Kaoru Takegawa - Academia.edu (original) (raw)

Papers by Kaoru Takegawa

Agricultural and biological chemistry, Jun 1, 1987

Endo-fiN -acetylglucosaminidase, purified to homogeneity from• the culture filtrate of a Flavobac... more Endo-fiN -acetylglucosaminidase, purified to homogeneity from• the culture filtrate of a Flavobacterium sp., liberated the carbohydrate chains from yeast invertase. About 90% of the carbohydrate associated with this glycoprotein was removed by the endo-fiN -acetylglucosaminidase. The native and carbohydrate-depleted enzymes were compared and found to exhibit similar catalytic activities, thermal stabilities and pH-activity profiles. However, the carbohydrate-depleted invertase was more susceptible to proteases with relatively broad specificities such as subtilisin and pronase, as found on examination of the enzyme activity and electrophoresis. On the other hand, trypsin did not have such an effect on the enzyme activities of the native and carbohydrate-depleted enzymes. The endo-fiN -acetylglucosaminidase also released carbohydrate chains from the purified fiN -acetylhexosaminidase of Penicillium oxalicum. Although the native and carbohydrate-depleted fiN -acetylhexosaminidases did not differ significantly in their stabilities or pH-activity profiles, the carbohydrate-depleted form was more susceptible to proteolysis by subtilisin, pronase and trypsin. From these results, it would appear that the carbohydrate of a glycosylated enzyme plays a role in protecting the enzyme from proteolysis.

Acta Biochimica Polonica, Dec 31, 2002

Journal of General and Applied Microbiology, 2018

ergy metabolism and signal transduction, it is thought that Nudix enzymes play an important role ... more ergy metabolism and signal transduction, it is thought that Nudix enzymes play an important role in metabolic regulation and stress response. Nudix hydrolases are found in all types of living organisms; they are encoded by a different number of genes depending upon the organism, and vary in their substrate specificity. Analysis of the complete M. xanthus genomic sequence indicates that it contains 12 Nudix hydrolases. In this study, we cloned M. xanthus Nudix hydrolase genes (Table S1) in the pCold expression vector (Takara Bio.), expressed them in Escherichia coli Novablue, and determined their hydrolytic activity towards Ap n A and their requirements for metal cofactors. In addition, we addressed the role of Nudix hydrolases in M. xanthus by testing their specificity to 11 substrates [ATP, ADP, GTP, GDP, ADPribose, GDP-glucose, NADH, Ap 4 A, Ap 5 A, ppGpp, and 8-oxo-2¢-deoxyguanosine-5¢-triphosphate (8-oxo-dGTP)]. Analysis of the complete M. xanthus genomic sequence revealed that this species encoded 12 Nudix hydrolases; among them, eight contained the conserved Nudix box GX 5 EX 7 REX 2 EEXGU and four (MXAN_1246, 3769, 5418, and 6513) had highly similar motifs (Fig. S1). Except for MXAN_4891, 11 His-tagged Nudix hydrolases were expressed in E. coli and designated Nud1 to Nud11 according to the order of gene accession numbers. To determine the requirements for divalent metal cofactors by M. xanthus Nudix enzymes, we measured their hydrolytic activity towards Ap 4 A in the presence of different metal cations. M. xanthus Nudix hydrolases were stimulated by Co 2+ , Mn 2+ , or Mg 2+ (Fig. 1), whereas Ca 2+ , Fe 2+ , and Fe 3+ did not affect the hydrolytic activity towards Ap 4 A (data not shown). Nudix hydrolases require Mg 2+ or Mn 2+ for activity (McLennan, 2006); however, Co 2+ was the most effective in stimulating Ap 4 A hydrolysis by M. xanthus Nud1, Nud6, and Nud11.

Applied Microbiology and Biotechnology, May 3, 2019

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well u... more 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.

Genome Announcements, Oct 29, 2015

By screening for microbes that exhibit ␤-D-galactofuranosidase (Galf-ase) activity, a Streptomyce... more By screening for microbes that exhibit ␤-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.

Journal of Biochemistry, Nov 30, 2009

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural d... more 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 micromol 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.

Current Microbiology, Nov 10, 2017

contains supplementary material, which is available to authorized users.

Bioscience, Biotechnology, and Biochemistry, Feb 23, 2008

Many plant secondary metabolites show strong biological activities and are potentially also toxic... more 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.

Yeast, 2005

We describe the development of a new transformation system, using multiple auxotrophic marker gen... more 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.

Journal of Biochemistry, Jun 13, 2007

Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) fam... more Arthrobacter endo-beta-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 beta-glycosyl azide (Man(5)GlcNAc-beta-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-beta-N-acetylglucosaminidases.

Journal of Bacteriology, Aug 12, 2011

A Myxococcus xanthus cytoplasmic bacterial tyrosine kinase, BtkA, showed phosphorylation activity... more A Myxococcus xanthus cytoplasmic bacterial tyrosine kinase, BtkA, showed phosphorylation activity in the presence of Exo. Phosphorylated BtkA was expressed late after starvation induction and early after glycerol induction. The btkA mutant was unable to complete maturation to heat-and sonication-resistant spores under both starvation-and glycerol-induced developmental conditions. Myxococcus xanthus is a Gram-negative soil bacterium which exhibits a complex life cycle and social behavior (22). Upon nutritional starvation, more than 10 5 cells migrate to an aggregation center and form a fruiting body, within which rodshaped cells differentiate into spherical myxospores. A survey of the M. xanthus genome database indicated the presence of two bacterial protein tyrosine kinases (BY kinases), a Gramnegative BY kinase (MXAN_1025) and a Gram-positive BY kinase (MXAN_3228), the latter of which is here called BtkA. BY kinases from Gram-negative bacteria are usually large, membrane-spanning, multidomain proteins composed of an N-terminal transmembrane region and a C-terminal cytosolic tyrosine kinase domain (5). In Gram-positive bacteria, these BY kinases are often split into two distinct proteins, which are encoded by genes that are usually located next to each other (19). BY kinases are involved in the biosynthesis and transport of exopolysaccharide (1, 21). In this study, we found that a cytosolic BY kinase, BtkA, of M. xanthus is required for the formation of mature spores. Functional motifs of BtkA. BtkA consists of 231 amino acid residues with a calculated molecular mass of 24.7 kDa. BtkA exhibited 22% to 31% identity with these BY kinases (Fig. 1). BtkA contains amino acid sequences that resemble the Walker A (GXGK[T/S]) and Walker B (hhhD, where h represents a hydrophobic amino acid) motifs normally found in nucleotidebinding proteins but not in eukaryotic tyrosine kinases (13). In addition, the Walker AЈ (DXDXR) motif, important for Mg 2ϩ binding, was located in BtkA. A tyrosine cluster containing multiple tyrosine residues was not located at the C-terminal end of BtkA, but two tyrosine residues existed at the C-terminal end of BtkA. Protein kinase activity of BtkA is stimulated by Exo. In Gram-positive BY kinases, two distinct proteins, a periplasmic receptor and a cytoplasmic protein kinase, encoded by two different genes are both required for phosphorylation (19). These proteins are encoded by genes that are usually located next to each other. An upstream gene (exo; MXAN_3227) of

Applied Microbiology and Biotechnology, Oct 21, 2009

Journal of Cell Science, 2013

Rheb GTPase and the Tsc1-Tsc2 protein complex, which serves as a GTPase-activating protein for Rh... more Rheb GTPase and the Tsc1-Tsc2 protein complex, which serves as a GTPase-activating protein for Rheb, have crucial roles in the regulation of cell growth in response to extracellular conditions. In Schizosaccharomyces pombe, Rheb and Tsc1-Tsc2 regulate cell cycle progression, the onset of meiosis and the uptake of amino acids. In cells lacking Tsc2 (Dtsc2), the amino acid transporter Aat1, which is normally expressed on the plasma membrane under starvation conditions, is confined to the Golgi. Here, we show that the loss of either pub1 + , encoding an E3 ubiquitin ligase, or any1 + , encoding a b-arrestin-like protein, allows constitutive expression of Aat1 on the plasma membrane in Dtsc2 cells, suggesting that Pub1 and Any1 are required for localization of Aat1 to the Golgi. Subsequent analysis revealed that, in the Golgi, Pub1 and Any1 form a complex that ubiquitylates Aat1. Physical interaction of Pub1 and Any1 is more stable in Dtsc2 cells than in wild-type cells and is independent of Tor2 activity. These results indicate that the TSC-Rheb signaling pathway regulates the localization of amino acid transporters via Pub1 and Any1 in a Tor2-independent manner. Our study demonstrates that, unlike in budding yeast (in which Rsp5 and ARTs, a pair of proteins analogous to Pub1 and Any1, respectively, primarily act to reduce expression of the transporters on plasma membrane when nutrients are abundant), the primary role of fission yeast Pub1 and Any1 is to store the transporter in the Golgi under nutrient-rich conditions.

Yeast, 2001

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist... more 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, itshowed 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.

Yeast, 2001

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the tran... more 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 UDPgalactose 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

Microbiology, May 1, 2006

The mechanism by which soluble proteins, such as carboxypeptidase Y, reach the vacuole in Sacchar... more The mechanism by which soluble proteins, such as carboxypeptidase Y, reach the vacuole in Saccharomyces cerevisiae is very similar to the mechanism of lysosomal protein sorting in mammalian cells. Vps10p is a receptor for transport of soluble vacuolar proteins in S. cerevisiae. vps10 + , a gene encoding a homologue of S. cerevisiae PEP1/VPS10, has been identified and deleted from the fission yeast Schizosaccharomyces pombe. Deletion of the vps10 + gene resulted in missorting and secretion of Sch. pombe vacuolar carboxypeptidase Cpy1p, indicating that it is required for targeting Cpy1p to the vacuole. Sch. pombe Vps10p (SpVps10p) is a type I transmembrane protein and its C-terminal cytoplasmic tail domain is essential for Cpy1p transport to the vacuole. Cells expressing green fluorescent protein-tagged SpVps10p produced a punctate pattern of fluorescence, indicating that SpVps10p was largely localized in the Golgi compartment. In addition, Sch. pombe vps26 + , vps29 + and vps35 + , encoding homologues of the S. cerevisiae retromer components VPS26, VPS29 and VPS35, were identified and deleted. Fluorescence microscopy demonstrated that SpVps10p mislocalized to the vacuolar membrane in these mutants. These results indicate that the vps26 + , vps29 + and vps35 + gene products are required for retrograde transport of SpVps10p from the prevacuolar compartment back to the Golgi in Sch. pombe cells.

Plant Physiology, Dec 20, 2010

Plant and Cell Physiology, 2003

Scientific Reports, Feb 13, 2020

This Article contains typographical errors in the Materials and Methods section under subheading ... more This Article contains typographical errors in the Materials and Methods section under subheading ' Accession Numbers'. "The nucleotide sequences of the ORF1119, ORF4395 and ORF4971 genes have been deposited in the DDBJ/ EMBL/GenBank under the accession nos. LC306881, LC306883 and LC306885, respectively. " should read: "The nucleotide sequences of the ORF1119, ORF4395 and ORF4971 genes have been deposited in the DDBJ/ EMBL/GenBank under the accession nos. LC317050, LC317052 and LC317053, respectively. "

Agricultural and biological chemistry, Jun 1, 1987

Endo-fiN -acetylglucosaminidase, purified to homogeneity from• the culture filtrate of a Flavobac... more Endo-fiN -acetylglucosaminidase, purified to homogeneity from• the culture filtrate of a Flavobacterium sp., liberated the carbohydrate chains from yeast invertase. About 90% of the carbohydrate associated with this glycoprotein was removed by the endo-fiN -acetylglucosaminidase. The native and carbohydrate-depleted enzymes were compared and found to exhibit similar catalytic activities, thermal stabilities and pH-activity profiles. However, the carbohydrate-depleted invertase was more susceptible to proteases with relatively broad specificities such as subtilisin and pronase, as found on examination of the enzyme activity and electrophoresis. On the other hand, trypsin did not have such an effect on the enzyme activities of the native and carbohydrate-depleted enzymes. The endo-fiN -acetylglucosaminidase also released carbohydrate chains from the purified fiN -acetylhexosaminidase of Penicillium oxalicum. Although the native and carbohydrate-depleted fiN -acetylhexosaminidases did not differ significantly in their stabilities or pH-activity profiles, the carbohydrate-depleted form was more susceptible to proteolysis by subtilisin, pronase and trypsin. From these results, it would appear that the carbohydrate of a glycosylated enzyme plays a role in protecting the enzyme from proteolysis.

Acta Biochimica Polonica, Dec 31, 2002

Journal of General and Applied Microbiology, 2018

ergy metabolism and signal transduction, it is thought that Nudix enzymes play an important role ... more ergy metabolism and signal transduction, it is thought that Nudix enzymes play an important role in metabolic regulation and stress response. Nudix hydrolases are found in all types of living organisms; they are encoded by a different number of genes depending upon the organism, and vary in their substrate specificity. Analysis of the complete M. xanthus genomic sequence indicates that it contains 12 Nudix hydrolases. In this study, we cloned M. xanthus Nudix hydrolase genes (Table S1) in the pCold expression vector (Takara Bio.), expressed them in Escherichia coli Novablue, and determined their hydrolytic activity towards Ap n A and their requirements for metal cofactors. In addition, we addressed the role of Nudix hydrolases in M. xanthus by testing their specificity to 11 substrates [ATP, ADP, GTP, GDP, ADPribose, GDP-glucose, NADH, Ap 4 A, Ap 5 A, ppGpp, and 8-oxo-2¢-deoxyguanosine-5¢-triphosphate (8-oxo-dGTP)]. Analysis of the complete M. xanthus genomic sequence revealed that this species encoded 12 Nudix hydrolases; among them, eight contained the conserved Nudix box GX 5 EX 7 REX 2 EEXGU and four (MXAN_1246, 3769, 5418, and 6513) had highly similar motifs (Fig. S1). Except for MXAN_4891, 11 His-tagged Nudix hydrolases were expressed in E. coli and designated Nud1 to Nud11 according to the order of gene accession numbers. To determine the requirements for divalent metal cofactors by M. xanthus Nudix enzymes, we measured their hydrolytic activity towards Ap 4 A in the presence of different metal cations. M. xanthus Nudix hydrolases were stimulated by Co 2+ , Mn 2+ , or Mg 2+ (Fig. 1), whereas Ca 2+ , Fe 2+ , and Fe 3+ did not affect the hydrolytic activity towards Ap 4 A (data not shown). Nudix hydrolases require Mg 2+ or Mn 2+ for activity (McLennan, 2006); however, Co 2+ was the most effective in stimulating Ap 4 A hydrolysis by M. xanthus Nud1, Nud6, and Nud11.

Applied Microbiology and Biotechnology, May 3, 2019

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well u... more 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.

Genome Announcements, Oct 29, 2015

By screening for microbes that exhibit ␤-D-galactofuranosidase (Galf-ase) activity, a Streptomyce... more By screening for microbes that exhibit ␤-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.

Journal of Biochemistry, Nov 30, 2009

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural d... more 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 micromol 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.

Current Microbiology, Nov 10, 2017

contains supplementary material, which is available to authorized users.

Bioscience, Biotechnology, and Biochemistry, Feb 23, 2008

Many plant secondary metabolites show strong biological activities and are potentially also toxic... more 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.

Yeast, 2005

We describe the development of a new transformation system, using multiple auxotrophic marker gen... more 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.

Journal of Biochemistry, Jun 13, 2007

Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) fam... more Arthrobacter endo-beta-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 beta-glycosyl azide (Man(5)GlcNAc-beta-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-beta-N-acetylglucosaminidases.

Journal of Bacteriology, Aug 12, 2011

A Myxococcus xanthus cytoplasmic bacterial tyrosine kinase, BtkA, showed phosphorylation activity... more A Myxococcus xanthus cytoplasmic bacterial tyrosine kinase, BtkA, showed phosphorylation activity in the presence of Exo. Phosphorylated BtkA was expressed late after starvation induction and early after glycerol induction. The btkA mutant was unable to complete maturation to heat-and sonication-resistant spores under both starvation-and glycerol-induced developmental conditions. Myxococcus xanthus is a Gram-negative soil bacterium which exhibits a complex life cycle and social behavior (22). Upon nutritional starvation, more than 10 5 cells migrate to an aggregation center and form a fruiting body, within which rodshaped cells differentiate into spherical myxospores. A survey of the M. xanthus genome database indicated the presence of two bacterial protein tyrosine kinases (BY kinases), a Gramnegative BY kinase (MXAN_1025) and a Gram-positive BY kinase (MXAN_3228), the latter of which is here called BtkA. BY kinases from Gram-negative bacteria are usually large, membrane-spanning, multidomain proteins composed of an N-terminal transmembrane region and a C-terminal cytosolic tyrosine kinase domain (5). In Gram-positive bacteria, these BY kinases are often split into two distinct proteins, which are encoded by genes that are usually located next to each other (19). BY kinases are involved in the biosynthesis and transport of exopolysaccharide (1, 21). In this study, we found that a cytosolic BY kinase, BtkA, of M. xanthus is required for the formation of mature spores. Functional motifs of BtkA. BtkA consists of 231 amino acid residues with a calculated molecular mass of 24.7 kDa. BtkA exhibited 22% to 31% identity with these BY kinases (Fig. 1). BtkA contains amino acid sequences that resemble the Walker A (GXGK[T/S]) and Walker B (hhhD, where h represents a hydrophobic amino acid) motifs normally found in nucleotidebinding proteins but not in eukaryotic tyrosine kinases (13). In addition, the Walker AЈ (DXDXR) motif, important for Mg 2ϩ binding, was located in BtkA. A tyrosine cluster containing multiple tyrosine residues was not located at the C-terminal end of BtkA, but two tyrosine residues existed at the C-terminal end of BtkA. Protein kinase activity of BtkA is stimulated by Exo. In Gram-positive BY kinases, two distinct proteins, a periplasmic receptor and a cytoplasmic protein kinase, encoded by two different genes are both required for phosphorylation (19). These proteins are encoded by genes that are usually located next to each other. An upstream gene (exo; MXAN_3227) of

Applied Microbiology and Biotechnology, Oct 21, 2009

Journal of Cell Science, 2013

Rheb GTPase and the Tsc1-Tsc2 protein complex, which serves as a GTPase-activating protein for Rh... more Rheb GTPase and the Tsc1-Tsc2 protein complex, which serves as a GTPase-activating protein for Rheb, have crucial roles in the regulation of cell growth in response to extracellular conditions. In Schizosaccharomyces pombe, Rheb and Tsc1-Tsc2 regulate cell cycle progression, the onset of meiosis and the uptake of amino acids. In cells lacking Tsc2 (Dtsc2), the amino acid transporter Aat1, which is normally expressed on the plasma membrane under starvation conditions, is confined to the Golgi. Here, we show that the loss of either pub1 + , encoding an E3 ubiquitin ligase, or any1 + , encoding a b-arrestin-like protein, allows constitutive expression of Aat1 on the plasma membrane in Dtsc2 cells, suggesting that Pub1 and Any1 are required for localization of Aat1 to the Golgi. Subsequent analysis revealed that, in the Golgi, Pub1 and Any1 form a complex that ubiquitylates Aat1. Physical interaction of Pub1 and Any1 is more stable in Dtsc2 cells than in wild-type cells and is independent of Tor2 activity. These results indicate that the TSC-Rheb signaling pathway regulates the localization of amino acid transporters via Pub1 and Any1 in a Tor2-independent manner. Our study demonstrates that, unlike in budding yeast (in which Rsp5 and ARTs, a pair of proteins analogous to Pub1 and Any1, respectively, primarily act to reduce expression of the transporters on plasma membrane when nutrients are abundant), the primary role of fission yeast Pub1 and Any1 is to store the transporter in the Golgi under nutrient-rich conditions.

Yeast, 2001

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist... more 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, itshowed 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.

Yeast, 2001

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the tran... more 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 UDPgalactose 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

Microbiology, May 1, 2006

The mechanism by which soluble proteins, such as carboxypeptidase Y, reach the vacuole in Sacchar... more The mechanism by which soluble proteins, such as carboxypeptidase Y, reach the vacuole in Saccharomyces cerevisiae is very similar to the mechanism of lysosomal protein sorting in mammalian cells. Vps10p is a receptor for transport of soluble vacuolar proteins in S. cerevisiae. vps10 + , a gene encoding a homologue of S. cerevisiae PEP1/VPS10, has been identified and deleted from the fission yeast Schizosaccharomyces pombe. Deletion of the vps10 + gene resulted in missorting and secretion of Sch. pombe vacuolar carboxypeptidase Cpy1p, indicating that it is required for targeting Cpy1p to the vacuole. Sch. pombe Vps10p (SpVps10p) is a type I transmembrane protein and its C-terminal cytoplasmic tail domain is essential for Cpy1p transport to the vacuole. Cells expressing green fluorescent protein-tagged SpVps10p produced a punctate pattern of fluorescence, indicating that SpVps10p was largely localized in the Golgi compartment. In addition, Sch. pombe vps26 + , vps29 + and vps35 + , encoding homologues of the S. cerevisiae retromer components VPS26, VPS29 and VPS35, were identified and deleted. Fluorescence microscopy demonstrated that SpVps10p mislocalized to the vacuolar membrane in these mutants. These results indicate that the vps26 + , vps29 + and vps35 + gene products are required for retrograde transport of SpVps10p from the prevacuolar compartment back to the Golgi in Sch. pombe cells.

Plant Physiology, Dec 20, 2010

Plant and Cell Physiology, 2003

Scientific Reports, Feb 13, 2020

This Article contains typographical errors in the Materials and Methods section under subheading ... more This Article contains typographical errors in the Materials and Methods section under subheading ' Accession Numbers'. "The nucleotide sequences of the ORF1119, ORF4395 and ORF4971 genes have been deposited in the DDBJ/ EMBL/GenBank under the accession nos. LC306881, LC306883 and LC306885, respectively. " should read: "The nucleotide sequences of the ORF1119, ORF4395 and ORF4971 genes have been deposited in the DDBJ/ EMBL/GenBank under the accession nos. LC317050, LC317052 and LC317053, respectively. "