Biomedical subjects

Y Jigami

Publications and source records attributed to Y Jigami.

At least 19 recordsLinked to original sources

PubMed2001

Schizosaccharomyces pombe och1(+) encodes alpha-1,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides.

The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N-linked oligosaccharides on many of its glycoproteins. We identified an S. pombe och1 mutant that did not synthesize the outer chains on acid phosphatase. The S. pombe och1(+) gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated alpha-1,6-linked mannose into pyridylaminated Man(9)GlcNAc(2), indicating that och1(+) encodes an alpha-1,6-mannosyltransferase. Our results indicate that SpOch1p is a key enzyme of outer chain elongation. The substrate specificity of SpOch1p was different from that of S. cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p.

Amino Acid Sequence↗

PubMed2001

Hut1 proteins identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe are functional homologues involved in the protein-folding process at the endoplasmic reticulum.

The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER.

Amino Acid Sequence↗

PubMed2001

Overexpression of HUT1 gene stimulates in vivo galactosylation by enhancing UDP-galactose transport activity in Saccharomyces cerevisiae.

Transfer of activated sugar-nucleotides from the cytoplasm to the lumen of the Golgi is an essential requirement for glycosylation of glycoproteins, proteoglycans and glycosphingolipids. Although mannosylation is the major modification in the yeast Saccharomyces cerevisiae, several reports suggest the presence of galactose residues on yeast proteins and sphingolipids. We have detected alpha-galactosylated O-linked chitinase by lectin blotting from cells that functionally express the gma12(+) gene, encoding alpha 1,2-galactosyltransferase from Schizosaccharomyces pombe. This result implies the presence of a UDP-galactose transporter in S. cerevisiae. A conserved gene, HUT1, which encodes a putative multi-transmembrane protein, was cloned and characterized for its possible involvement in galactosylation. The HUT1 gene is not essential and is expressed at a relatively low level under the physiological conditions we examined. The disruption of this gene did not show any apparent impairments in glycosylation. However, a temperature- and concentration-dependent increase in UDP--galactose transport activity was detected from cells overexpressing HUT1 in the presence of gma12(+). The surface of these cells was confirmed to carry galactose residues by staining with FITC-conjugated alpha-galactose-specific lectin. These results suggest a role for Hut1p in the transport of UDP--galactose from the cytosol into the Golgi lumen in S. cerevisiae.

Amino Acid Sequence↗

PubMed2000

Characterization of Yeast Yea4p, a uridine diphosphate-N-acetylglucosamine transporter localized in the endoplasmic reticulum and required for chitin synthesis.

Chitin is an essential cell wall component, synthesis of which is regulated throughout the cell cycle in the yeast Saccharomyces cerevisiae. We cloned an S. cerevisiae gene, YEA4, whose product is homologous to the Kluyveromyces lactis uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transporter. An epitope-tagged Yea4p localized mainly in the 10,000 x g pellet (P2), suggesting endoplasmic reticulum (ER) localization. Membrane vesicles from the P2 fraction showed an 8-fold higher UDP-GlcNAc transport activity in cells harboring a multicopy YEA4 plasmid than in cells harboring vector alone. The activity distribution is identical with the protein distribution in P2, whether the gene is overexpressed or not, suggesting its native localization in P2. Immunolocalization of epitope-tagged Yea4p further revealed ER localization. The increase in transport activity due to the YEA4 overexpression is specific for UDP-GlcNAc, but not for UDP-galactose and GDP-mannose. Deltayea4-disrupted cells showed a reduced rate of UDP-GlcNAc transport, contained less chitin, and were larger and rounder in shape than the wild type cells. Our results indicate that YEA4 encodes an ER-localized UDP-GlcNAc transporter that is required for cell wall chitin synthesis in S. cerevisiae.

Amino Acid Sequence↗

PubMed2000

Identifying tagged transposon insertion sites in yeast by direct genomic sequencing.

Tagged transposons are powerful tools for large-scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time-consuming and problematic. We show that the DNA amplification step can be bypassed. Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon-specific primer, BigDye-labelled terminators, and an automated sequencer. Direct genomic sequencing can also save time on the genetic analysis phase of transposon-based projects.

Base Sequence↗

PubMed2000

Schizosaccharomyces pombe gmd3(+)/alg11(+) is a functional homologue of Saccharomyces cerevisiae ALG11 which is involved in N-linked oligosaccharide synthesis.

The oligosaccharide of glycoproteins in the fission yeast Schizosaccharomyces pombe is unique in containing galactose. We isolated four mutants that had reduced amounts of galactose residues on their cell surface glycoproteins by fluorescence-activated cell sorter. The isolated four recessive mutants, gmd1 to gmd4, showed a defect in glycosylation of acid phosphatase, a cell surface glycoprotein. In gmd3 mutant cells, the amounts of both mannose and galactose residues were decreased on the cell surface galactomannoproteins, suggesting an underglycosylation of galactomannoproteins. The gmd3(+) gene encodes a protein that has significant similarity with Saccharomyces cerevisiae Alg11p and is likely to be involved in N-linked core oligosaccharide synthesis. ALG11 suppressed the gmd3 mutation, indicating that gmd3(+) gene is a functional homologue of the ALG11 gene. We therefore designated gmd3(+) as alg11(+).

Acid Phosphatase↗

PubMed2000

Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus, Aspergillus oryzae.

We cloned all the glycolytic genes from Aspergillus oryzae and analyzed their transcriptional regulation by the carbon source in the medium. The deduced amino-acid sequences of the glycolytic genes showed high identity (approximately 41-93%) to those from other lower eukaryotes. Genomic Southern hybridization indicated that all the genes existed as a single copy in the genome. Comparison of mRNA levels between mycelia grown on glucose and on pyruvate showed that most of the A. oryzae glycolytic genes were induced by glucose in the medium. The overall expression profiles of the A. oryzae glycolytic genes resembled those of Saccharomyces cerevisiae. The expression of one of the phosphofructokinase genes (pfkB), however, was repressed by glucose while both PFK1 and PFK2 were induced in S. cerevisiae. These findings indicate that further analysis of the transcriptional regulation of the A. oryzae glycolytic genes will be useful for investigating the evolutionary change of transcription regulation in lower eukaryotes and to construct promoters for industrial applications.

Aspergillus oryzae↗

PubMed2000

Defects in protein glycosylation cause SHO1-dependent activation of a STE12 signaling pathway in yeast.

In haploid Saccharomyces cerevisiae, mating occurs by activation of the pheromone response pathway. A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity. Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT14, and OCH1, genes required for mannose utilization and protein glycosylation, activated a pheromone-response-pathway-dependent reporter (FUS1) in cells lacking a basal signal (ste4). Pathway activation was suppressed by the addition of mannose to hexose isomerase mutants pgi1-101 and pmi40-101, which bypassed the requirement for mannose biosynthesis in these mutants. Pathway activation was also suppressed in dpm1-101 mutants by plasmids that contained RER2 or PSA1, which produce the substrates for Dpm1. Activation of FUS1 transcription in the mannose utilization/protein glycosylation mutants required some but not all proteins from three different signaling pathways: the pheromone response, invasive growth, and HOG pathways. We specifically suggest that a Sho1 --> Ste20/Ste50 --> Ste11 --> Ste7 --> Kss1 --> Ste12 pathway is responsible for activation of FUS1 transcription in these mutants. Because loss of pheromone response pathway components leads to a synthetic growth defect in mannose utilization/protein glycosylation mutants, we suggest that the Sho1 --> Ste12 pathway contributes to maintenance of cell wall integrity in vegetative cells.

Cell Wall↗

PubMed2000

Molecular cloning and characterization of rpbA encoding RNA polymerase II largest subunit from a filamentous fungus, Aspergillus oryzae.

We have cloned rpbA encoding the RNA polymerase II largest subunit (polIIL) from a filamentous fungus, Aspergillus oryzae. The rpbA product included eight highly conserved regions and the carboxyl-terminal domain (CTD). A. oryzae polIIL CTD with 184 amino acids was composed of 25 CTD consensus repeats, which was a similar number to those of lower eukaryotes. The amino acids in each repeat of A. oryzae polIIL, however, conformed less to the CTD consensus than those of polIILs from other lower eukaryotes.

Amino Acid Sequence↗

PubMed1999

The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae.

Glycolytic gene expression is mediated by the Gcr1p-Gcr2p transcriptional activation complex. A screen for multicopy suppressors of gcr2 yielded SGC1. SGC1's suppression activity was specific to gcr2, it did not extend to gcr1. Disruption of SGC1 moderately affected glycolytic enzyme activities, although no growth defect was evident. Sgc1p exhibits a bHLH motif which is characteristic of E-box DNA-binding proteins. DNA footprinting experiments demonstrated Sgc1p's ability to bind at an E-box. However, its binding specificity was less than 10-fold, which is also characteristic of E-box binding proteins. LexA fusion experiments demonstrated that Sgc1p has weak intrinsic activating activity independent of GCR1 and GCR2. We propose that Sgc1p binds at E-boxes of glycolytic genes and contributes to their activation.

DNA-Binding Proteins↗

PubMed1999

YND1, a homologue of GDA1, encodes membrane-bound apyrase required for Golgi N- and O-glycosylation in Saccharomyces cerevisiae.

The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and approximately 50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Deltaynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Deltagda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Deltaynd1Deltagda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells.

Apyrase↗

PubMed1999

Mannosylphosphate transfer to yeast mannan.

Mannoproteins located in the outermost layer of yeast cell wall determine the wall's porosity and thereby regulate leakage of proteins from the periplasmic space and entrance of macromolecules from the environment. In several yeasts, including Saccharomyces cerevisiae, the glycan portion of mannoproteins is composed not only of neutral oligosaccharides containing mannose and N-acetylglucosamine, but also of acidic oligosaccharides containing mannosylphosphate. The mannosylphosphate residues confer a net negative charge on the cell wall, and so change the properties and environment of the cell surface. Progress on mannosylphosphorylation and its regulation in S. cerevisiae is summarized. Two genes required for mannosylphosphate transfer, MNN4 and MNN6, have been cloned, and a functional analysis of these genes suggests a mechanism for mannosylphosphate transfer. Possible functions for mannosylphosphate transfer in yeast are also discussed. These include supply of GMP for sugar nucleotide transport in the Golgi, cross-linking of mannoproteins to beta-glucan, and a cellular stress response to environmental changes. Glycans in pathogenic yeast and protozoa are also modified with mannosylphosphate, and the potential contribution of this modification to the pathogenicity of these organisms is evaluated.

Cloning, Molecular↗

PubMed1999

The trp1- delta FA designer deletion for PCR-based gene functional analysis in Saccharomyces cerevisiae.

PCR-based gene deletion and modification are now common techniques for rapid gene manipulation in the yeast Saccharomyces cerevisiae. The techniques work best when the host strain lacks sequence homology to the PCR-amplified selectable markers. One of the most versatile sets of PCR deletion/modification vectors is the pFA system described by Longtine et al.(1998), which is based on both heterologous (kanMX6 and HIS3MX6) and homologous (TRP1) markers. Here we describe the trp1-DeltaFA designer deletion allele that removes precisely from the genome TRP1 sequences carried in the pFA vectors. The trp1-DeltaFA allele can be introduced easily into TRP1 and most trp1 starting strains, and its use increases the frequency of correct integrants when using the pFA system's TRP1-based constructs. Unlike trp1-Delta1, trp1-DeltaFA does not remove neighbouring GAL3 upstream activating sequences and therefore does not interfere with GAL gene induction.

Aldose-Ketose Isomerases↗

PubMed1999

A human gene, hSGT1, can substitute for GCR2, which encodes a general regulatory factor of glycolytic gene expression in Saccharomyces cerevisiae.

To determine whether similar regulatory mechanisms control the expression of glycolytic genes in yeast and human cells, we screened a human brain cDNA library for clones which complement the growth defect of the gcr2 mutant of Saccharomyces cerevisiae, and isolated hSGT1 (human suppressor of GCR two). Further work confirmed that the rescue of growth was associated with recovery of glycolytic enzyme activities, and that hSGT1 did not complement the growth defect of a gcr1 mutant. A hybrid protein comprising hSgt1p and the DNA-binding domain of Gal4p (GBD) activated a GAL1-lacZ reporter gene fusion, suggesting that the cloned gene may be a transcriptional activator. Two-hybrid experiments in yeast also indicate that hSgt1p interacts with Gcr1p. Northern analysis showed that hSGT1 is highly expressed in muscle and heart. Although the predicted amino acid sequence of hSgt1p does not display significant similarity to Gcr2p, we speculate that their functions may be analogous.

DNA-Binding Proteins↗

PubMed1999

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae.

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP-galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled alpha-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12(+) and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the Delta mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than alpha1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S. pombe alpha1,2 GalT to prove that it transfers galactose to both N - and O -linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O -mannosyl residue attached to polypeptides and produces Galalpha1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S. cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S. cerevisiae.

Biological Transport↗

PubMed1999

Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris.

To get high level secretion of human lysozyme in Pichia pastoris, the following three signal sequences and one prepro sequence were evaluated: chicken lysozyme signal peptide, leucine-rich artificial signal peptide, Saccharomyces invertase signal peptide, and Saccharomyces prepro sequence of alpha factor (MF-alpha Prepro). Transformants harboring a lysozyme gene with MF-alpha Prepro secreted 20-fold more lysozyme than those harboring the lysozyme gene with any one of the other three signal sequences. Three mutant leader sequences derived from MF-alpha Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-alpha Prepro. In contrast, MF-alpha Prepro with the EAEAEA sequence directed the secretion of an equivalent level of lysozyme having the extra amino acids (EAEAEA) in its N-terminus. For the effective secretion of native human lysozyme, MF-alpha Prepro without any spacer sequences was most suitable. The secreted protein by MF-alpha Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis.

Amino Acid Sequence↗

PubMed1998

Differences in in vivo acceptor specificity of two galactosyltransferases, the gmh3+ and gma12+ gene products from Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, gmh1+, gmh2+ and gmh3+ genes encode alpha-1,2-galactosyltransferase homologues. In an in vitro galactosyltransferase assay, the gmh3+ gene product showed galactose transfer activity toward a-methyl-D-mannoside as an acceptor. The disruption of gma12+, the major galactosyltransferase gene [Chappell, T. G., Hajibagheri, M. A. N., Ayscough, K., Pierce, M. & Warren, G. (1994) Mol. Biol. Cell 5, 519-528], and of gmh3+ in S. pombe caused decreases in the total remaining galactosyltransferase activity and cell surface galactose content. Disruption of gma12+ and gmh3+ also caused an increase in electrophoretic mobility of acid phosphatase, indicating their involvement in the galactosylation of cell surface glycoproteins. The gmh3delta gma12delta double mutant cells had more severe galactose-less phenotypes than single gene mutant cells. HPLC analysis of O-linked mannoprotein oligosaccharides from wild-type and disrupted strains revealed that the gma12+ gene product is responsible for the galactosylation of O-linked oligosaccharide, whereas gmh3+ has no involvement in the process. In contrast, both the gmh3+ and gma12+ gene products are involved in the galactosylation of the N-linked core oligosaccharide Man9GlcNAc2. From these results, it is evident that there are some functional differences between the enzymes in the process of galactosylation. It appears that the gmh3+ gene product transfers galactose to N-linked oligosaccharide, while the gma12+ gene product transfers galactose to both N-linked and O-linked oligosaccharides.

Base Sequence↗

PubMed1998

Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae.

A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for alpha-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro alpha-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an alpha-1, 2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.

Amino Acid Sequence↗