The VPS 34 gene product (Vps34p) is required for protein sorting to the lysosome-like vacuole of yeast Saccharomyces cerevisiae . Vps34p shares significant sequence similarity with catalytic subunit bovine phosphatidylinositol (PI) 3-kinase [the 110-kilodalton (p110) PI 3-kinase], which known interact activated cell surface receptor tyrosine kinases. Yeast strains deleted or carrying vps point mutations lacked detectable activity and exhibited severe defects in vacuolar sorting....

A membrane-associated complex composed of the Vps15 protein kinase and Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for delivery proteins to yeast vacuole. An active Vps15p required recruitment Vps34p membrane subsequent stimulation PtdIns activity. Consistent with this, mutations altering highly conserved residues in lipid domain lead a dominant-negative phenotype resulting from titration activating proteins. In contrast, catalytically inactive mutants do not produce...

Abstract The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms such an aberrant modification have been largely unknown. We here report critical roles a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which upregulated great majority cancers and encodes glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown GALNT6 by small interfering RNA significantly enhanced cell adhesion function...

The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI (VPS34) than mammalian (p110). protein two conserved domains: catalytic site with ATP-binding near C terminus calcium-dependent lipid-binding domain N terminus. plant does not rescue vps34 deletion mutant, but chimeric gene in which coding for C-terminal third of VPS34 is replaced by corresponding mutant....

The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) was enhanced by inclusion organic solvents in the reaction mixture. In aqueous solution, yield relative to starting substrate 32% using Man9GlcNAc2Asn as donor and 0.5 M GlcNAc acceptor. However, media containing 30% (v/v) acetone, dioxane, N,N-dimethylformamide, or dimethyl sulfoxide with acceptor, attained yields 89, 13, 28, 75%, respectively, analyzed high performance anion exchange...

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate.These phosphoinositides have been shown to function in vesiclemediated protein sorting, they serve as second-messenger signaling molecules for regulating cell growth.To further elucidate the mechanism regulation phosphoinositides, we cloned genes encoding five putative from Dictyostelium discoideum.Database analysis indicates that D. discoideum...

The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here genome sequence IFO 4308 was determined annotated. Analysis may provide insight into properties this that make it superior use in shochu production, leading to further development industrial applications.

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

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 isomerase (Erg2p), C-5 desaturase (Erg31p, Erg32p), C-22 (Erg5p) and C-24 (28) reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse sequence reactions from zymosterol ergosterol. Disruption mutants these genes were unable synthesize ergosterol, most tolerant the polyene drugs amphotericin B nystatin. erg31+ or erg32+ did cause...

Abstract In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces (3,5) P2 from 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations FAB1A FAB1B fused with green fluorescent protein revealed FAB1A/B-green proteins localize to endosomes root epidermal Arabidopsis....

Summary The cells walls of filamentous fungi in the genus A spergillus have galactofuranose ( G al f )‐containing polysaccharides and glycoconjugates, including O ‐glycans, N fungal‐type galactomannan glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple monomers onto other components nidulans . Using reverse‐genetic biochemical approaches, identified AN8677 gene encoded a...

ABSTRACT We have cloned the gene, vps34+, from fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. Vps34 shares 43% sequence identity Saccharomyces cerevisiae and 28% p110 catalytic subunit of mammalian 3-kinase. When vps34+ gene is disrupted, strains are temperature-sensitive for growth mutant cells contain enlarged vacuoles. Furthermore, while wild-type exhibit substantial levels activity, this activity not...

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog mammalian Rab11 GTP-binding protein. mutant temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, these were exacerbated by FK506-treatment, specific inhibitor calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 cytoplasmic staining that was concentrated at growth...

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin not essential for viability but required cytokinesis and Cl(-) homeostasis. genetic screen mutations that are synthetically lethal with deletion, we isolated mutant, cis1-1/apm1-1, an allele the apm1(+) gene encodes homolog mammalian micro1A subunit clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well apm1-deleted (Deltaapm1) cells showed distinct...

Abstract We describe a simple and efficient procedure for transformation of Schizosaccharomyces pombe. Sz. pombe colonies grown on minimal (SD) plates were directly removed suspended in 100 µl reaction mixture containing 70 PLATE solution (50% polyethylene glycol‐4000, m M lithium acetate, 10 Tris–HCl, pH 4.9, 1 EDTA), plasmid DNA (1 µg), carrier (100 µg) sterile distilled water. After incubation at 30 °C h followed by heat shock treatment 42 15 min, the was spread selection plate. The...

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

This letter describes the spectroscopic measurements of fluorescently stained cells. Variable phase-contrast fluorescence spectrometry was used on cells to achieve high two-dimensional spatial resolution. Phase shift interferometry by autocorrelation interference made it possible measure spectra in field view without separation wavelengths, as case a conventional dispersive spectrometer. In this letter, authors describe experimental characteristics generated from and verify that area can be...

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, organelles, are enclosed within autophagosomes and degraded upon lysosome–vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe . Using marker, 13 Atg homologues were also found to be required yeast. budding yeast, autophagy-deficient mutants...

Endo-β-N-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of N-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents glycoproteins. However, there are only few known ENGases that can catalyze the human complex type and although commercially available, they expensive. Here, we report cloning two ENGase encoding cDNAs from basidiomycete fungus Coprinopsis cinerea, Endo-CC1 Endo-CC2. We successfully expressed recombinant His6-tagged Endo-CC2 in Escherichia...