Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Rheb GTPaseであるTsc1-Tsc2複合体は細胞外の環境に応答して細胞成長調節において重要な役割を果たしている。分裂酵母においてTsc1-Tsc2複合体は細胞周期の進行やアミノ酸の取り込みなどを調節することが報告されていた。我々はE3ユビキチンリガーゼであるPub1とβ-アレスチン様タンパクであるAny1が細胞膜アミノ酸トランスポーターであるAat1の局在を制御していることを見出した。またPub1とAny1が分裂酵母細胞内で相互作用していることからTSC-Rhebシグナル経路がPub1やAny1を通じて細胞膜のアミノ酸トランスポーターの局在を制御していることを明らかにした。

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

分裂酵母の糖鎖中には多量のガラクトースが含まれている。糖鎖構造解析の結果、ガラクトース残基はα1,2-およびα1,3-結合で付加している。分裂酵母ゲノム中に存在する全てのα1,2-ガラクトース転移酵素遺伝子を破壊した株を作成したところ、まだα1,3-結合のガラクトースが残っていることを見出した。そこでさらにゲノムを解析したところ、仮想糖転移酵素が３遺伝子存在することがわかり、これらを破壊した10重破壊株を作成したところ、糖鎖のガラクトース残基は完全に欠失することを明らかにした。また大腸菌で生産させたこれらの遺伝子産物が実際にα1,3-ガラクトース転移活性を示すこともわかった。以上の結果から分裂酵母に存在する新規α1,3-ガラクトース転移酵素の諸性質を明らかにすることができた。

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and β-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for β-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of β-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells.

DOI： 10.1038/s41598-024-64531-5

Web of Science

Scopus

PubMed

researchmap

Other Link： https://www.nature.com/articles/s41598-024-64531-5

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.micres.2024.127653

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2023.12.004

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2023.03.002.

Language：English   Publishing type：Research paper (scientific journal)  

N-linked oligosaccharides in the fission yeast Schizosaccharomyces pombe contain large amounts of d-galactose (Gal), which mainly comprises α1,2- and α1,3-linked Gal except for pyruvylated β1,3-linked Gal (PvGalβ) at the non-reducing end. The PvGalβ unit of N-glycans is important for regulating nonsexual flocculation and invasive growth, but the mechanistic basis for β-galactosylation in fission yeast is poorly understood. To gain insight into this mechanism, we have characterized three genes previously identified to be involved in PvGalβ biosynthesis (pvg2, pvg3, and pvg5), with a focus on pvg3, which is predicted to contain a domain conserved in galactosyltransferase family 31 (GT31) proteins. Fluorescent microscopy revealed that Pvg3 is stably localized at the Golgi membrane, regardless of the presence of pvg2+ or pvg5+, suggesting that Pvg2 and Pvg5 are essential for the function of Pvg3 as a β1,3-galactosyltransferase, and not for its localization to the Golgi. Mutation of the GT31 family DXD motif and GT-A fold in Pvg3 resulted in loss of catalytic activity in vivo, supporting the idea that Pvg3 is a GT-A type β1,3-galactosyltransferase. Docking simulations further indicated that Pvg3 can recognize donor and acceptor substrates suitable for β-(1 → 3) bond formation. Yeast two-hybrid assay showed that Pvg5 physically interacts with Pvg3 and the pyruvyltransferase Pvg1. Collectively, these results provide insight into β-galactosylation catalyzed by Pvg3 and the supporting role of Pvg5 in PvGalβ biosynthesis.

DOI： 10.1016/j.jbiosc.2023.03.002

Web of Science

Scopus

PubMed

researchmap

Language：English  

DOI： 10.1111/cbdd.14194

Web of Science

PubMed

Language：Japanese   Publisher：公益財団法人 発酵研究所  

DOI： 10.60396/iforc.37.0_134

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.jbiosc.2022.07.014.

Language：English   Publishing type：Research paper (scientific journal)  

Schizosaccharomyces japonicus is a dimorphic yeast, transiting between unicellular and hyphal growth. The glycoproteins of fission yeast contain, in addition to mannose (Man), a large number of galactose (Gal) residues. Previously, we reported that the cell-surface O-glycans of S. japonicus comprise mainly tri-saccharides (Gal-Man-Man) as a main component, in contrast to the tetra-saccharides observed in other Schizosaccharomyces species. Here we have investigated the function of cell-surface Gal residues in S. japonicus. Because disruption of gms1+, encoding the UDP-Gal transporter required for galactomannan synthesis, abolishes cell-surface galactosylation in Schizosaccharomyces pombe, we constructed a deletion mutant of the homologous gene in S. japonicus gms1Δ [gms1 (S.j)] and determined the N- and O-linked oligosaccharide structures present on the cell surface. Disruption of gms1 (S.j) resulted in a complete lack of Gal on the cell surface, indicating that Gms1 plays an essential role in supplying UDP-Gal from the cytoplasm to the Golgi lumen. Analytical microscopy of gms1Δ demonstrated that the lack of cell-surface Gal did not affect cell growth or morphology during vegetative growth. However, hyphal development was blocked in gms1Δ, even in the presence of the topoisomerase I inhibitor camptothecin, which is known to induce hyphal differentiation in wild-type S. japonicus. Collectively, these findings show that Gal-containing oligosaccharides are required for cell wall integrity during filamentous growth in S. japonicus.

DOI： 10.1016/j.jbiosc.2022.07.014

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Both pyruvylation and sialylation onto the terminus of oligosaccharides of N-glycoproteins seem to be structurally and functionally similar with a property of conferring negative charge. However, detailed molecular characteristics of pyruvylation and sialylation in vivo were elusive. Here, to investigate an effect of terminal pyruvylation to N-glycan on in vivo biodistribution and kinetics, we prepared human serum albumin (HSA) modified with pyruvylated N-glycan (PvG), conjugated with HiLyte Fluor 750 (FL750-PvGHSA). In vivo imaging by using FL750-PvGHSA revealed that terminally pyruvylated N-glycoalbumin was excreted like sialylated N-glycoalbumin, suggesting that pyruvylation mimics sialylation in in vivo biodistribution and kinetics of N-glycoproteins. Terminal pyruvylation onto N-glycans can be a potential tool for a novel glycoengineering strategy.

DOI： 10.1016/j.bmc.2022.116943

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bmc.2022.116943.

Language：English   Publishing type：Research paper (scientific journal)  

Endo-β-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

DOI： 10.1016/j.jbiosc.2022.03.011

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi.org/10.3390/cells11091519

Language：English   Publisher：Cells  

The mitotic exit network (MEN) is a conserved signalling pathway essential for the termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Instead, we identified two essential kinases OpHcd1 and OpHcd2 (homologue candidate of ScCdc15) that are homologous to SpSid1 and SpCdc7, respectively, components of the septation initiation network (SIN) of the fission yeast Schizosaccharomyces pombe. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. Unlike Cdc15 in S. cerevisiae, the association of OpHcd1 and OpHcd2 with the yeast centrosomes (named spindle pole bodies, SPBs) is restricted to the SPB in the mother cell body. SPB localisation of OpHcd2 is regulated by the status of OpTem1 GTPase, while OpHcd1 requires the polo-like kinase OpCdc5 as well as active Tem1 to ensure the coordination of mitotic exit (ME) signalling and cell cycle progression. Our study suggests that the divergence of molecular mechanisms to control the ME-signalling pathway as well as the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

DOI： 10.3390/cells11091519

Web of Science

Scopus

PubMed

Language：English  

AAA ATPases widely exist in many organisms and function in various organelles. However, there is little information about AAA ATPase functioning in endocytosis. In Aspergillus oryzae, we previously discovered a putative AAA ATPase AipA thatwould be involved in endocytosis. Here,we further examined the function of AipA and AoAbp1 in endocytosis using enhanced green fluorescent protein (EGFP)-tagged arginine permease AoCan1 as an endocytic marker. In the ΔaipA strain, endocytosis ofAoCan1-EGFPwasmore facilitated than the control strain, suggesting that AipA negatively regulates endocytosis. In contrast, in the ΔAoabp1 strain, endocytosis of AoCan1-EGFP was delayed compared with the control strain, suggesting that AoAbp1 positively functions in endocytosis. In addition, in the ΔaipAΔAoabp1 strain, endocytosis of AoCan1-EGFP was delayed. AipA localized at the endocytic collar of the hyphal tip, only in the presence of AoAbp1, suggesting AipA functions downstream of AoAbp1 in endocytosis. Moreover, we investigated the aipA-overexpressing strain, and found that endocytosis of AoCan1-EGFP was inhibited. Furthermore, we examined strains expressing <aipA>＾＾＾K542A or <aipA>＾＾＾E596Q,which decreased ATPase activity, in the backgrounds of complementation or overexpression, respectively, and found that AoCan1-EGFP endocytosis was promoted. These results suggested that AAA ATPase activity of AipA is important for its function in endocytosis.

DOI： 10.1016/j.funbio.2021.11.007

Web of Science

Scopus

PubMed

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.funbio.2021.11.007.

CiNii Research

Language：English   Publisher：Applied Microbiology and Biotechnology  

Abstract: The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive β-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support β-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. Key points: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.

DOI： 10.1007/s00253-021-11649-5

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1007/s00253-021-11649-5.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.3389/ffunb.2021.72139

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1093/glycob/cwab028

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.2323/jgam.2020.07.002.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.bbamem.2020.183507.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2020.09.006.

Language：English   Publisher：Journal of Bioscience and Bioengineering  

Although β-D-galactofuranosidases (Galf-ases) that hydrolyze β-D-galactofuranose (Galf)-containing oligosaccharides have been characterized in various organisms, to date no Galf-specific Galf-ase-encoding genes have been reported in Aspergillus fungi. Based on the amino acid sequences of previously identified bacterial Galf-ases, here we found two candidate Galf-specific Galf-ase genes AN2395 (gfgA) and AN3200 (gfgB) in the genome of Aspergillus nidulans. Indeed, recombinant GfgA and GfgB proteins exhibited Galf-specific Galf-ase activity, but no detectable α-L-arabinofuranosidase (Araf-ase) activity. Phylogenetic analysis of GfgA and GfgB orthologs indicated that there are two types of Aspergillus species: those containing one ortholog each for GfgA and GfgB; and those containing only one ortholog in total, among which Aspergillus fumigatus there is a representative with a single ortholog Galf-ase Afu2g14520. Unlike GfgA and GfgB, the recombinant Afu2g14520 protein showed higher Araf-ase activity than Galf-ase activity. An assay of substrate specificity revealed that although GfgA and GfgB are both exo-type Galf-ases and hydrolyze β-(1,5) and β-(1,6) linkages, GfgA hydrolyzes β-(1,6)-linked Galf-oligosaccharide more effectively as compared with GfgB. Collectively, our findings indicate that Galf-ases in Aspergillus species may have a role in cooperatively degrading Galf-containing oligosaccharides depending on environmental conditions.

DOI： 10.1016/j.jbiosc.2020.09.006

Scopus

Language：English  

PubMed

Language：English   Publisher：Frontiers in Fungal Biology  

Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.

DOI： 10.3389/ffunb.2021.721398

Scopus

Language：English   Publisher：Applied Microbiology and Biotechnology  

Abstract: The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74+ (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74+ gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe. Key points: • Gpp74p is involved in the localization of glycosyltransferases to the Golgi. • The cytosolic tails of glycosyltransferases are important for Golgi localization. • Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.

DOI： 10.1007/s00253-020-10881-9

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2020.578862

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.funbio.2020.06.004

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2020.06.003.

Language：English   Publisher：Journal of Bioscience and Bioengineering  

The N- and O-linked oligosaccharides from fission yeast Schizosaccharomyces pombe not only contain large amounts of D-mannose (Man) but also contain large amounts of D-galactose (Gal). Although the galactomannans of S. pombe are mainly composed of α1,2- or α1,3-linked Gals, some of the terminal α1,2-linked Gals are found to be linked to pyruvylated β1,3-linked galactose (PvGal). We have determined the structural characteristics of the N-glycans and O-glycans in three Schizosaccharomyces species (S. japonicus, S. octosporus, and S. cryophilus) using lectin blot, 1H NMR spectroscopy, and size-fractionation high performance liquid chromatography (HPLC), and found that the galactosylation of oligosaccharides was a common feature in fission yeasts. In addition, each of the terminal Galα1,2-, Galβ1,3- and non-substituted Man residues exhibited distinct characteristics. A BLAST search of gene databases in Schizosaccharomyces identified genes homologous to pvg1 encoding pyruvyltransferase of S. pombe. These genes, when expressed in an S. pombe pvg1Δ strains, led to the pyruvylation of non-reducing terminal β-linked Gal, suggesting the biosynthetic pathway of PvGal-containing oligosaccharides is highly conserved in fission yeasts.

DOI： 10.1016/j.jbiosc.2020.03.008

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.5458/jag.jag.JAG-2020_0005

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2020.03.008.

Language：English   Publisher：Biochemical and Biophysical Research Communications  

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5–7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.

DOI： 10.1016/j.bbrc.2019.12.079

Scopus

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2019.12.079.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2019.12.006.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.RA119.011817.

Language：English   Publisher：mSphere  

ABSTRACT The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/ β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatus. Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus. The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice. IMPORTANCE β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.

DOI： 10.1128/mSphere.0770-19

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Carbohydrate Research  

Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP β-D-Galf, giving a KM value of 250 μM, and the highest enzymatic efficiency ever observed of 14 mM−1 s−1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.

DOI： 10.1016/j.carres.2019.05.011

Scopus

Language：English   Publisher：Applied Microbiology and Biotechnology  

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well understood. Here, to elucidate the molecular mechanism underlying the alkaline stress response in S. pombe, we performed DNA microarray analysis. We found that a homolog of human catechol O-methyltransferase 2 (COMT2) is highly upregulated in S. pombe cells exposed to alkaline conditions. We designated the S. pombe homolog as cmt2+ and also identified its paralog, cmt1+, in the S. pombe genome. Reverse transcription PCR confirmed that both cmt1+ and cmt2+ are upregulated within 1 h of exposure to alkaline stress and downregulated within 30 min of returning to an acidic environment. Moreover, we verified that recombinant Cmt proteins exhibit catechol O-methyltransferase activity. To further characterize the expression of cmt1+ and cmt2+, we carried out an EGFP reporter assay using their promoter sequences, which showed that both genes respond not only to alkaline but also to salt stress. Collectively, our findings indicate that the cmt promoter might be an advantageous expression system for use in S. pombe under alkaline culture conditions.

DOI： 10.1007/s00253-019-09858-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.carres.2019.05.011.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00253-019-09858-0.

Language：English   Publisher：Carbohydrate Research  

β-D-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and degradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the synthesis of p-nitrophenyl β-D-galactofuranoside and its disaccharides by chemo-enzymatic methods including use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis to purify p-nitrophenyl β-D-galactofuranoside, a promising substrate for β-D-galactofuranosidase from Streptomyces species.

DOI： 10.1016/j.carres.2019.01.005

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Scientific Reports  

Endo-β-N-acetylglucosaminidase (ENGase) catalyzes hydrolysis of N-linked oligosaccharides. Although many ENGases have been characterized from various organisms, so far no fucose-containing oligosaccharides-specific ENGase has been identified in any organism. Here, we screened soil samples, using dansyl chloride (Dns)-labeled sialylglycan (Dns-SG) as a substrate, and discovered a strain that exhibits ENGase activity in the culture supernatant; this strain, named here as strain HMA12, was identified as a Sphingobacterium species by 16S ribosomal RNA gene analysis. By draft genome sequencing, five candidate ENGase encoding genes were identified in the genome of this strain. Recombinant proteins, purified from Escherichia coli expressing candidate genes ORF1152, ORF1188, ORF3046 and ORF3750 exhibited fucose-containing oligosaccharides-specific ENGase activity. These ENGases exhibited optimum activities at very acidic pHs (between pH 2.3-2.5). BLAST searches using sequences of these candidate genes identified two fungal homologs of ORF1188, one in Beauveria bassiana and the other in Cordyceps militaris. Recombinant ORF1188, Beauveria and Cordyceps ENGases released the fucose-containing oligosaccharides residues from rituximab (immunoglobulin G) but not the high-mannose-containing oligosaccharides residues from RNase B, a result that not only confirmed the substrate specificity of these novel ENGases but also suggested that natural glycoproteins could be their substrates.

DOI： 10.1038/s41598-017-17467-y

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Resource Announcements  

The genome sequence of the Bacillus sp. strain HMA207, the culture supernatant of which exhibited -D-galactosidase activity to release pyruvylated galactose (PvGal), was examined to identify a PvGal-ase-encoding gene. We report here the result of whole-genome shotgun sequencing, which revealed putative PvGal-ase genes.

DOI： 10.1128/MRA.01169-18

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.2323/jgam.2017.07.001

Language：English   Publisher：Current Microbiology  

Polyphosphate kinase 1 (Ppk1) catalyzes reverse transfer of the terminal phosphate from ATP to form polyphosphate (polyP) and from polyP to form ATP, and is responsible for the synthesis of most of cellular polyPs. When Ppk1 from Myxococcus xanthus was incubated with 0.2 mM polyP60−70 and 1 mM ATP or ADP, the rate of ATP synthesis was approximately 1.5-fold higher than that of polyP synthesis. If in the same reaction the proportion of ADP in the ATP/ADP mixture exceeded one-third, the equilibrium shifted to ATP synthesis, suggesting that M. xanthus Ppk1 preferentially catalyzed ATP formation. At the same time, GTP and GDP were not recognized as substrates by Ppk1. In the absence of polyP, Ppk1 generated ATP and AMP from ADP, and ADP from ATP and AMP, suggesting that the enzyme catalyzed the transfer of a phosphate group between ADP molecules yielding ATP and AMP, thus exhibiting adenylate kinase activity.

DOI： 10.1007/s00284-017-1391-y

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00284-017-1391-y.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/genomeA.00228-18

Scopus

Language：English   Publisher：Biochemical and Biophysical Research Communications  

Fission yeast Pik1p is one of three phosphatidylinositol 4-kinases associated with the Golgi complex, but its function is not fully understood. Deletion of pot1+ causes telomere degradation and chromosome circularization. We searched for the gene which becomes synthetically lethal with pot1Δ. We obtained a novel pik1 mutant, pik1-1, which is synthetically lethal with pot1Δ. We found phosphoinositol 4-phosphate in the Golgi was reduced in pik1-1. To investigate the mechanism of the lethality of the pot1Δ pik1-1 double mutant, we constructed the nmt-pot1-aid pik1-1 strain, where Pot1 function becomes low by drugs, which leads to telomere loss and chromosome circularization, and found pik1-1 mutation does not affect telomere resection and chromosome circularization. Thus, our results suggest that pik1+ is required for the maintenance of circular chromosomes.

DOI： 10.1016/j.bbrc.2018.02.001

Scopus

Language：English   Publisher：Genome Announcements  

The genome sequence of the soil bacterium Sphingobacterium sp. strain HMA12, the culture supernatant of which exhibited endo-β-N-acetylglucosaminidase (ENGase) activity, was examined for ENGase-encoding genes. Here, we report the characterization of new genes of ENGases, obtained by whole-genome shotgun sequencing, that are capable of specifically hydrolyzing fucose-containing oligosaccharides.

DOI： 10.1128/genomeA.01525-17

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2018.02.001.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/genomeA.01525-17.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2017.08.008.

Language：English   Publisher：Journal of Bioscience and Bioengineering  

The molecular mechanism of tolerance to alkaline pH is well studied in model fungi Aspergillus nidulans and Saccharomyces cerevisiae. However, how fission yeast Schizosaccharomyces pombe survives under alkaline stress remains largely unknown, as the genes involved in the alkaline stress response pathways of A. nidulans and S. cerevisiae were not found in the genome of this organism. Since uptake of iron and copper into cells is important for alkaline tolerance in S. cerevisiae, here we examined whether iron and copper uptake processes were involved in conferring tolerance to alkaline stress in S. pombe. We first revealed that S. pombe wild-type strain could not grow at a pH higher than 6.7. We further found that the growths of mutants harboring disruption in the iron uptake-related gene frp1+, fio1+ or fip1+ were severely inhibited under ambient pH stress condition. In contrast, derepression of these genes, by deletion of their repressor gene fep1+, caused cells to acquire resistance to pH stress. Together, these results suggested that uptake of iron is essential for ambient pH tolerance in S. pombe. We also found that copper is required for the pH stress response because disruptants of ctr4+, ctr5+, ccc2+ and cuf1+ genes, all of which are needed for regulating intracellular Cu+, displayed ambient pH sensitivity. Furthermore, supplementing Fe2+ and Cu2+ ions to the culture media improved growth under ambient pH stress. Taken together, our results suggested that uptake of iron and copper is the crucial factor needed for the adaptation of S. pombe to ambient pH stress.

DOI： 10.1016/j.jbiosc.2017.08.008

Scopus

Language：English   Publisher：Scientific Reports  

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.

DOI： 10.1038/s41598-017-16284-7

Scopus

Language：English   Publisher：Scientific Reports  

Recent research in filamentous fungi has revealed that the motility of an endocytic organelle early endosome (EE) has a versatile role in many physiological functions. Here, to further examine the motility of EEs in the industrially important fungus Aspergillus oryzae, we visualized these organelles via the Rab5 homolog AoRab5 and identified AoHok1, a putative linker protein between an EE and a motor protein. The Aohok1 disruptant showed retarded mycelial growth and no EE motility, in addition to an apical accumulation of EEs and peroxisomes. We further demonstrated that the Aohok1 disruptant exhibited less sensitivity to osmotic and cell wall stresses. Analyses on the protein secretory pathway in ΔAohok1 cells showed that, although distribution of the endoplasmic reticulum and Golgi was not affected, formation of the apical secretory vesicle cluster Spitzenkörper was impaired, probably resulting in the observed reduction of the A. oryzae major secretory protein α-amylase. Moreover, we revealed that the transcript level of α-amylase-encoding gene amyB was significantly reduced in the Aohok1 disruptant. Furthermore, we observed perturbed conidial and sclerotial formations, indicating a defect in cell differentiation, in the Aohok1 disruptant. Collectively, our results suggest that EE motility is crucial for α-amylase production and cell differentiation in A. oryzae.

DOI： 10.1038/s41598-017-16163-1

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2017.08.008.

Language：English   Publisher：Biochemical and Biophysical Research Communications  

Acyl-CoA binding protein (ACBP) plays important roles in the metabolism of lipids in eukaryotic cells. In the industrially important filamentous fungus Aspergillus oryzae, although we have previously demonstrated that the A. oryzae ACBP (AoACBP) localizes to punctate structures and exhibits long-range motility, which is dependent on autophagy-related proteins, the physiological role of AoACBP remains elusive. Here, we describe identification and characterization of another ACBP from A. oryzae; we named this ACBP as AoAcb2 and accordingly renamed AoACBP as AoAcb1. The deduced amino acid sequence of AoAcb2 lacked a signal peptide. Phylogenetic analysis classified AoAcb2 into a clade that was same as the ACBP Acb1 of the model yeast Saccharomyces cerevisiae, but was different from that of AoAcb1. In contrast to punctate localization of AoAcb1, AoAcb2 was found to be dispersedly distributed in the cytoplasm, as was previously observed for the S. cerevisiae Acb1. Since we could not generate an Aoacb2 disruptant, we created an Aoacb2 conditional mutant that exhibited less growth under Aoacb2-repressed condition, suggesting that Aoacb2 is an essential gene for growth. Moreover, we observed that A. oryzae AoAcb2, but not A. oryzae AoAcb1, was secreted under carbon-starved condition, suggesting that AoAcb2 might be secreted via the unconventional protein secretion (UPS) pathway, just like S. cerevisiae Acb1. We also demonstrated that the unconventional secretion of AoAcb2 was dependent on the t-SNARE AoSso1, but was independent of the autophagy-related protein AoAtg1, suggesting that the unconventional secretion of AoAcb2, unlike that of S. cerevisiae Acb1, via the UPS pathway, is not regulated by the autophagy machinery. Thus, the filamentous fungus A. oryzae harbors two types of ACBPs, one of which appears to be essential for growth and undergoes unconventional secretion.

DOI： 10.1016/j.bbrc.2017.08.166

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-017-16163-1.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-017-16284-7.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2017.04.015

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Genome Announcements  

The genome sequence of Streptomyces sp. strain JHA26, the culture supernatant of which exhibited β-D-galactofuranosidase (Galf-ase) activity, was analyzed to search for a Galf-ase-encoding gene. We report here the results of wholegenome shotgun sequencing and reveal the identity of a new Galf-ase gene.

DOI： 10.1128/genomeA.00190-17

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Glycobiology  

Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-D-galactofuranose as a sugar donor, and p-nitrophenyl-β-Dgalactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the β-galactofuranose residue via a β-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-D-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the δAfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of β1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-D-galactofuranose: β-D-galactofuranoside β1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.

DOI： 10.1093/glycob/cwx028

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09168451.2017.1300518.

Language：English   Publisher：Biotechnology Letters  

Objectives: To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities. Results: Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline’s stability and Endo-CC’s transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 μg GlcNAc-RNase B, 200 μg SG-oxazoline and 3 μg Endo-CCN180H in 20 μl 20 mM Tris/HCl pH 7.5 at 30 °C for 30–60 min. Conclusions: This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

DOI： 10.1007/s10529-016-2230-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2016.10.018.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

DOI： 10.1080/09168451.2017.1394813

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

As a constituent of polysaccharides and glycoconjugates, β-D-galactofuranose (Galf) exists in several pathogenic microorganisms. Although we recently identified a β-D-galactofuranosidase (Galf-ase) gene, ORF1110, in the Streptomyces strain JHA19, very little is known about the Galf-ase gene. Here, we characterized a strain, named JHA26, in the culture supernatant of which exhibited Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) as a substrate. Draft genome sequencing of the JHA26 strain revealed a putative gene, termed ORF0643, that encodes Galf-ase containing a PA14 domain, which is thought to function in substrate recognition. The recombinant protein expressed in Escherichia coli showed the Galf-specific Galf-ase activity and also released galactose residue of the polysaccharide galactomannan prepared from Aspergillus fumigatus, suggesting that this enzyme is an exo-type Galf-ase. BLAST searches using the amino acid sequences of ORF0643 and ORF1110 Galf-ases revealed two types of Galf-ases in Acti-nobacteria, suggesting that Galf-specific Galf-ases may exhibit discrete substrate specificities.

DOI： 10.1080/09168451.2017.1300518

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Archives of Biochemistry and Biophysics  

Myxococcus xanthus lysyl-tRNA synthetase (LysS) produces diadenosine tetraphosphate (Ap4A) from ATP in the presence of Mn2+; in the present study, it also generated Ap4 from ATP and triphosphate. When ATP and Ap4 were incubated with LysS and pyrophosphatase, first Ap4A, Ap5A, and ADP, and then Ap5, Ap6A, and Ap3A were generated. The results suggest that in the first step, LysS can form lysyl-AMP and lysyl-ADP intermediates from Ap4 and release triphosphate and diphosphate, respectively, whereas in the second step, it can produce Ap5 from lysyl-ADP with triphosphate, and Ap6A from lysyl-ADP with Ap4. In addition, in the presence of Ap4 and pyrophosphatase, but absence of ATP, LysS also generates diadenosine oligophosphates (ApnAs: n = 3–6). These results indicate that LysS has the ability to catalyze the formation of various ApnAs from Ap4 in the presence of pyrophosphatase. Furthermore, the formation of Ap4A by LysS was inhibited by tRNALys in the presence of 1 mM ATP. To the best of our knowledge, this is the first report of Ap5A and Ap6A synthesis by lysyl-tRNA synthetase.

DOI： 10.1016/j.abb.2016.07.002

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Scientific Reports  

Pyruvylation onto the terminus of oligosaccharide, widely seen from prokaryote to eukaryote, confers negative charges on the cell surface and seems to be functionally similar to sialylation, which is found at the end of human-Type complex oligosaccharide. However, detailed molecular mechanisms underlying pyruvylation have not been clarified well. Here, we first determined the crystal structure of fission yeast pyruvyltransferase Pvg1p at a resolution of 2.46 Å. Subsequently, by combining molecular modeling with mutational analysis of active site residues, we obtained a Pvg1p mutant (Pvg1p H168C) that efficiently transferred pyruvyl moiety onto a human-Type complex glycopeptide. The resultant pyruvylated human-Type complex glycopeptide recognized similar lectins on lectin arrays as the α2,6-sialyl glycopeptides. This newly-generated pyruvylation of human-Type complex oligosaccharides would provide a novel method for glyco-bioengineering.

DOI： 10.1038/srep26349

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Genome Announcements  

To comprehensively identify glycosyl hydrolase genes in the genome of Bacillus clausii strain AKU0647, which produces endo- β-N-acetylglucosaminidase A (Endo-A), we conducted whole-genome shotgun sequencing. We identified several other putative glycosyl hydrolase genes apart from the Endo-A gene, and report these findings here.

DOI： 10.1128/genomeA.00310-16

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：PLoS ONE  

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms. Copyright:

DOI： 10.1371/journal.pone.0137230

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：PLoS ONE  

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3⁺-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3⁺ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3⁺-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles. Copyright:

DOI： 10.1371/journal.pone.0130542

Scopus

Language：English   Publisher：Archives of Biochemistry and Biophysics  

Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap<inf>4</inf>A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn²⁺ for the synthesis of Ap<inf>4</inf>A. Surprisingly, Zn²⁺ inhibited enzyme activity in the presence of Mn²⁺. When incubated with ATP, Mn²⁺, lysine, and inorganic pyrophosphatase, LysS first produced Ap<inf>4</inf>A and ADP, then converted Ap<inf>4</inf>A to diadenosine triphosphate (Ap<inf>3</inf>A), and finally converted Ap<inf>3</inf>A to ADP, the end product of the reaction. Recombinant LysS retained Ap<inf>4</inf>A synthase activity without lysine addition. Additionally, when incubated with Ap<inf>4</inf>A (minus pyrophosphatase), LysS converted Ap<inf>4</inf>A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported.

DOI： 10.1016/j.abb.2015.05.014

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Biochemical and Biophysical Research Communications  

Abstract The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells.

DOI： 10.1016/j.bbrc.2015.02.031

Scopus

Language：English   Publisher：Journal of Biochemistry  

Myxococcus xanthus has two bacterial protein-tyrosine (BY) kinases, BtkA and BtkB. Autophosphorylation in C-terminal tyrosine-rich clusters and poly(Glu, Tyr) kinase activities of cytoplasmic catalytic domains of BtkA and BtkB were activated by the intracellular juxtamembrane regions of the second transmembrane helices. Protein kinase activity against poly(Glu, Tyr) of cytoplasmic fragment of BtkB (CF-BtkB) containing an activator region was not inhibited by serine/threonine protein kinase inhibitors. However, addition of tyrosine protein kinase inhibitors, genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), at a concentration of 0.2 mM, inhibited the CF-BtkB kinase activity by 20 and 64%, respectively. A CF-BtkB mutant constructed by replacing all C-terminal tyrosine residues with phenylalanines, did not undergo autophosphorylation. Further, this mutation did not significantly affect poly(Glu, Tyr) kinase activity, suggesting that M. xanthus BtkB kinase activity is not dependent on autophosphorylation in the C-terminal tyrosine cluster. A conserved motif (ExxRxxR) of BY kinases is involved in the self-association of catalytic domains of BY kinases, necessary to accomplish trans-phosphorylation. An ExxRxxR motif mutant of CF-BtkB led to loss of autophosphorylation and poly(Glu, Tyr) kinase activities. These observations provide insights into the regulation mechanism of M. xanthus BY kinase activity.

DOI： 10.1093/jb/mvv053

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Genome Announcements  

By screening for microbes that exhibit β-D-galactofuranosidase (Galf-ase) activity, a Streptomyces sp. strain, named JHA19, was isolated from a soil sample from Kagawa University, Japan, in 2010. Here, we report the results of whole-genome shotgun sequencing and found that the strain has four predicted Galf-ase genes.

DOI： 10.1128/genomeA.01171-15

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：FEMS Yeast Research  

The cell surface of Schizosaccharomyces pombe is negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell-cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1+, pet2+, and pet3+) in S. pombe, identified by sequence homology search against the Arabidopsis thaliana PEP transporter AtPPT1. Schizosaccharomyces pombe strain carrying a disruption in pet1+ (pet1Δ) or in pet2+ (pet2Δ), but not the strain carrying a disruption in pet3+ (pet3Δ), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed the pet1Δ phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed the pet1Δ phenotype. Furthermore, the reduction in cell surface negative charge in pet1Δ cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi.

DOI： 10.1111/1567-1364.12207

Scopus

Language：English   Publisher：FEBS Letters  

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap<inf>4</inf>A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap<inf>n</inf>A and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap<inf>4</inf>A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap<inf>4</inf>A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap<inf>n</inf>A and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap<inf>n</inf>A hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap<inf>n</inf>A hydrolase, respectively.

DOI： 10.1016/j.febslet.2014.07.031

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Biochemistry  

A Myxococcus xanthus DspA contains 12 subdomains characteristic of eukaryotic-like protein kinases but with an atypical sequence, RDxSPHN, in the catalytic loop, different from the consensus motifs observed in Ser/Thr kinases (RDxKxxN) or Tyr kinases (RDx(A/R)A(A/R)N). DspA phosphorylated myelin basic protein (MBP) on Ser and Thr residues. Mutations of the SPHN motif within the catalytic loop to KPHN or KPEN for Ser/Thr kinases, AARN for Tyr kinases and TPHN or TSHN for Dictyostelium Tyr kinases markedly reduced autophosphorylation and kinase activities. Phosphorylation assays, Western blot analysis and mutational analysis revealed that DspA is a dual-specificity kinase that autophosphorylates on two Thr residues (Thr-199 and Thr-201) in the activation loop and two Tyr residues (Tyr-35 and Tyr-111). RD kinases such as DspA are activated by phosphorylation in the activation loop. Replacement of Thr-199 or/and Thr-201 in the DspA activation loop by alanine also almost abolished autophosphorylation and kinase activities. In addition, mutation of either Tyr-35 or Tyr-111 to phenylalanine decreased kinase activities against MBP, and double mutation abolished kinase activity. These results suggested that DspA is activated by dual autophosphorylation of Thr residues in the activation loop, and autophosphorylation on two Tyr residues of DspA are required for high-level kinase activity. © 2013 The Authors.

DOI： 10.1093/jb/mvt101

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Molecular Microbiology  

Summary: The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of β-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitroGalf antigen synthase assay revealed that GfsA has β1,5- or β1,6-galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-d-Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β-galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O-glycans in the Golgi. © 2013 John Wiley & Sons Ltd.

DOI： 10.1111/mmi.12416

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

DOI： 10.1271/bbb.130387

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

In the fission yeast Schizosaccharomyces pombe, the gld1 + gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 + was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1-EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source. © 2013 Springer-Verlag Berlin Heidelberg.

DOI： 10.1007/s00253-013-4812-2

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

Schizosaccharomyces pombe has eight hexose transporter genes, ght1 + to ght8 +. Here we report that ght2 +, which is highly expressed in the presence of glucose, is essential for UDP-galactose synthesis from extracellular galactose when cells grow on glucose. The galactosylation defect of a uge1Δ mutant defective in synthesis of UDP-galactose from glucose was suppressed in galactose-containing medium, but disruption of ght2 + in the uge1Δ mutant reversed suppression of the galactosylation defect. Expression of Saccharomyces cerevisiae GAL2 in uge1Δght2Δ cells suppressed the defective galactosylation phenotype in galactose-containing medium. These results indicate that galactose is transported from the medium to the cytosol in a Ght2-dependent manner, and is then converted into UDP-galactose. © 2012 Springer-Verlag Berlin Heidelberg.

DOI： 10.1007/s00253-012-4637-4

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Nucleic Acids Research  

The Schizosaccharomyces pombe genome is one of the smallest among the free-living eukaryotes. We further reduced the S. pombe gene number by large-scale gene deletion to identify a minimal gene set required for growth under laboratory conditions. The genome-reduced strain has four deletion regions: 168.4 kb in the left arm of chromosome I, 155.4 kb in the right arm of chromosome I, 211.7 kb in the left arm of chromosome II and 121.6 kb in the right arm of chromosome II. The deletions corresponded to a loss of 223 genes of the original ∼5100. The quadruple-deletion strain, with a total deletion size of 657.3 kb, showed a decreased ability to uptake glucose and some amino acids in comparison with the parental strain. The strain also showed increased gene expression of the mating pheromone M-factor precursor and the nicotinamide adenine dinucleotide phosphate -specific glutamate dehydrogenase. There was also a 2.7-fold increase in the concentration of cellular adenosine triphosphate, and levels of the heterologous proteins, enhanced green fluorescent protein and secreted human growth hormone were increased by 1.7- and 1.8-fold, respectively. The transcriptome data from this study have been submitted to the Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE38620 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token= vjkxjewuywgcovc&acc=GSE38620). © 2013 The Author(s) 2013.

DOI： 10.1093/nar/gkt233

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：AMB Express  

Organohalide-respiring Desulfitobacterium strains are believed to play an important role in the bioremediation and natural attenuation of chlorinated aliphatic and aromatic hydrocarbons. However, several studies have reported that chloroform significantly inhibits microbial reductive dechlorination of chloroethene. In this study, we examined the effect of chloroform on several Desulfitobacterium strains, including ortho-chlorophenol-dechlorinating Desulfitobacterium dehalogenans JW/IU-1 and Desulfitobacterium hafniense DCB-2, and also the chloroethene-dechlorinating strain D. hafniense TCE1. In medium containing 3-chloro-4-hydroxyphenylacetate as an electron acceptor, chloroform inhibited the growth of strains JW/IU-1 and DCB-2. Although chloroform did not directly inhibit dechlorination of 3-chloro-4-hydroxyphenylacetate by resting cells, cells cultivated with chloroform showed decreased dechlorination activity. Moreover, transcription of the gene encoding the reductive dehalogenase CprA decreased significantly in cells cultivated with chloroform. These results indicate that chloroform inhibits the growth and dechlorination activity of strains JW/IU-1 and DCB-2 via inhibition of cprA transcription. In contrast, cultivation of strain TCE1 in the presence of chloroform gave rise to a PceA reductive dehalogenase gene-deletion variant of strain TCE1; a similar phenomenon was observed in our previous study of chloroethene-dechlorinating D. hafniense strain Y51. Our results suggest that chloroform extensively inhibits the dechlorination activity of Desulfitobacterium strains, and that the inhibitory mechanism appears to differ between ortho-chlorophenol dechlorinators and chloroethene dechlorinators. © 2013 Bel-Rhlid et al.

DOI： 10.1186/2191-0855-3-30

Scopus

Language：English   Publisher：Journal of Biological Chemistry  

The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of D-galactose (Gal) in addition to D-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1+-otg3 + (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2+ gene product had Gal transfer activity toward a pyridylaminated Man 9GlcNAc2 oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3 + gene product exhibited Gal transfer activity toward the pyridylaminated Man9GlcNAc2 oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, 1H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3- galactosylation of S. pombe oligosaccharides. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M112.347351

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Microbiology (United Kingdom)  

Protein-tyrosine phosphorylation plays a significant role in multiple cellular functions in bacteria. Bacterial tyrosine phosphatases catalyse the dephosphorylation of tyrosyl-phosphorylated proteins. Myxococcus xanthus PhpA shares homology with DNA polymerase and histidinol phosphatase family members. Recombinant His-tagged PhpA requires Mn2+ or Co2+ for phosphatase activity, and shows strict specificity for phosphorylated tyrosine residues. The km values of PhpA for p-nitrophenyl phosphate (pNPP) and phosphotyrosine peptide, RRLIEDAEpYAARG, were 803 and 139 μM, respectively. The phosphatase activity of PhpA was inhibited by sodium orthovanadate with a ki of 33 μM. phpA gene expression was observed under both vegetative and developmental conditions, but peaked during late fruiting body formation. A phpA mutant exhibited an elevated level of tyrosine phosphorylation of a 79 kDa protein and cytoplasmic tyrosine kinase, BtkA. In M. xanthus, exopolysaccharide (EPS) is essential for cell-cell adhesion and fruiting body formation. phpA mutant cells exhibited enhanced capacity for cell- cell agglutination in agglutination buffer. Under starvation conditions, phpA mutation caused early aggregation and sporulation. The EPS production assay showed that the phpA mutant produced an increased amount of EPS in comparison with the wild-type. These results indicate that PhpA may negatively regulate the production of EPS in M. xanthus. © 2012 SGM.

DOI： 10.1099/mic.0.059824-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Biochemistry  

The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg 2+-or Mn2+-dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal-or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound. © 2012 The Authors.

DOI： 10.1093/jb/mvs067

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

The fission yeast Schizosaccharomyces pombe does not grow in media containing glycerol as a sole carbon source but uses glycerol in the presence of ethanol. Ethanol, but not glycerol, triggered upregulation of gld1 + and fbp1 + during glucose starvation even though gld1 + and fbp1 + are essential for growth on glycerol. This upregulation occurred at a very low concentration of ethanol. The transcriptional regulation of gld1 + was tested in the presence of various alcohols, and both ethanol and 1-propanol were found to induce gld1 + and to support growth in glycerol-containing media. We suggest that S. pombe has a novel ethanol and/or 1-propanol recognition mechanism that upregulates glycerol utilization during glucose starvation. © Springer-Verlag 2012.

DOI： 10.1007/s00253-012-3971-x

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Microbiology  

In Saccharomyces (Sacc.) cerevisiae, the final step of the complex sphingolipid biosynthetic pathway requires Ipt1p for synthesis of mannosyldiinositol phosphorylceramide [M(IP)2C]. No fission yeast equivalent to Ipt1p has been found in the Schizosaccharomyces (Schiz.) pombe genome, and the most abundant complex sphingolipid is mannosylinositol phosphorylceramide. To examine the effect of expressing Sacc. cerevisiae IPT1 (ScIPT1) in Schiz. pombe, the ScIPT1 gene was cloned into an inducible fission yeast integrative vector and expressed in wild-type Schiz. pombe. In the Schiz. pombe ScIPT1-expressing cells, M(IP)2C was detected, indicating that ScIpt1p functions in M(IP)2C synthesis in Schiz. pombe. Expression of ScIPT1 caused pleiotropic phenotypes, including aberrant morphology and mislocalization of ergosterols in the plasma membrane. Furthermore, growth of Schiz. pombe was severely impaired. We analysed the sphingolipid composition of ScIPT1-expressing cells following a prolonged lag phase, and found that M(IP)2C was not synthesized, indicating that Ipt1p had been inactivated. GFP-tagged ScIpt1 localized primarily in the Golgi apparatus in wild-type Schiz. pombe. Over time, ScIpt1p was eventually transported to the vacuolar lumen through the multivesicular body pathway. These results indicate that M(IP)2C is toxic to Schiz. pombe and that fission yeast possesses an unknown mechanism to effectively extrude toxic sphingolipids from cells. © 2012 SGM.

DOI： 10.1099/mic.0.056184-0

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The functions of two Schizosaccharomyces pombe Vps9- like genes, SPBC4F6.10/vps901+ and SPBC29A10.11c/ vps902+, were characterized. Genomic sequence analysis predicted that Vps901p contains a VPS9 domain, whereas cDNA analyses revealed that Vps901p contains a CUE domain (coupling of ubiquitin to ER degradation) in its C-terminal region. Deletion of vps901+ resulted in mis-sorting and secretion of S. pombe vacuolar carboxypeptidase Cpy1p, whereas deletion of vps902+ had no effect, suggesting that only Vps901p functions in vacuolar protein transport in S. pombe. Deletion of vps901+ further produced pleiotropic phenotypes, including vacuolar homotypic fusion and endocytosis defects. Heterologous expression of the budding yeast VPS9 gene corrected the CPY mis-sorting defect in vps901Δ cells. These findings suggest that the VPS9 domain of Vps901p is required for vacuolar protein trafficking in S. pombe.

DOI： 10.1271/bbb.110609

Scopus

Language：English   Publisher：Autophagy  

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Microbiology  

In Schizosaccharomyces pombe, neither intracellular sorting nor ubiquitination of amino acid permeases is well understood. In the present study, we show that intracellular sorting of the amino acid permease Aat1p in S. pombe depends on the presence of a nitrogen source in the growth medium. Under nitrogen-sufficient conditions, Aat1p appeared to be stably localized at the Golgi apparatus. In contrast, under nitrogen-insufficient conditions, Aat1p was sorted to the plasma membrane. Over time, plasma membrane-localized Aat1p was internalized and sorted to the lumen of the vacuole, where it was degraded. Sorting of Aat1p to the vacuolar lumen was dependent on the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular body. S. pombe has three genes (pub1 +, pub2 + and pub3 +) that are homologous to the ubiquitin ligase RSP5. Under nitrogen-sufficient conditions, Aat1-GFP was missorted to the plasma membrane in pub1D cells and ubiquitinated Aat1p was not detected. These results suggest that Pub1p-mediated ubiquitination is required for retention of Aat1 at the Golgi under nitrogen-sufficient conditions. The Aat1p lysine mutant Aat1 K18, 26, 27 was completely missorted to the plasma membrane under nitrogen-rich conditions. Furthermore, Aat1 K4, 18R, Aat1 K4, 26, 27R and Aat1 K18, 26, 27K mutants were severely blocked in endocytosis. These results indicate that ubiquitination is an important determinant for localization and regulation of the Aat1p permease in S. pombe. © 2012 SGM.

DOI： 10.1099/mic.0.053389-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPYY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPYY*was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPYY*was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY*degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells. © 2011 Springer-Verlag.

DOI： 10.1007/s00253-011-3662-z

Scopus

Language：English   Publisher：Eukaryotic Cell  

The fission yeast Schizosaccharomyces pombe exhibits invasive growth and nonsexual flocculation in response to nitrogen limitation. Gsf2, a flocculin of fission yeast, is required not only for nonsexual flocculation but also for invasive growth through the recognition of galactose residues on cell surface glycoconjugates. We found that pyruvylation negatively regulates nonsexual flocculation by capping the galactose residues of N-linked galactomannan. We investigated whether pyruvylation also regulates invasive growth. The pvg4 + gene originally was isolated as a multicopy suppressor of apvg4 mutant defective in the pyruvylation of N-linked oligosaccharides. However, we did not detect a defect in cell surface pyruvylation in the pvg4/mbx2 deletion mutant, as assessed by alcian blue staining and a Q-Sepharose binding assay. Instead, the deletion prevented invasive growth under conditions of low nitrogen and high glucose, and it reduced the adhesion and flocculation of otherwise flocculent mutants by reducing gsf2 + expression. mbx2 +-overexpressing strains exhibited nonsexual and calcium-dependent aggregation, which was inhibited in the presence of galactose but mediated by the induction ofgsf2 +. These findings indicate that Mbx2 mediates invasive growth and flocculation via the transcriptional activation ofgsf2 + in fission yeast. In addition, we found that fission yeast Mbx2 induces the nonsexual flocculation of budding yeast by the activation of FLO1. © 2012, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/EC.05276-11

Scopus

Language：English   Publisher：Trends in Glycoscience and Glycotechnology  

In the fission yeast Schizosaccharomyces pombe, galactose residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. Although galactose residues are not essential for growth of S. pombe, the galactosylation of protein is required for maintenance of normal cell shape, sexual agglutination, and tolerance toward various drugs. Cell surface glycoproteins play a key role in flocculation and filamentous invasive growth in yeasts. We identified fission yeast gsf2+, encoding a flocculin that binds to galactose residues located on cell surface glycoconjugates. Flocculation and invasive growth of S. pombe is tightly controlled by gsf2+ expression. Furthermore, pyruvylation of galactose residues negatively regulates flocculation by capping galactose residues in N-linked galactomannan. S. pombe appears to have a unique galactose-specific recognition system in which Gsf2p/flocculin plays an essential role in mediating cell-cell interactions. ©2011 FCCA.

DOI： 10.4052/tigg.24.24

Scopus

Language：English   Publisher：Molecular Microbiology  

Summary: 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. This non-sexual flocculation was Ca 2+-dependent, and was inhibited by addition of galactose, but not by mannose, glucose or sucrose. In the FLO8-overexpressing strain, a gene designated gsf2 + (galactose-specific flocculation) was specifically induced. The gsf2 + gene was also highly expressed in lkh1Δ, tup12Δ and gsf1 mutants, all of which exhibited non-sexual flocculation dependent on gsf2 +. We show that the N-terminal region of Gsf2 recognizes galactose in mediating cell-cell interaction. Disruption of gsf2 + also abolished the adhesion phenotype and invasive growth of the wild-type strain cultured in low ammonium medium. The newly identified flocculin Gsf2 in fission yeast was not only required for non-sexual flocculation but was also required for adhesion and filamentous growth through recognition of galactose residues on cell surface glycoconjugates. © 2011 Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2958.2011.07908.x

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Eukaryotic Cell  

The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications. © 2011, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/EC.05224-11

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

A microbial consortium that reductively dechlorinates trichloroethene, cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC) to ethene with methanogenesis was enriched from chloroethene-contaminated soil from Japan. Dechlorination activity was maintained for over 4 years. Using quantitative polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis targeting the "Dehalococcoides" 16S rRNA gene, four strains were detected. Their growth and dechlorination activities were classified into two types: one that grows by converting cis-DCE to ethene and the other that grows by converting cis-DCE to VC. Then, the vcrA and bvcA genes encoding cis-DCE/VC reductive dehalogenases were detected. Inhibitors of methanogenesis (2-bromoethanesulfonate) and sulfidogenesis (molybdate) led to accumulation of cis-DCE and of VC respectively. These results suggest that methanogens and sulfate-reducing bacteria can play a significant role in dechlorination by "Dehalococcoides".

DOI： 10.1271/bbb.110028

Scopus

Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Bacteriology  

A protein phosphatase, designated Pph3, from Myxococcus xanthus showed the enzymatic characteristics of PP2C-type serine/threonine protein phosphatases, which are metal ion-dependent, okadaic acid-insensitive protein phosphatases. The pph3 mutant under starvation conditions formed immature fruiting bodies and reduced sporulation. © 2011, American Society for Microbiology.

DOI： 10.1128/JB.01357-10

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins. © 2010 Springer-Verlag.

DOI： 10.1007/s00253-010-3031-3

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et ah, Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22A cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

DOI： 10.1271/bbb.100747

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Bioscience and Bioengineering  

The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase δ variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7+), SPBPB2B2.12c (gal10+) and SPBPB2B2.13 (gal1+), homologous to Saccharomyces cerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7δ strain, carrying an integrated ura4+ marker at the gal7+ locus, grew on 5-fluoroorotic acid (5-FOA)-containing medium. In contrast, the FG2-8 gal7δ strain could not grow on 5-FOA medium. In addition, expression of gal7+, SPBPB2B2.13, gal10+ and gal1+ genes increased in the wild-type strain when carried on a vector, and these transformants grew on galactose medium. We suggest that gal7+, gal10+, and gal1+ are localized close to a chromosomal terminal repressed by gene silencing in S. pombe. In contrast, gene silencing was defective in the FG2-8 strain making galactose assimilation possible. © 2010 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2010.10.007

Scopus

Language：English   Publisher：Plant Physiology  

In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P2 from PtdIns 3-P, and functions in vacuole/ lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis. © 2010 American Society of Plant Biologists.

DOI： 10.1104/pp.110.167981

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Biotechnology  

The early stages of N-linked glycosylation are highly conserved between fungal and mammalian cells. Such N-linked oligosaccharides are synthesized through the ordered assembly of a dolichyl pyrophosphate (Dol-PP)-linked Glc3Man9GlcNAc2 structure by the sequential actions of several glycosyltransferases located in the endoplasmic reticulum (ER). Of the glycosyltransferase genes, Saccharomyces cerevisiae ALG3 has been identified to encode the Dol-P-Man:Man5GlcNAc2-PP-Dol α1,3-mannosyltransferase, and an alg3 mutant has been shown to accumulate an Endo H-resistant M5B (Manα1,2-Manα1,2-Manα1,3(Manα1,6-)-Manβ1,4-GlcNAcβ1,4-GlcNAc) structure. Although Schizosaccharomyces pombe contains a homolog of the ALG3 gene (SPAC7D4.06c), the role of this gene in oligosaccharide biosynthesis is not at all clear. In this study, we deleted the alg3+ gene in the och1Δ mutant and analyzed the detailed oligosaccharide structures in alg3Δoch1Δ double mutant. The oligosaccharides were prepared from cell-surface glycoproteins by hydrazinolysis and fluorescent labeling with 2-aminopyridine. The labeled oligosaccharides were analyzed by high performance liquid chromatography, in combination with sequential glycosidase digestion and methylation analysis. These analyses revealed that the N-linked oligosaccharides of S. pombe alg3Δoch1Δ cells mainly consisted of two or three α-galactose-capped M5B structures. Finally, western blot analysis of recombinant human transferrin suggested that heterologously expressed glycoproteins in alg3Δoch1Δ cells have Endo H-resistant N-linked oligosaccharide structures similar to those of alg3Δoch1Δ cell-surface glycoproteins. © 2010 Elsevier B.V.

DOI： 10.1016/j.jbiotec.2010.09.942

Scopus

Language：English   Publisher：Yeast  

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast. © 2010 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.1794

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：FEBS Letters  

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids. © 2010 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2010.04.012

Scopus

Language：English   Publisher：Journal of Cell Science  

In Saccharomyces cerevisiae, three classes of sphingolipids contain myo-inositol - inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP) 2C]. No fission yeast equivalent of Ipt1p, the inositolphosphotransferase that synthesizes M(IP)2C from MIPC, has been found in the Schizosaccharomyces pombe genome. Analysis of the sphingolipid composition of wild-type cells confirmed that MIPC is the terminal and most abundant complex sphingolipid in S. pombe. Three proteins (Sur1p, Csg2p and Csh1p) have been shown to be involved in the synthesis of MIPC from IPC in S. cerevisiae. The S. pombe genome has three genes (SPAC2F3.01, SPCC4F11.04c and SPAC17G8.11c) that are homologues of SUR1, termed imt1+, imt2 + and imt3+, respectively. To determine whether these genes function in MIPC synthesis in S. pombe, single and multiple gene disruptants were constructed. Single imt disruptants were found to be viable. MIPC was not detected and IPC levels were increased in the triple disruptant, indicating that the three SUR1 homologues are involved in the synthesis of MIPC. GFP-tagged Imt1p, Imt2p and Imt3p localized to Golgi apparatus membranes. The MIPC-deficient mutant exhibited pleiotropic phenotypes, including defects in cellular and vacuolar morphology, and in localization of ergosterols. MIPC seemed to be required for endocytosis of a plasma-membrane-localized amino acid transporter, because sorting of the transporter from the plasma membrane to the vacuole was severely impaired in the MIPC-deficient mutant grown under nitrogen-limiting conditions. These results suggest that MIPC has multiple functions not only in the maintenance of cell and vacuole morphology but also in vesicular trafficking in fission yeast.

DOI： 10.1242/jcs.059139

Scopus

Language：English   Publisher：Cancer Research  

The structure of O-glycosylated proteins is altered in breast cancer cells, but the mechanisms of such an aberrant modification have been largely unknown. We here report critical roles of a novel druggable target, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Knockdown of GALNT6 by small interfering RNA significantly enhanced cell adhesion function and suppressed the growth of breast cancer cells. Western blot and immunostaining analyses indicated that wild-type GALNT6 protein could glycosylate and stabilize an oncoprotein mucin 1 (MUC1), which was upregulated with GALNT6 in breast cancer specimens. Furthermore, knockdown of GALNT6 or MUC1 led to similar morphologic changes of cancer cells accompanied by the increase of cell adhesion molecules β-catenin and E-cadherin. Our findings implied that overexpression of GALNT6 might contribute to mammary carcinogenesis through aberrant glycosylation and stabilization of MUC1 and that screening of GALNT6 inhibitors would be valuable for the development of novel therapeutic modalities against breast cancer. © 2010 American Association for Cancer Research.

DOI： 10.1158/0008-5472.CAN-09-3911

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

Although the fission yeast Schizosaccharomyces pombe has been used for high-level heterologous protein production, the productivity of secreted human serum transferrin (hTF) has been low, presumably, because the protein harbors twenty disulfide bonds and two Nglycosylation sites. In the present study, we found that overexpression of endogenous putative protein disulfide isomerase (PDI) improved productivity. Whole genome sequence analysis of S. pombe revealed five putative PDI genes and overexpression of two of them, SPAC17H9.14c and SPBC3D6.13c (SpPdi2p or SpPdi3p, respectively), significantly improved the productivity of secreted hTF. GFP-fused SpPdi2p and SpPdi3p were found to localize to the endoplasmic reticulum. Co-overexpression of SpPdi2p or SpPdi3p with hTF coupled with modifications to the growth medium reported in our previous study were able to increase the level of secreted hTF approximately 30-fold relative to conventional conditions. © 2009 Springer-Verlag.

DOI： 10.1007/s00253-009-2393-x

Scopus

Language：English   Publisher：Journal of Biochemistry  

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural distributions and biological functions remain to be clarified. To establish a robust analytical system that can separate, identify and quantify rare sugars, 12 d-hexoses - including five rare aldohexoses and three rare ketohexoses - were labelled with 2-aminobenzamide (2-AB), and the fluorescently tagged monosaccharides were separated by high-performance liquid chromatography (HPLC). Because the ketohexoses were much less reactive than were the aldohexoses, the reaction conditions were optimized to achieve the maximum yields (>75) for both aldohexoses and ketohexoses. The calibration curve determined for the rare ketohexose, d-psicose (Psi), was linear between 1 pmol and 1 mol and had a correlation coefficient of 0.9999. Using the developed method, the psicose content in a leaf of Itea virginica, in which the presence of psicose has previously been reported, was found to be 2.7 mg psicose/g leaf. The result proved feasibility of the method even for natural products. Because the system is a comprehensive tool, it should help answer questions concerning the biosyntheses and functions of rare hexose sugars. © 2009 The Authors.

DOI： 10.1093/jb/mvp199

Scopus

Language：English   Publisher：FEBS Letters  

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 exit the vegetative cell cycle and initiate sexual differentiation to produce spores. Most of the nitrogen used for de novo protein synthesis during sporulation derives from the autophagic protein degradation system. This review focuses on the recent advances in the role of autophagy in fission yeast. © 2009 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2009.12.037

Scopus

Language：English   Publisher：Microbiology  

Schizosaccharomyces species are currently the only known organisms with two types of genes encoding UDP-glucose/-galactose 4-epimerase, uge1+ and gal10+. A strain deleted for uge1+ exhibited a severe galactosylation defect and a decrease in activity and in UDP-galactose content when grown in glucose-rich medium (2% glucose), indicating that Uge1p is a major UDP-glucose/-galactose 4-epimerase under these growth conditions. In contrast, gal10+ was efficiently expressed and involved in galactosylation of cell-surface proteins in low-glucose medium (0.1% glucose and 2% glycerol), but not in galactose-containing medium. In a uge1Δgal10Δ strain, the galactosylation defect was suppressed and UDP-galactose content restored to wild-type levels in galactose-containing medium. Disruption of gal7+, encoding galactose-1-phosphate uridylyltransferase, in the uge1Δ gal10Δ strain reversed suppression of the galactosylation defect and reduced levels of UDP-galactose, indicating that galactose is transported from the medium to the cytosol and is converted into UDP-galactose via galactose 1-phosphate by Gal7p in Sch. pombe. © 2010 SGM.

DOI： 10.1099/mic.0.035279-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to α-d-mannose (Man), a large number of α-d-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by α-1,6-mannosylatransferase encoded by the och1 + gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Δ mutant consisted of Gal2-6Man9GlcNAc 2 with α-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1 +, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Δoch1Δ double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with α1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Δoch1Δ cells consisted of α1,2-linked Man-extended core oligosaccharides (Man8-12GlcNAc2) from which the fission yeast-specific α-linked Gal residues were completely absent. © 2009 Springer-Verlag.

DOI： 10.1007/s00253-009-2297-9

Scopus

Language：English   Publisher：Journal of Bacteriology  

Glycine sarcosine methyltransferase (Gsm) and sarcosine dimethylglycine methyltransferase (Sdm) catalyze glycine betaine synthesis from glycine. Disruption of the M. xanthus gsmA (MXAN 7068) or sdmA (MXAN 3190) gene, encoding Gsm or Sdm homologue proteins, respectively, generated mutants that exhibited a longer lag period of growth and delayed spore germination under osmostress. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/JB.01118-09

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe. © 2009 Springer-Verlag.

DOI： 10.1007/s00253-009-2151-0

Scopus

Language：English   Publisher：Microbiology  

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are 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 this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30% of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15% of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1D and atg12D mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation. © 2009 SGM.

DOI： 10.1099/mic.0.034389-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of General and Applied Microbiology  

DOI： 10.2323/jgam.55.499

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Microbiology and Biotechnology  

The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane. © 2009 Springer-Verlag.

DOI： 10.1007/s00253-009-2130-5

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Eukaryotic Cell  

Aspergillus nidulans possesses threepmt genes encoding protein O-D-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. ApmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/EC.00371-08

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Chromatography A  

A rare sugar is considered to be a monosaccharide rarely found in nature. To investigate their natural distribution and biological roles, a robust analytical system must be used to isolate, identify, and quantify them. Herein, we report the development of such a system that can specifically quantify and chromatographically separate four aldopentoses and eight aldohexoses tagged with 2-aminopyridine. Purified monosaccharides derivatized with a pyridylamino moiety (PA-monosaccharides) are first chromatographed over a high-performance anion-exchange resin. But, because two of the PA-aldohexoses used in this study, PA-talose and PA-idose, co-elute with the common saccharides, PA-glucose and PA-mannose, respectively, a second chromatographic step, reversed-phase high-performance liquid chromatography, is used to completely separate them. Thus, as shown by the results of this study, chromatographic separation of PA-monosaccharides is achievable and provides a quantitative measurement of common and rare isomeric aldopentoses and aldohexoses. © 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.chroma.2009.04.072

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：FEMS Yeast Research  

The KTR α1,2-mannosyltransferase gene family of Saccharomyces cerevisiae is responsible not only for outer-chain modifications of N-linked oligosaccharides but also for elongation of O-linked mannose residues. To identify genes involved in the elongation step of O-linked oligosaccharide chains in Schizosaccharomyces pombe, we characterized six genes, omh1 +-omh6+, that share significant sequence similarity to the S. cerevisiae KTR family. Six deletion strains were constructed, each carrying a single disrupted omh allele. All strains were viable, indicating that none of the omh genes was essential. Heterologous expression of a chitinase from S. cerevisiae in the omh mutants revealed that O-glycosylation of chitinase had decreased in omh1Δ cells, but not in the other mutants, indicating that the other omh genes do not appear to be required for O-glycan synthesis. Addition of the second α1,2-linked mannose residue was blocked in omh1Δ cells. An Omh1-GFP fusion protein was found to be localized in the Golgi apparatus. These results indicate that Omh1p plays a major role in extending α1,2-linked mannose in the O-glycan pathway in S. pombe. © 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

DOI： 10.1111/j.1567-1364.2008.00458.x

Scopus

Language：English   Publisher：FEBS Letters  

Myxococcus xanthus PdeA and PdeB, enzymes homologous to class III 3′,5′-cyclic nucleotide phosphodiesterases, hydrolyzed 3′,5′- and 2′,3′-cyclic AMP (cAMP) to adenosine, and also demonstrated phosphatase activity toward nucleoside 5′-tri-, 5′-di-, 5′- and 3′-monophosphates with highest activities for nucleoside 5′-monophosphates. The substrate specificities of PdeA and PdeB show no similarity to that of any known cNMP phosphodiesterase, nucleotidase, or phosphatase. The enzyme activities of PdeA and PdeB were stimulated by 50 μM Mn2+ or Co2+. The Km values of PdeA and PdeB for 3′,5′-cAMP, 2′,3′-cAMP, 5′-ATP, and 5′-AMP were in the low micromolar range (1.4-12.5 μM). © 2008 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2008.12.044

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Bioscience and Bioengineering  

Two Ser/Thr protein kinases, SpkA and SpkB, selected from Myxococcus xanthus based on amino acid sequence similarities with the catalytic subunits of cAMP-dependent protein kinases (PKA) were synthesized using a cell-free protein synthesis system. In various protein kinase assays, purified StkA and StkB showed their highest protein kinase activities in a PKA assay using the selective PKA substrate Kemptide and in a protein kinase C (PKC) assay using the selective PKC substrate neurogranin(28-43), respectively. SpkA had apparent Km values of 45 μM and 37 μM for Kemptide and ATP, respectively. Phosphorylation of Kemptide was inhibited by a specific PKA inhibitor peptide, PKI5-24, and the IC50 and Ki values for inhibition of the SpkA activity were 117 nM and 36 nM, respectively. © 2008 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2008.08.002

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：FEBS Letters  

We have identified the Schizosaccharomyces pombe SPBC3E7.06c gene (fnx2+) from a homology search with the fnx1+ gene involving in G0 arrest upon nitrogen starvation. Green fluorescent protein-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H+-ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Δfnx1 and Δfnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that fnx1+ and fnx2+ are involved in vacuolar amino acid uptake in S. pombe. © 2008 Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2008.05.017

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin-GFP- carboxypeptidase S (Ub-GFP-CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.

DOI： 10.1271/bbb.70666

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

Many plant secondary metabolites show strong biological activities and are potentially also toxic to plants, while plants producing such active compounds are usually insensitive to their own metabolites, suggesting that they have species-specific detoxification mechanisms. In order to clarify the detoxification mechanism of alkaloids, we used cultured cells of Coptis japonica, which are capable of producing a yellow benzylisoquinoline alkaloid, berberine, and accumulate it in the vacuole. Unlike other plant cells that do not produce berberine, C. japonica shows strong tolerance to this alkaloid. We established a fission yeast strain that was sensitive to berberine and performed functional screening using a C. japonica cDNA library. One cDNA clone, which conferred clear berberine tolerance, encoded galactinol synthase (CjGolS). The possible role of CjGolS in berberine tolerance is discussed.

DOI： 10.1271/bbb.70495

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Microbiology  

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31+ or erg32+ did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergΔ mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Δ but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergA mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins. © 2008 SGM.

DOI： 10.1099/mic.0.2007/011155-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Physics Letters  

The authors propose a double filtering method to measure translational velocity for tracking fluorescently stained cells. This method employs two diffraction gratings installed in the infinity space through which the parallel pencil beam of the fluorescence passes. With this method, the change in light intensity whose period is proportional to the translational velocity of the sample can be obtained at the imaging surface. By using a sample that has a random distribution of fluorescence intensity, the authors verified that translational velocity measurements could be achieved using the proposed method. © 2007 American Institute of Physics.

DOI： 10.1063/1.2793695

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Biochemistry  

Arthrobacter endo-β-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) family 85, catalyses the hydrolysis and transglycosylation of asparagine-linked oligosaccharides of glycoproteins with retention of anomeric configuration. Glu-173 of Endo-A is a catalytically essential amino acid residue, and the corresponding residue is conserved in all GH family 85 members. The catalytic activity of Endo-A E173A mutant was rescued by the addition of sodium azide or sodium formate. Furthermore, the produced β-glycosyl azide (Man 5GlcNAc-β-N 3) retained the anomeric configuration, indicating that Glu-173 is the catalytic acid-base residue of Endo-A. This is the first identification of the catalytic residue for GH family 85 endo-β-N-acetylglucosaminidases. © 2007 The Japanese Biochemical Society.

DOI： 10.1093/jb/mvm124

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Microbiology  

The multivesicular body (MVB) sorting pathway is required for a number of biological processes, including downregulation of cell-surface proteins and protein sorting into the vacuolar lumen. The function of this pathway requires endosomal sorting complexes required for transport (ESCRT) composed of class E vacuolar protein sorting (Vps) proteins in Saccharomyces cerevisiae, many of which are conserved in Schizosaccharomyces pombe. Of these, sst4/vps27 (homologous to VPS27) and sst6 (similar to VPS23) have been identified as suppressors of sterility in ste12Δ (sst), although their functions have not been uncovered to date. In this report, these two sst genes are shown to be required for vacuolar sorting of carboxypeptidase Y (CPY) and an MVB marker, the ubiquitin-GFP-carboxypeptidase S (Ub-GFP-CPS) fusion protein, despite the lack of the ubiquitin E2 variant domain in Sst6p. Disruption mutants of a variety of other class E vps homologues also had defects in sorting of CPY and Ub-GFP-CPS. Sch. pombe has a mammalian AMSH homologue, sst2. Phenotypic analyses suggested that Sst2p is a class E Vps protein. Taken together, these results suggest that sorting into multivesicular bodies is dependent on class E Vps proteins, including Sst2p, in Sch. pombe. © 2007 SGM.

DOI： 10.1099/mic.0.2007/006072-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Optical Review  

This paper describes a method for measuring the three-dimensional (3D) refractive-index distribution in a single cell. The method can be used to observe the distribution of cell components without fluorescence staining. The two-dimensional optical path length distributions from multiple directions are obtained by non-contact rotation of the cell. These optical path lengths are converted into the line integrals of the refractive index, and the 3D refractive-index distribution is reconstructed by means of computed tomography. The refractive-index distribution in a breast cancer cell can be measured using a phase-shifting Mach-Zehnder interferometer in conjunction with proximal two-beam optical tweezers. © 2007 The Optical Society of Japan.

DOI： 10.1007/s10043-007-0161-7

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

Schizosaccharomyces pombe has four α-amylase homologs (Aah1p-Aah4p) with a glycosylphosphatidylinositol (GPI) modification site at the C-terminal end. Disruption mutants of aah genes were tested for mislocalization of vacuolar carboxypeptidase Y (CPY), and aah3Δ was found to secrete CPY. The conversion rate from pro- to mature CPY was greatly impaired in aah3Δ, and fluorescence microscopy inidicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. These results indicate that aah3Δ had a defect in the retrograde transport of Vps10p, and that Aah3p is the first S. pombe specific protein required for vacuolar protein sorting.

DOI： 10.1271/bbb.60609

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Physics Letters  

This letter reports a method for displacement measurement of the depth migration of transparent cells. This proposed optical spatial filtering method allows visualization of the transparent cells and determination of depth migration as a horizontal displacement positive or negative first order diffracted light on the detector surface. When the sample is displaced upward or downward from the focal plane, first and negative first order diffracted light form images at a different point as a light circle. The coordinates of these two light circles on the detector surface change places when the displacement of depth migration moves to the opposite direction. © 2006 American Institute of Physics.

DOI： 10.1063/1.2405396

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Proceedings of SPIE - The International Society for Optical Engineering  

We study on the imaging technology of three-dimensional distribution for sugar chain on single living cell-membrane. This technology can observe the entire cell surface. To observe the cell surface, the local area image of cell-membrane is taken by TIRF (total internal reflection fluorescence) microscopy. And by scanning the whole cell surface area, we can obtain the image of the entire cell membrane. These observed local area images can be converted into an entire surface image by the pattern matching processing. For this scanning technology, we propose the proximal two beam optical tweezers to rotate the single floating cell. This proximal two beam optical tweezers can rotate the floating single cell in the nutrient medium by light pressure. Two beams illuminate the single cell at proximal two points from below and above. The cell is trapped at the center of these two focal points. At the same time, light pressures that are generated at two focal points are made to act as rotational torque. Conventionally TIRF microscope is well known as the observation technology for the cell-membrane using the evanescent light as the exciting light. We can observe the local area images of the fluorescently labeled sugar chain that binds the glycoprotein. Using the proposed optical system, we can obtain the fluorescent distribution images on the cell-membrane.

DOI： 10.1117/12.685893

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Physics Letters  

Technology for the attitudinal manipulation of medical equipment for local dosing to single living cells is presented. The authors developed a "juggling probe" which can manipulate a bacillary probe with multiple degrees of freedom and high accuracy without physical contact by using light pressure. With this technology, the authors cause two beams to focus on the probe from both the top and bottom faces. When the two focal points coincide, the probe becomes trapped immediately in a stable fixed attitude. Furthermore, the probe remains at rest in an arbitrary tilted attitude simply by controlling the distance between the two focal points. © 2006 American Institute of Physics.

DOI： 10.1063/1.2357922

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：FEMS Yeast Research  

A new, heat shock-inducible expression system based on an endogenous hsp16+ promoter was developed for use in the fission yeast Schizosaccharomyces pombe. Analysis of GFP expression profiles indicated that a 1.2-kb segment of the hsp16+ promoter region was sufficient to drive expression of heterologous protein. The hsp16+ promoter was found to be activated not only by heat shock but also by other stresses including cadmium, ethanol, and oxidative stress. Two expression vectors, pHIL and pHIU, were constructed using the 1.2-kb hsp16+ promoter for inducible gene expression in Sch. pombe. This new expression system utilizes a simple induction protocol and promises to be a useful tool for analyzing gene expression in Sch. pombe. © 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.

DOI： 10.1111/j.1567-1364.2006.00093.x

Scopus

Language：English   Publisher：Microbiology  

ATP-binding cassette (ABC) proteins transport a wide variety of substrates, including sugars, amino acids, metal ions, lipids, peptides and proteins, across membranes, and most ABC proteins contain transmembrane domains (ABC transporters). Sequencing of the Schizosaccharomyces pombe genome has allowed identification of all genes encoding ABC transporters in fission yeast. To date, six such genes have been characterized, and an additional five genes encoding ABC transporters were identified from the genome sequence. In an attempt to characterize all of the ABC transporters in fission yeast, all 11 genes were disrupted. While all the genes were found to be dispensable for cell viability, some disruptants lacked apparent phenotypes. GFP-tagged ABC transporters were localized to membranes as follows: plasma membrane (2), vacuolar membrane (4), mitochondrial membrane (2), endoplasmic reticulum membrane (2), and endosome and Golgi membranes (1). Two Cluster II. 1 proteins, Abc2p (SPAC3F10.11c) and Abc4p (SPAC30.04c), were found to be localized to vacuolar membranes, and to be responsible for accumulation of a characteristic red pigment in the vacuole of an adenine biosynthetic mutant. The doubly disrupted mutant abc2Δ abc4Δ exhibited drug sensitivity, and a decreased accumulation of monochlorobimane, suggesting that both of the proteins encoded by these genes are involved in detoxification of xenobiotics, and vacuolar sequestration of glutathione S-conjugates. © 2006 SGM.

DOI： 10.1099/mic.0.28952-0

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPIanchored proteins in S. pombe, four α-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3 + resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3 + deletion, but the ascal wall of aah3Δ cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Δ cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.

DOI： 10.1271/bbb.50693

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Plant and Cell Physiology  

We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. In the L. japonicus expressed sequence tag (EST) clone databases of Kazusa DNA Research Institute, another syntaxin-related clone (LjSYP32-2) was also detected, and the nucleotide and amino acid sequences of these two clone are very similar to each other. Real-time PCR and promoter analysis indicated that expression of LjSYP32-1 was dominant compared with LjSYP32-2 in the various plant organs. Promoter analysis and in situ hybridization revealed that LjSYP32-1 was expressed significantly in the inner cortex cell layer surrounding the infected zone of young nodules and in the meristem area of developing lateral root. To explore the function and physiological role of LjSYP32-1 in nodules and other plant organs, stable transformation lines of L. japonicus expressing either sense or antisense LjSYP32-1 were prepared. The antisense plants showed a significantly retarded plant growth phenotype, suggesting a role for LjSYP32-1 in supporting plant growth. In the same transgenic lines, the plants were capable of forming nodules, but the acetylene reduction activity was reduced by around 50% per plant. The nodules were much smaller and some nodules were fused to each other by sharing the inner cortex. The rate of occurence of such irregular nodules was twice that observed in wild-type plants. The data suggest that LjSYP32-1 contributes to the support of plant growth and normal nodule tissue differentiation. © The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.

DOI： 10.1093/pcp/pcj054

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Applied Physics Letters  

A precise method to rotate single cells is reported. In this method, the light pressure in the optical axis direction is harnessed as a rotating torque. Two proximal points in each cell are illuminated from different directions using two beams, and a light pressure is created that acts as a rotating torque. Using this proposed method, we could control the rotational direction of a microsphere regardless of the refractive index distribution in a noncontact operation. The microsphere could be rotated using proximal two-beam optical tweezers, and the rotational velocity could be controlled by changing the light intensity. © 2006 American Institute of Physics.

DOI： 10.1063/1.2190268

Scopus

Language：English   Publisher：Genes to Cells  

We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1+ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1+ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling. © 2006 The Author(s) Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2443.2006.00935.x

Scopus

Language：English   Publisher：Carbohydrate Research  

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-d-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-β-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives. © 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2005.11.015

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Nucleic Acids Research  

The technologies for chromosome modification developed to date are not satisfactorily universal, owing to the typical requirements for special enzymes and sequences. In the present report, we propose a new approach for chromosome modification in Schizosaccharomyces pombe that does not involve any special enzymes or sequences. This method, designated the 'Latour system', has wide applicability with extremely high efficiency, although both the basic principle and the operation are very simple. We demonstrate the ability of the Latour system to discriminate essential genes, with a long chromosomal area of 100 kb containing 33 genes deleted simultaneously and efficiently. Since no foreign sequences are retained after deletion using the Latour system, this system can be repeatedly applied at other sites. Provided that a negative selectable marker is available, the Latour system relies solely upon homologous recombination, which is highly conserved in living organisms. For this reason, it is expected that the system will be applicable to various yeasts. © The Author 2006. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/nar/gnj011

Scopus

Language：English   Publisher：Microbiology  

Methionine synthase (EC2.1.1.14) catalyses the final step in methionine synthesis, i.e. methylation of homocysteine. A search of the Schizosaccharomyces pombe genomic database revealed a gene designated SPAC9.09, encoding a protein with significant homology to methionine synthase. Disruption of SPAC9.09 caused methionine auxotrophy, and thus the gene was identified as a methionine synthase and designated met26. The met26 mutant was found to exhibit a remarkable growth defect in the absence of adenine even in medium supplemented with methionine. This phenotype was not observed in other methionine auxotrophs. In the budding yeast Saccharomyces cerevisiae, which has been reported to utilize homocysteine in cysteine synthesis, lack of a functional methionine synthase did not cause a requirement for adenine. The introduction of genes from Sac. cerevisiae constituting the cystathionine pathway (CYS4 and CYS3) into Sch. pombe Δmet26 cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in Δmet26 cells was higher than in other methionine auxotrophs and that introduction of the Sac. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in the met26 mutant. © 2006 SGM.

DOI： 10.1099/mic.0.28398-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Cell Structure and Function  

Ypt7p, a fission yeast (Schizosaccharomyces pombe) homologue of Rab7 GTPase, mediates fusion of endosomes to vacuoles and homotypic vacuole fusion. Here, we report that Ypt7p plays important roles in sporulation. Most ypt7Δ asci produced less than four spores, which were apparently immature and germinated at low frequency. Furthermore, ypt7Δ cells were defective in development of the forespore membranes. Vacuoles in sporulating cells were found to undergo extensive homotypic vacuole fusion to form a few large compartments occupying the entire cytoplasm of asci. This extensive vacuole fusion depended on Ypt7p. © 2005 by Japan Society for Cell Biology.

DOI： 10.1247/csf.30.43

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

A potential correlation between mitochondrial and vacuolar functions is known to exit in yeast. Fission yeast atm1+, SPAC15A10.01, encodes a putative half-type ABC transporter with an N-terminal mitochondrial-targeting signal. In an attempt to evaluate the possible involvement of mitochondrion in vacuole function, a functional analysis of atm1+ was performed by gene disruption. Growth of the atm1 mutant was inhibited in the presence of oxidizing agents, and S. cerevisiae Atm1p was found to complement this growth defect. atm1Δ cells exhibited defects in fluid-phase endocytosis and vacuolar fusion under hypotonic stress. GFP-tagged Atm1p was observed to be localized in the mitochondria. These data strongly suggest that fission yeast Atm1p was not only involved in protection against oxidative stress, but also played a role in vacuolar functions.

DOI： 10.1271/bbb.69.2109

Scopus

Language：English   Publisher：Proceedings of the SICE Annual Conference  

Currently, we study about spectroscopy-tomography of living single-cells whose diameter is about 10 μm to assist the quantitative diagnosis of early cancer. This technology is composed of two elemental technologies, high spatial resolution spectrometry and a precise single-cell rotating method. In these two technologies, we propose proximal two-beam optical tweezers as the precise rotating method. In this method, we harness the light pressure generated by light absorption as rotating torque. We decided to illuminate the proximal two points in each from different directions using two beams. In this case, the light pressure generated by light absorption is made to act as rotating torque. Using this proposed method, we can control the rotational velocity of a microsphere regardless of refractive index distribution by non-contact operation. In addition, rotational speed is controlled by optical PWM operation. This proposed optical PWM operation is that the received light intensity is changed by the illumination time. This method can be developed into the 6-DOF control of single-cell. © 2005 SICE.

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1271/bbb.69.2109.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The moc1/sds23 gene was isolated to induce sexual development of a sterile strain due to overexpression of adenylate cyclase in Schizosaccharomyces pombe. Here, we studied the functional conservation between moc1/ sds23 and its two orthologs SDS23 and SDS24 in Saccharomyces cerevisiae. We observed that the temperature sensitivity, salt tolerance, cell morphology, and sterility of the Δmoc1 mutant in S. pombe were recovered by expressing either S. cerevisiae SDS23 or SDS24. We found that deletion of both SDS23 and SDS24 resulted in the production of a large vacuole that was reversed by the expression of S. pombe moc1/sds23. In these ways we found that S. pombe Moc1/Sds23 and S. cerevisiae SDS23p or SDS24p are functional homologs. In addition we found that the Δsds23 Δsds24 diploid strain reduces cell separation in forming pseudohyphal-like growth in S. cerevisiae. Thus S. pombe moc1/sds23 and S. cerevisiae SDS23 or SDS24 are interchangeable with each other, but their disruptants are phenotypically dissimilar.

DOI： 10.1271/bbb.69.1422

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1271/bbb.69.1422.

Language：English   Publisher：Biochemical and Biophysical Research Communications  

Protein O-glycosylation is an essential protein modification in eukaryotic cells. In Saccharomyces cerevisiae, O-mannosylation is initiated in the lumen of the endoplasmic reticulum by O-mannosyltransferase gene products (Pmt1p-7p). A search of the Schizosaccharomyces pombe genome database revealed a total of three O-glycoside mannosyltransferase homologs (ogm1+, ogm2 +, and ogm4+), closely related to Saccharomyces cerevisiae PMT1, PMT2, and PMT4. Although individual ogm genes were not found to be essential, ogm1Δ and ogm4Δ mutants exhibited aberrant morphology and failed to agglutinate during mating. The phenotypes of the ogm4Δ mutant were not complemented by overexpression of ogm1+ or ogm2+, suggesting that each of the Ogm proteins does not have overlapping functions. Heterologous expression of a chitinase from S. cerevisiae in the ogm mutants revealed that O-glycosylation of chitinase had decreased in ogm1Δ cells. A GFP-tagged Fus1p from S. cerevisiae was specifically not glycosylated and accumulated in the Golgi in ogm4Δ cells. These results indicate that O-glycosylation initiated by Ogm proteins plays crucial physiological roles and can serve as a sorting determinant for protein transport of membrane glycoproteins in S. pombe. © 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.03.033

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2005.03.033.

Language：English   Publisher：Journal of Bacteriology  

We have previously reported that a receptor-type adenylyl cyclase (CyaA) of Myxococcus xanthus undergoes an osmosensor mainly during spore germination (Y. Kimura et al., J. Bacteriol. 184:3578-3585, 2002). In the present study, we cloned another receptor-type adenylyl cyclase gene (cyaB) and characterized the function of the cyaB-encoded protein. Disruption of cyaB generates a mutant that showed growth retardation at high ionic (NaCl) or high nonionic (sucrose) osmolarity. When vegetative cells were stimulated with 0.15 M NaCl, the increases in intracellular cyclic AMP levels of cyaB mutant cells were lower than those of wild-type cells. Under nonionic osmostress, the cyaB mutant exhibited reduced spore germination; however, the germination rate of the cyaB mutant was significantly higher than that of the cyaA mutant. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/JB.187.10.3593-3598.2005

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/JB.187.10.3593-3598.2005.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1002/yea.1201.

Language：English   Publisher：Yeast  

We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12+, tyr1+ and ade7+) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed. Copyright © 2005 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.1201

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1247/csf.30.43.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1356-9597.2004.00744.x.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/yea.1134.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.e03-09-0659.

Language：English   Publisher：Yeast  

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 end4Δ as well as ypt7Δ, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant. Deletion of end4 resulted in pleiotropic phenotypes consistent with F-actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects. Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant. These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post-internalization step after the intersection of the endocytic and the vacuolar protein-sorting pathways in fission yeast. Copyright © 2004 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.1134

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1271/bbb.68.1059.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00438-003-0971-7.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/yea.1104.

Language：English   Publisher：Yeast  

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 μl reaction mixture containing 70 μl PLATE solution (50% polyethylene glycol-4000, 100 mM lithium acetate, 10 mM Tris-HCl, pH 4.9, and 1 mM EDTA), 10 μl plasmid DNA (1 μg), 10 μl carrier DNA (100 μg) and 10 μl sterile distilled water. After incubation at 30°C for 1 h followed by heat shock treatment at 42°C for 15 min, the reaction mixture was spread on a selection plate. The transformation efficiency obtained using the procedure was approximately 8000 transformants/μg DNA. The method is simple and time-saving, making it especially useful for a large number of samples and when a high transformation efficiency is not required. Copyright © 2004 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.1104

Scopus

Language：English   Publisher：Molecular Biology of the Cell  

Calcineurin is a highly conserved regulator of Ca2+ signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl- homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1+ gene that encodes a homolog of the mammalian μ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Δapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Δapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Δapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

DOI： 10.1091/mbc.E03-09-0659

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jb/mvh026.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-β-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids. Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-β-N-acetylglucosaminidase activity. Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity. On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced. These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity. Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-β-N-acetylglucosaminidases of various origins.

DOI： 10.1271/bbb.68.1059

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1271/bbb.68.545.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1271/bbb.68.306.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4+ cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1+ as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4+ could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.

DOI： 10.1271/bbb.68.545

Scopus

Language：English   Publisher：Molecular Genetics and Genomics  

The vacuolar H+ -ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted. Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca2+. The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants. Moreover, deletion of the S. pombe vma1 or vma3+ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects. These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S. pombe. © Springer-Verlag 2004.

DOI： 10.1007/s00438-003-0971-7

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1356-9597.2004.00699.x.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the 5. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.

DOI： 10.1271/bbb.68.306

Scopus

Language：English   Publisher：Journal of Biochemistry  

Pyridoxal (PL) reductase encoded by the plr1+ gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5′-phosphate (PLP), a coenzyme form of vitamin B6, or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1Δ) strain was constructed and its phenotype was examined. The plr1Δ cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1Δ strain became flocculent at the late stationary phase for an unknown reason. The plr1Δ cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1+ gene, which maintained the flow of "PL → PN → PNP → PLP" in the salvage synthesis of PLP. The total vitamin B6 and pyridoxamine 5′-phosphate contents in the plr1Δ cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B6 compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1Δ cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1+ gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis.

DOI： 10.1093/jb/mvh026

Scopus

Language：English   Publisher：Genes to Cells  

Schizosaccharomyces pombe pmr1+ gene is homologous to Saccharomyces cerevisiae PMR1 gene, which encodes the P-type Ca2+/Mn2+-ATPase. Addition of Mn2+, as well as Ca2+, to the medium induced pmr1+ gene expression in a calcineurin-dependent manner. The pmr1 knockout (Δpmr1) cells exhibited hypersensitivity to EGTA. A screen for high gene dosage-suppressors of the EGTA-hypersensitive phenotype of Δpmr1 led to the identification of pdt1+ gene, which encodes an Nramp-related metal transporter. The Δpmr1 cells showed round cell morphology. Although Δpdt1 cells appeared normal in the regular medium, it showed round cell morphology similar to that of the Δpmr1 cells when Mn2+ was removed from the medium. The removal of Mn2+ also exacerbated the round morphology of the Δpmr1 cells. The Δpmr1 Δpdt1 double mutants grew very slowly and showed extremely aberrant cell morphology with round, enlarged and depolarized shape. The addition of Mn2+, but not Ca2+, to the medium completely suppressed the morphological defects, while both Mn2+ and Ca2+ markedly improved the slow growth of the double mutants. These results suggest that Pmr1 and Pdt1 cooperatively regulate cell morphogenesis through the control of Mn2+ homeostasis, and that calcineurin functions as a Mn2+ sensor as well as a Mn2+ homeo-stasis regulator.

DOI： 10.1111/j.1356-9597.2004.00699.x

Scopus

Language：English   Publisher：Biochemical and Biophysical Research Communications  

Intracellular vesicle trafficking is mediated by a set of SNARE proteins in eukaryotic cells. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. A search of the Schizosaccharomyces pombe genome database revealed a total of 17 SNARE-related genes. Although no homologs of Vam3p, Nyv1p, and Vam7p have been found in S. pombe, we identified one SNARE-like protein that is homologous to S. cerevisiae Pep12p. However, the disruptants transport vacuolar hydrolase CPY (SpCPY) to the vacuole normally, suggesting that the Pep12 homolog is not required for vacuolar protein transport in S. pombe cells. To identify the SNARE protein(s) involved in Golgi-to-vacuole protein transport, we have deleted four SNARE homolog genes in S. pombe. SpCPY was significantly missorted to the cell surface on deletion of one of the SNARE proteins, Fsv1p (SPAC6F12.03c), with no apparent S. cerevisiae ortholog. In addition, sporulation, endocytosis, and in vivo vacuolar fusion appear to be normal in fsv1Δ cells. These results showed that Fsv1p is mainly involved in vesicle-mediated protein transport between the Golgi and vacuole in S. pombe cells. © 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2003.09.179

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.bbrc.2003.09.179.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1271/bbb.67.1772.

Language：English   Publisher：Yeast  

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33+ gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33+ gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells. Copyright © 2003 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.1011

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1002/yea.1011.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1007/s00294-002-0357-0.

Language：English   Publisher：Current Genetics  

To investigate the intracellular transport mechanism of the vacuolar carboxypeptidase of Schizosaccharomyces pombe (SpCPY), SpCPY was expressed in Saccharomyces cerevisiae and its biosynthesis and sorting were examined. When Sac. cerevisiae prc1Δ, devoid of intrinsic (Sc) CPY activity, was transformed with a plasmid carrying the Sch. pombe cpy1+ gene, CPY activity was restored. Pulse-chase experiments revealed that SpCPY is initially synthesized in a pro-precursor form and then converted to a heterodimer, the mature form, in Sac. cerevisiae cells. SpCPY was not processed into intermediate or mature forms in pep4 mutant cells, indicating that SpCPY was proteolytically cleaved in a PEP4-dependent manner in Sac. cerevisiae. Several vps mutants, which are defective in vacuolar protein-sorting, exhibited a defect in the maturation of SpCPY. Moreover, the maturation of SpCPY was severely inhibited in a vps10 strain, although the pro- segment of SpCPY does not contain a QRPL-like sequence, which is the putative targeting signal of ScCPY. When SpCPY was expressed in a wild-type strain, more than 90% of ScCPY was normally sorted to the vacuole, indicating that SpCPY does not compete with ScCPY for vacuolar sorting.In contrast, expression of SpCPY resulted in a missorting of a ScCPY-invertase fusion protein to the cell surface. These results suggested that there are two different binding sites for SpCPY and ScCPY on Vps10p and that the binding of SpCPY to Vps10p interferes with the binding of a ScCPY-invertase fusion protein.

DOI： 10.1007/s00294-002-0357-0

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1002/yea.953.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The ste12+ gene of Schizosaccharomyces pombe codes for a phosphatidylinositol (PI) 3-phosphate 5′-kinase, which is required for efficient mating. Suppressor mutants for sterility of ste12Δ cells were screened for. Most of the mutant genes turned out to be recessive. Six genes were cloned and the open reading frames responsible for the suppressor activity were identified. They included genes coding for proteins with domains homologous to calcium transporters, casein kinase II, UBC13, AMSH, Vps23p, and Vps27p of Saccharomyces cerevisiae. Disruption of these genes resulted in suppression of the defects of the ste12Δ cells, including low mating efficiency and formation of large vacuoles. Since many of these gene products are homologous to the proteins involved in vesicle transport, sterility caused by inactivation of ste12 may be due to a disordered vesicle transport system. © 2003 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

DOI： 10.1271/bbb.67.1772

Scopus

Language：English   Publisher：Methods in Enzymology  

DOI： 10.1016/S0076-6879(03)01006-1

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1091/mbc.01-09-0463.

Language：English   Publisher：Journal of Bacteriology  

An adenylyl cyclase gene (cyaA) present upstream of an osmosensor protein gene (mokA) was isolated from Myxococcus xanthus, cyaA encoded a polypeptide of 843 amino acids with a predicted molecular mass of 91,187 Da. The predicted cyaA gene product had structural similarity to the receptor-type adenylyl cyclases that are composed of an amino-terminal sensor domain and a carboxy-terminal catalytic domain of adenylyl cyclase. In reverse transcriptase PCR experiments, the transcript of the cyaA gene was detected mainly during development and spore germination. A cyaA mutant, generated by gene disruption, showed normal growth, development, and germination. However, a cyaA mutant placed under conditions of ionic (NaCl) or nonionic (sucrose) osmostress exhibited a marked reduction in spore formation and spore germination. When wild-type and cyaA mutant cells at developmental stages were stimulated with 0.2 M NaCl or sucrose, the mutant cells increased cyclic AMP accumulation at levels similar to those of the wild-type cells. In contrast, the mutant cells during spore germination had mainly lost the ability to respond to high-ionic osmolarity. In vegetative cells, the cyaA mutant responded normally to osmotic stress. These results suggested that M. xanthus CyaA functions mainly as an ionic osmosensor during spore germination and that CyaA is also required for osmotic tolerance in fruiting formation and sporulation.

DOI： 10.1128/JB.184.13.3578-3585.2002

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1128/JB.184.13.3578-3585.2002.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Acta Biochimica Polonica  

So far only little data have been available concerning the eliciting capacity of well defined glycan molecules isolated from plant pathogens. This study brings new information about changes in plant cells caused by fungal pathogens. Sugar fractions derived from glycoproteins isolated from the fungus Fusarium sp. M7-1 have been tested here as signaling molecules. The ability of three O-glycan fractions (named in this work inducer I, II, III) to trigger responses in Rubus protoplasts has been examined. It was found that inducer III was the most efficient as it elicited changes in the levels of phenylpropanoid pathway intermediates in relation to phenylalanine-ammonia lyase (PAL) activation.

DOI： 10.18388/abp.2002_3762

Scopus

Language：English   Publisher：Biochimica et Biophysica Acta - General Subjects  

A disaccharide substrate of Manβ1-4GlcNAc-oxazoline 2 was designed and synthesized as a novel probe for detection of the transglycosylating activity of endoglycosidases. A regio- and stereoselective transglycosylation reaction of 2 to GlcNAcβ1-O-pNP or Dns-Asn(GlcNAc)-OH catalyzed by endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) and endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has been demonstrated for the first time, resulting in the core trisaccharide derivative Manβ1-4GlcNAcβ1-4GlcNAcβ1-O-pNP 8 (or -(Dns)Asn-OH). Interestingly, the transglycosylation proceeds irreversibly; the resulting trisaccharide 8 was not hydrolyzed by Endo-M and Endo-A. Based on these results, a new mechanism including an oxazolinium ion intermediate has been proposed for the endoglycosidase-catalyzed hydrolysis or transglycosylation. © 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0304-4165(01)00164-7

Scopus

Language：English   Publisher：Trends in Glycoscience and Glycotechnology  

In recent years, the fission yeast Schizosaccharomyces pombe has attracted interest as a promising unicellular eukaryote model for studies on the biosynthetic pathway and oligosaccharide structures of glycoproteins. The glycoproteins of S. pombe contain a large amount of galactose in addition to mannose, indicating that S. pombe is equipped with mechanisms for galactosylation of glycoprotein, like mammalian cells. To elucidate the physiological role of galactosylation, we isolated an S. pombe mutant (gmsl) defective in protein galactosylation. We found that disruption of the ginsl+ gene (Δgmsl) in S. pombe led to a complete loss of cell surface galactosylation, due to a defect in the transport of UDP-galactose as substrate for galactosyltransferase from cytosol into the lumen of the Golgi apparatus. Therefore, the Δgmsl strain is very useful for the analysis of the phenotypes of S. pombe cells lacking galactose residues and for the elucidation of the galactosylation mechanism. Although galactose residues are not essential for growth of S. pombe cells, the galactosylation of protein is required for the maintenance of normal cell shape, sexual agglutination, tolerance toward various drugs, and non-sexual flocculation in S. pombe.

DOI： 10.4052/tigg.13.519

Scopus

Language：English   Publisher：Yeast  

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Δgms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+ was viable; however, it showed an aberrant cell morphology and increased sensitivities to digestion with β-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Δgms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Δgms1 cells, while other phenotypes were weakly suppressed. The Δgms1 (h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe. Copyright © 2001 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.740

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/s0304-4165(01)00164-7.

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The gene encoding the endo-β-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.

DOI： 10.1271/bbb.65.1542

Scopus

Language：English   Publisher：Yeast  

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP-galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP-galactose transporter. We isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins in Sz. pombe, and found that the gms1+ gene encodes a UDP-galactose transporter. In the prediction of secondary structure of the Gms1 protein, an eight-membrane-spanning structure was obtained. Fluorescent microscopy revealed the functional Gms1-GFP fusion protein to be stably localized at the Golgi membrane. Sequencing analysis of the coding region of Gms1p derived from galactosylation-defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively. The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP-galactose transport activity but no change in the localization to the Golgi membrane. The C-terminal truncated Gms1p mutants demonstrated that the C-terminal hydrophilic region was dispensable for targeting and function as UDP-galactose transporter at the Golgi membrane. We suggest that the putative eighth (the most C-terminus-proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane. Copyright © 2001 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.725

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1002/yea.740.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1271/bbb.65.1542.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1002/yea.725.

Language：English   Publisher：Journal of Bacteriology  

Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for L-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.

DOI： 10.1128/JB.183.7.2226-2233.2001

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1006/bbrc.2001.4836

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1128/JB.183.7.2226-2233.2001

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1006/bbrc.2001.4631

Language：English   Publisher：Journal of Bacteriology  

A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain, mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum.

DOI： 10.1128/JB.183.4.1140-1146.2001

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1128/JB.183.4.1140-1146.2001

Language：English   Publisher：Biochemical and Biophysical Research Communications  

A novel chemoenzymatic approach to synthesize neoglycoproteins containing high-mannose-type oligosaccharides is described. p-Isothiocyanatophenyl-β-D-glucopyranoside (Glc-ITC) was transferred to the reducing end of the high-mannose-type oligosaccharides using a transglycosylation activity of endo-β-N-acetylglucosaminidase A (Endo-A). A novel oligosaccharide, Man6GlcNAc-Glc-ITC, was synthesized as a coupling reagent for lysyl and N-terminal residues of the protein moiety. The neoglycoconjugate was coupled with several nonglycosylated proteins such as ribonuclease A, lysozyme, and α-lactalbumin. Between one and four high-mannose-type oligosaccharides were incorporated per molecule of these proteins. This method should be very useful for the synthesis of neoglycoproteins with homogeneous high-mannose-type oligosaccharides. © 2001 Academic Press.

DOI： 10.1006/bbrc.2001.4631

Scopus

Language：English   Publisher：Journal of Bioscience and Bioengineering  

The transglycosylation activity of Arthrobacter endo-β-N-acetylglucosaminidase (Endo-A) was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-5-bromo-4-chloro-3-indolyl-β-glucoside (Man6GlcNAc-Glc-β-X). Various endo-β-N-acetylglucosaminidases hydrolyzed this oligosaccharide, producing Man6GlcNAc and Glc-β-X. The E. coli strains coexpressing Endo-A and β-glucosidase formed blue colonies in the presence of Man6GlcNAc-Glc-β-X. Therefore, endo-β-N-acetylglucosaminidase activity could be directly detected by the plate assay. This simple plate assay is useful for screening microorganisms for endo-β-N-acetylglucosaminidase activity.

DOI： 10.1016/S1389-1723(01)80021-9

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1006/bbrc.1999.1963.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Nippon Nogeikagaku Kaishi  

DOI： 10.1271/nogeikagaku1924.74.1237

Scopus

Language：English   Publisher：Biochemical Journal  

The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1Δ, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1Δ. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.

DOI： 10.1042/0264-6021:3440211

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/s0014-5793(99)00596-7.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1128/JB.181.4.1356-1359.1999.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1271/bbb.63.1075.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Bacteriology  

We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild- type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.

DOI： 10.1128/jb.181.4.1356-1359.1999

Scopus

Language：English   Publisher：Bioscience, Biotechnology and Biochemistry  

The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants. We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis. One of these mutants, the nss1 mutant, was characterized in detail. The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations. The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects. The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S. cerevisiae. All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin. Nystatin was found to cause extensive enlargement of the vacuole in wild-type S. cerevisiae cells. These results are discussed with special reference to the vacuolar function of S. cerevisiae. © 1999, Taylor & Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.63.1075

Scopus

Language：English   Publisher：Journal of Bioscience and Bioengineering  

Chemo-enzymatic addition of a high-mannose type oligosaccharide to eel calcitonin (CT), a calcium-regulating hormone, was examined. The endo-β-N- acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) transglycosylated the Man6-GlcNAc moiety from an ovalbumin-derived high- mannose type glycosyl asparagine, Asn(Man6-GlcNAc2)-OH, to a calcitonin derivative, [Asn(GlcNAc)3)-CT, in which the N-acetyl-D-glucosamine (GlcNAc) is attached to the third L-asparagine (Asn) residue of the peptide, and a calcitonin derivative containing a high-mannose type oligosaccharide, [Asn(Man6-GlcNAc2)3]-CT, was synthesized. The optimal reaction conditions for the synthesis of [Asn(Man6-GlcNAc2)3]-CT from Asn(Man6-GlcNAc2)-OH and [Asn(GlcNAc)3]-CT catalyzed by Endo-A were examined. The transglycosylation yield relative to the concentration of the [Asn(GlcNAc)3- CT added was 32.7%, and 4.42 mg of [Asn(Man6-GlcNAc2)3]-CT was prepared.

DOI： 10.1016/S1389-1723(99)89008-2

Scopus

Language：English   Publisher：Journal of Bacteriology  

For the fission yeast Schizosaccharomyces pombe, adaptation to high- osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1+) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive S. pombe mutants, including wis1, sty1, and gpd1. In this study, we attempted to further isolate novel osmolarity- sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants. One of them, the hos1 mutant, was characterized in detail. The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1+ gene, which encodes a small GTP-binding protein. Disruption of the ryh1+ gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the Δryh1 mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of S. pombe.

DOI： 10.1128/jb.180.19.5038-5043.1998