- 2393 Ikenobe Miki-cho Kagawa, 761-0795, JAPAN
Mitsuaki Tabuchi
Kagawa University, Faculty of Agriculture, Faculty Member
MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC... more
MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.
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Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory... more
Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory mechanism of sphingolipid biosynthesis, we generated strains upon replacing the chromosomal LIP1 promoter with a Tet‐off promoter, which enables the expression in Dox‐dependent manner. The lip1‐1 strain, obtained through the promoter substitution, exhibits severe growth inhibition and remarkable decrease in sphingolipid synthesis in the presence of Dox. Using this strain, we investigated the effect of a decrease in ceramide synthesis on TOR complex 2 (TORC2)‐Ypk1 signaling, which senses the complex sphingolipid level at the plasma membrane and promotes sphingolipid biosynthesis. In lip1‐1 cells, Ypk1 was activated via both upstream kinases, TORC2 and yeast PDK1 homologues, Pkh1/2, thereby inducing hyperphosphorylation of Lag1, but not of another Ypk1‐substrate, Orm1, which is a known negative regulator of the first step of sphingolipid metabolism, in the presence of Dox. Therefore, our data suggest that the metabolic enzyme activities at each step of the sphingolipid biosynthetic pathway are controlled through a fine regulatory mechanism.
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Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside... more
Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modificatio...
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PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential... more
PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor fo...
Research Interests: Biochemistry, Bacteriology, Biology, Medicine, Biological Sciences, and 15 moreSaccharomyces cerevisiae, Fission yeast, Enzyme, Molecular cloning, Schizosaccharomyces Pombe, Gene fusion, Amino Acid Profile, Carboxypeptidases, Base Sequence, Open Reading Frame, Polyacrylamide Gel Electrophoresis, Protein Sorting, Molecular Sequence Data, Sodium Dodecyl Sulfate, and Medical and Health Sciences
Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that... more
Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in pla...
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Research Interests: Botany, Biology, Membrane Proteins, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Phylogeny, Escherichia coli, Glutathione, Plants, Pathogen, Proteins, Gene Regulatory Networks, CHEMICAL SCIENCES, Host Pathogen Interactions, Amino Acid Sequence, Botànica, Peroxiredoxins, Molecular Sequence Data, and Medical and Health Sciences
Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1... more
Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3' exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3' untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type-specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosom...
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Research Interests: Immunology, Biology, Membrane Proteins, Innate immunity, Medicine, and 15 moreMolecular Immunology, Humans, Cerebral Cortex, Mice, Animals, Antimicrobial activity, Gene, Molecular weight, Amino Acid Sequence, Base Sequence, Gene Family, Iron binding proteins, Molecular Sequence Data, Nucleotide sequence, and Macrophage activation
The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the... more
The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4 ts mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol ...
Research Interests: Cellular Biology, Biology, Cell Biology, Medicine, Biological Sciences, and 15 moreHumans, Mice, Animals, Genetic Screening, Cell Viability, Inositol, Lipid metabolism, Actin Cytoskeleton, Calmodulin, Calcineurin, Phosphatidylinositol, Cell Survival, Plasma Membrane, Phospholipase C, and Medical and Health Sciences
Research Interests: Genetics, Biology, Japan, Infectious Diseases, Medicine, and 15 moreChlamydia pneumoniae, Biological Sciences, Humans, United States, Polymerase Chain Reaction, Genome, Gene, Infectious, Chlamydia, Whole Genome Sequencing, Genetic Polymorphism, Genome sequence, Outer Membrane Protein, Molecular Sequence Data, and Medical and Health Sciences
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Research Interests: Biochemistry, Kinetics, Mass Spectrometry, Biology, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Escherichia coli, Biological, Enzyme, CHEMICAL SCIENCES, Molecular cloning, Adenylate Kinase, Cysteine, Amino Acid Sequence, Base Sequence, Amino Acid Substitution Rates, Recombinant Proteins, Molecular Sequence Data, and Medical and Health Sciences
To evaluate a newly developed immunochromatographic test (the MySet test) for the detection of Chlamydophila pneumoniae-specific immunoglobulin M antibodies, the results obtained by the MySet test were compared with those obtained by two... more
To evaluate a newly developed immunochromatographic test (the MySet test) for the detection of Chlamydophila pneumoniae-specific immunoglobulin M antibodies, the results obtained by the MySet test were compared with those obtained by two serological tests. The sensitivity and specificity of the MySet test were 100% and 92.9%, respectively. The MySet test is rapid and simple to use and is thought to be a useful tool for the selection of appropriate antibiotic therapy.
Research Interests: Immunology, Biology, Adolescent, Lipopolysaccharide, Humans, and 15 moreChild, Female, Male, Antimicrobial resistance, Enzyme, Antibody, Adult, Immunoassay, Community Acquired Pneumonia, Diagnostic Test, Child preschool, Enzyme Immunoassay, immunoglobulin M, Antibiotic treatment, and Antibiotic Therapy
Background Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species... more
Background Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs. Results In this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1), and 2 candidate heme transporters--heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1)--in non-polarized and pol...
Research Interests: Biochemistry, Biology, Medicine, Dogs, Humans, and 15 moreEndocytosis, Animals, Cell Polarity, Research article, Iron, Heme Oxygenase, DMT, Heme, Ferroportin, Membrane transport proteins, Subcellular Localization, DNA binding proteins, Plasma Membrane, Protein Transport, and Biochemistry and cell biology
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Research Interests: Biochemistry, Functional Analysis, Chemistry, Biology, Membrane Proteins, and 15 moreMedicine, Gene expression, Biological Sciences, Humans, Mice, Animals, Fission yeast, Biochemical, DMT, CHEMICAL SCIENCES, Base Sequence, Ion Transport, Iron binding proteins, Medical and Health Sciences, and In Vitro Techniques
Research Interests: Biology, Membrane Proteins, Macrophages, Medicine, Cell line, and 15 moreGene Cloning, Humans, Human nature, Antimicrobial activity, Glutathione, Gene, Introns, Human Genome, Base Sequence, Gene Family, Fusion Protein, Biochemistry and cell biology, Iron binding proteins, Macrophage activation, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Molecular Biology, Biology, Medicine, Gene expression, and 15 moreSequence Analysis, Escherichia coli, Enzyme, Glycosylation, Recombinant DNA, Enzyme activity, SIGNAL PEPTIDE, Amino Acid Sequence, Base Sequence, Open Reading Frame, Recombinant Proteins, Biochemistry and cell biology, Molecular Sequence Data, Arthrobacter, and Nucleotide sequence
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Endosomal recycling of the mammalian iron transporter DMT1 is assumed to be important for efficient and rapid uptake of iron across the endosomal membrane in the transferrin cycle. Here, we show that the retromer, a complex that mediates... more
Endosomal recycling of the mammalian iron transporter DMT1 is assumed to be important for efficient and rapid uptake of iron across the endosomal membrane in the transferrin cycle. Here, we show that the retromer, a complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network, is required for endosomal recycling of DMT1-II, an alternative splicing isoform of DMT1. Bacterially expressed Vps26-Vsp29-Vsp35 trimer, a retromer cargo recognition complex, specifically binds to the cytoplasmic tail domain of DMT1-II in vitro. In particular, this binding is dependent on a specific hydrophobic motif of DMT1-II, which is required for its endosomal recycling. DMT1-II colocalizes with the Vps35 subunit of the retromer in TfR-positive endosomes. Depletion of the retromer by siRNA against Vps35 leads to mis-sorting of DMT1-II to LAMP2-positive structures, and expression of siRNA-resistant Vps35 can rescue this effect. These findings demonstrate that...