled water. After incubation at 30 degrees C for 1 h followed

Arthrobacter protophormiae produced a high level of extracellular endo-β- N -acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N -acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by p

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well-characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high-level expression in Sc. pombe have been developed and many foreign p

To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow

Autophagy is a non-selective degradation process in eukaryotic cells. The genome sequence of the fission yeast Schizosaccharomyces pombe has revealed that many of the genes required for autophagy are common between the fission yeast and budding yeast, suggesting that the basic machinery of autophagy is conserved between these species. Autophagy in fission yeast is specifically induced by nitrogen starvation based on monitoring a GFP-Atg8p marker. Upon nitrogen starvation, fission yeast cells exi

Endo-β-N-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of N-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents of glycoproteins. However, there are only a few known ENGases that can catalyze the hydrolysis of human complex type oligosaccharides, and although commercially available, they are expensive. Here, we report the cloning of two ENGase encoding cDNAs from the basidiomycete fungus Coprinopsis cinerea, Endo-CC1 and Endo-CC2. We successfully express

Endo-beta-N-acetylglucosaminidase from Flavobacterium sp. released about 30% of the N-linked sugar chains from the glucose oxidase of Aspergillus niger. To elucidate the role of the carbohydrate moiety, the enzymatic properties of native and carbohydrate-depleted glucose oxidases were compared. It was found that their catalytic activities and thermal and pH stabilities were identical. However, the carbohydrate-depleted glucose oxidase was more rapidly precipitated by the addition of trichloroace

The vacuole of Saccharomyces cerevisiae plays essential roles not only for osmoregulation and ion homeostasis but also down-regulation (degradation) of cell surface proteins and protein and organellar turnover. Genetic selections and genome-wide screens in S. cerevisiae have resulted in the identification of a large number of genes required for delivery of proteins to the vacuole. Although the complete genome sequence of the fission yeast Schizosaccharomyces pombe has been reported, there have b

Although various mutant strains of the fission yeast Schizosaccharomyces pombe exhibit non-sexual flocculation, little is known about the mechanistic basis for this phenomenon, nor have genes encoding the implicated flocculin been identified. In the budding yeast Saccharomyces cerevisiae, the transcription factor Flo8 controls expression of some of the genes involved in non-sexual flocculation. We have found that overexpression of S. cerevisiae FLO8 induced non-sexual flocculation in S. pombe. T