研究者詳細 - 表　弘志

論文 - 表　弘志

分割表示 >> ／全件表示 84 件中 1 - 84 件目

Polyamine release and vesicular polyamine transporter expression in megakaryoblastic cells and platelets. 国際誌

Mizuki Uehara, Ayaka Fukumoto, Hiroshi Omote, Miki Hiasa

Biochimica et biophysica acta. General subjects 1868 ( 6 ) 130610 - 130610 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Polyamines not only play essential roles in cell growth and function of living organisms but are also released into the extracellular space and function as regulators of chemical transduction, although the cells from which they are released and their mode of release are not well understood. The vesicular polyamine transporter (VPAT), encoded by the SLC18B1 is responsible for the vesicular storage of spermine and spermidine, followed by their vesicular release from secretory cells. Focusing on VPAT will help identify polyamine-secreting cells and new polyamine functions. In this study, we investigated the possible involvement of VPAT in vesicular release of polyamines in MEG-01 clonal megakaryoblastic cells and platelets. RT-PCR, western blotting, and immunohistochemistry revealed VPAT expression in MEG-01 cells. MEG-01 cells secreted polyamines upon A23187 stimulation in the presence of Ca2+, which is temperature-dependent and sensitive to bafilomycin A1. A23187-induced polyamine secretion from MEG-01 cells was reduced by treatment with reserpine, VPAT inhibitors, or VPAT RNA interference. Platelets also expressed VPAT, displaying a punctate distribution, and released spermidine upon A23187 and thrombin stimulation. These findings have demonstrated VPAT-mediated vesicular polyamine release from MEG-01 cells, suggesting the presence of similar vesicular polyamine release mechanisms in platelets.

DOI： 10.1016/j.bbagen.2024.130610

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors 査読

Loc Dinh Hoang, Eriko Aoyama, Miki Hiasa, Hiroshi Omote, Satoshi Kubota, Takuo Kuboki, Masaharu Takigawa

24 ( 24 ) 17294 - 17294 2023年12月

促進拡散型グルコーストランスポーターGLUT12の輸送機能と組織局在査読

松尾俊介, 日浅未来, 表弘志

168 ( 6 ) 611 - 620 2020年12月

The mitochondrial inner membrane protein LETM1 modulates cristae organization through its LETM domain. 査読国際誌

Seiko Nakamura, Aiko Matsui, Shiori Akabane, Yasushi Tamura, Azumi Hatano, Yuriko Miyano, Hiroshi Omote, Mizuho Kajikawa, Katsumi Maenaka, Yoshinori Moriyama, Toshiya Endo, Toshihiko Oka

Communications biology 3 ( 1 ) 99 - 99 2020年3月

記述言語：英語

LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.

DOI： 10.1038/s42003-020-0832-5

Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter. 査読国際誌

Iwai Y, Kamatani S, Moriyama S, Omote H

Journal of biochemistry 165 ( 6 ) 479 - 486 2019年6月

Purification and reconstitution of polyspecific H⁺/organic cation antiporter human MATE1. 査読国際誌

Kawasaki T, Matsumoto T, Iwai Y, Kawakami M, Juge N, Omote H, Nabekura T, Moriyama Y

Biochimica et biophysica acta. Biomembranes 1860 ( 11 ) 2456 - 2464 2018年11月

Outward open conformation of a Major Facilitator Superfamily multidrug/H⁺ antiporter provides insights into switching mechanism. 査読国際誌

Nagarathinam K, Nakada-Nakura Y, Parthier C, Terada T, Juge N, Jaenecke F, Liu K, Hotta Y, Miyaji T, Omote H, Iwata S, Nomura N, Stubbs MT, Tanabe M

Nature communications 9 ( 1 ) 4005 - 4005 2018年10月

Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli 査読

Mamiyo Kawakami, Narinobu Juge, Yuri Kato, Hiroshi Omote, Yoshinori Moriyama, Takaaki Miyaji

Journal of Proteome Research 17 ( 3 ) 1108 - 1119 2018年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Chemical Society

Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

DOI： 10.1021/acs.jproteome.7b00777

Reconstitution and transport analysis of eukaryotic transporters in the post-genomic era 査読

Hiroshi Omote, Yoshinori Moriyama

Methods in Molecular Biology 1700 343 - 352 2018年

記述言語：英語掲載種別：論文集(書籍)内論文出版者・発行元：Humana Press Inc.

Measuring transport activity through reconstituted proteoliposomes is a key technique to resolve numerous problems found in the traditional methods. The system includes overexpression, purification, and reconstitution of transporters. Mixing of purified transporter with lipid and dilution below the critical micelle concentration result in rapid generation of proteoliposomes. Incubation of proteoliposomes in the presence of a driving force initiates substrate uptake. After starting the reaction, samples are passed through a gel filtration column to separate proteoliposomes from the reaction mixture. Here, we describe step-by-step procedures for such reconstitution assays.

DOI： 10.1007/978-1-4939-7454-2_19

Vesicular nucleotide transporter mediates ATP release and migration in neutrophils 査読

Yuika Harada, Yuri Kato, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa

Journal of Biological Chemistry 293 ( 10 ) 3770 - 3779 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Biochemistry and Molecular Biology Inc.

Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT/ mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo. These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.

DOI： 10.1074/jbc.M117.810168

Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling 査読

Yoshinori Moriyama, Miki Hiasa, Shohei Sakamoto, Hiroshi Omote, Masatoshi Nomura

PURINERGIC SIGNALLING 13 ( 3 ) 387 - 404 2017年9月

記述言語：英語出版者・発行元：SPRINGER

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target.

DOI： 10.1007/s11302-017-9568-1

Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain 査読

Yuri Kato, Miki Hiasa, Reiko Ichikawa, Nao Hasuzawa, Atsushi Kadowaki, Ken Iwatsuki, Kazuhiro Shima, Yasuo Endo, Yoshiro Kitahara, Tsuyoshi Inoue, Masatoshi Nomura, Hiroshi Omote, Yoshinori Moriyama, Takaaki Miyaji

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 ( 31 ) E6297 - E6305 2017年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl-. A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT-/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

DOI： 10.1073/pnas.1704847114

Function and expression of a splicing variant of vesicular glutamate transporter 1 査読

Satomi Moriyama, Masafumi Iharada, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1859 ( 5 ) 931 - 940 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Vesicular glutamate transporter (VGLUT) is an active transporter responsible for vesicular storage of glutamate in synaptic vesicles and plays an essential role in glutamatergic neurotransmission. VGLUT consists of three isoforms, VGLUT1, VGLUT2, and VGLUT3. The VGLUT1 variant, VGLUTlv, with an additional 75-base pair sequence derived from a second intron between exons 2 and 3, which corresponds to 25 amino acid residues in the 1st loop of VGLUT1, is the only splicing variant among VGLUTs, although whether VGLUTlv protein is actually translated at the protein level remains unknown. In the present study, VGLUTly was expressed in insect cells, solubilized, purified to near homogeneity, and its transport activity was examined. Proteoliposomes containing purified VGLUTlv were shown to accumulate glutamate upon imposition of an inside-positive membrane potential (Delta psi). The Delta psi-driven glutamate uptake activity requires Cl- and its pharmacological profile and kinetics are comparable to those of other VGLUTs. The retinal membrane contained two VGLUT1 moieties with apparent molecular masses of 65 and 57 kDa. VGLUT1v-specific antibodies against an inserted 25-amino acid residue sequence identified a 65-kDa immunoreactive polypeptide. Immunohistochemical analysis indicated that VGLUTly immunoreactivity is present in photoreceptor cells and is associated with synaptic vesicles. VGLUT1v immunoreactivity is also present in pinealocytes, but not in other areas, including the brain. These results indicated that VGLUTly exists in a functional state in rat photosensitive cells and is involved in glutamatergic chemical transmission. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamem.2017.02.002

Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells 査読

Tomoya Takeuchi, Yuika Harada, Satomi Moriyama, Kazuyuki Furuta, Satoshi Tanaka, Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama, Miki Hiasa

JOURNAL OF BIOLOGICAL CHEMISTRY 292 ( 9 ) 3909 - 3918 2017年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A(1)-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A(1) and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

DOI： 10.1074/jbc.M116.756197

Structure, Function, and Drug Interactions of Neurotransmitter Transporters in the Postgenomic Era 査読

Hiroshi Omote, Takaaki Miyaji, Miki Hiasa, Narinobu Juge, Yoshinori Moriyama

ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, VOL 56 56 385 - 402 2016年

記述言語：英語掲載種別：論文集(書籍)内論文出版者・発行元：ANNUAL REVIEWS

Vesicular neurotransmitter transporters are responsible for the accumulation of neurotransmitters in secretory vesicles and play essential roles in chemical transmission. The SLC17 family contributes to sequestration of anionic neurotransmitters such as glutamate, aspartate, and nucleotides. Identification and subsequent cellular and molecular biological studies of SLC17 transporters unveiled the principles underlying the actions of these transporters. Recent progress in reconstitution methods in combination with postgenomic approaches has advanced studies on neurotransmitter transporters. This review summarizes the molecular properties of SLC17-type transporters and recent findings regarding the novel SLC18 transporter.

DOI： 10.1146/annurev-pharmtox-010814-124816

Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter 査読

Natsuko Togawa, Narinobu Juge, Takaaki Miyaji, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 309 ( 2 ) C71 - C80 2015年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Membrane potential (Delta psi)-driven and Cl--dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na+-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Delta psi- and Cl--dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4'-diisothiocyanostilbene- 2,2'-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Delta psi- and Cl--dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.

DOI： 10.1152/ajpcell.00048.2015

Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter 査読

Narinobu Juge, Sawako Moriyama, Takaaki Miyaji, Mamiyo Kawakami, Haruka Iwai, Tomoya Fukui, Nathan Nelson, Hiroshi Omote, Yoshinori Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 112 ( 11 ) 3356 - 3361 2015年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Extrusion of chloroquine (CQ) from digestive vacuoles through the Plasmodium falciparum CQ resistance transporter (PfCRT) is essential to establish CQ resistance of the malaria parasite. However, the physiological relevance of PfCRT and how CQ-resistant PfCRT gains the ability to transport CQ remain unknown. We prepared proteoliposomes containing purified CQ-sensitive and CQ-resistant PfCRTs and measured their transport activities. All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. CQ-resistant PfCRT exhibited decreased affinity for CQ, resulting in increased CQ uptake. Furthermore, CQ competitively inhibited amino acid transport. Thus, PfCRT is a H+-coupled polyspecific nutrient and drug exporter.

DOI： 10.1073/pnas.1417102112

AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis 査読

Takaaki Miyaji, Takashi Kuromori, Yu Takeuchi, Naoki Yamaji, Kengo Yokosho, Atsushi Shimazawa, Eriko Sugimoto, Hiroshi Omote, Jian Feng Ma, Kazuo Shinozaki, Yoshinori Moriyama

NATURE COMMUNICATIONS 6 5928 2015年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4; 4 protein exhibit membrane potential- and Cl- dependent ascorbate uptake. The AtPHT4; 4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4; 4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4; 4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

DOI： 10.1038/ncomms6928

Identification of a mammalian vesicular polyamine transporter 査読

Miki Hiasa, Takaaki Miyaji, Yuka Haruna, Tomoya Takeuchi, Yuika Harada, Sawako Moriyama, Akitsugu Yamamoto, Hiroshi Omote, Yoshinori Moriyama

SCIENTIFIC REPORTS 4 6836 2014年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-D-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H+. SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT).

DOI： 10.1038/srep06836

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity 査読

Shohei Sakamoto, Takaaki Miyaji, Miki Hiasa, Reiko Ichikawa, Akira Uematsu, Ken Iwatsuki, Atsushi Shibata, Hisayuki Uneyama, Ryoichi Takayanagi, Akitsugu Yamamoto, Hiroshi Omote, Masatoshi Nomura, Yoshinori Moriyama

SCIENTIFIC REPORTS 4 6689 2014年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic beta-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

DOI： 10.1038/srep06689

Involvement of the Leaf-Specific Multidrug and Toxic Compound Extrusion (MATE) Transporter Nt-JAT2 in Vacuolar Sequestration of Nicotine in Nicotiana tabacum 査読

Nobukazu Shitan, Shota Minami, Masahiko Morita, Minaho Hayashida, Shingo Ito, Kojiro Takanashi, Hiroshi Omote, Yoshinori Moriyama, Akifumi Sugiyama, Alain Goossens, Masataka Moriyasu, Kazufumi Yazaki

PLOS ONE 9 ( 9 ) e108789 2014年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

DOI： 10.1371/journal.pone.0108789

Components of Foods Inhibit a Drug Exporter, Human Multidrug and Toxin Extrusion Transporter 1 査読

Tatsuya Kawasaki, Hideyuki Ito, Hiroshi Omote

BIOLOGICAL & PHARMACEUTICAL BULLETIN 37 ( 2 ) 292 - 297 2014年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PHARMACEUTICAL SOC JAPAN

Human multidrug and toxic compounds extrusion transporter 1 (hMATE1/SLC47A1) is a H+-coupled organic cation exporter responsible for the final step of excretion of various xenobiotics at the kidney and liver. In this study, effects of dietary constituents on hMATE1 mediated drug transport were examined to evaluate possible food-drug interactions. Bergamottin inhibited hMATE1 mediated tetraethyl ammonium transport activity, with a K-i of 98.7 mu M. Coumarins, flavonols, and catechin inhibited hMATE1 activity. Among 23 compounds tested, isorhamnetin was the strongest inhibitor of hMATE1 with the K-i of 0.32 mu M in a competitive manner. Since isorhamnetin is abundant in Ginkgo biloba that is widely used for herbal supplements, the findings suggest the potential hMATE1 related food-drug interactions.

DOI： 10.1248/bpb.b13-00815

Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets 査読

Miki Hiasa, Natsuko Togawa, Takaaki Miyaji, Hiroshi Omote, Akitsugu Yamamoto, Yoshinori Moriyama

Physiological Reports 2 ( 6 ) 2014年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Physiological Society

Nucleotides are stored in the dense granules of platelets. The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation. However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood. The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized. We hypothesized that the vesicular nucleotide transporter (VNUT) is also involved in the vesicular storage of nucleotides in platelets. In this article, we present three lines of evidence that VNUT is responsible for the vesicular storage of nucleotides in platelets and that vesicular ATP transport is crucial for platelet function, detection and characterization of VNUT activity in platelets isolated from healthy humans and MEG-01 cells, RNA interference experiments on MEG-01 cells, and studies on nucleotide transport and release with a selective inhibitor.

DOI： 10.14814/phy2.12034

Vesicular GABA transporter (VGAT) transports β-alanine. 査読

Juge N, Omote H, Moriyama Y

Journal of neurochemistry 127 ( 4 ) 482 - 486 2013年11月

Inhibitors of ATP release inhibit vesicular nucleotide transporter 査読

Yuri Kato, Hiroshi Omote, Takaaki Miyaji

Biological and Pharmaceutical Bulletin 36 ( 11 ) 1688 - 1691 2013年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Vesicular nucleotide transporter (VNUT) is responsible for vesicular ATP storage in ATP-secreting cells. In the present study, we examined the effects on VNUT-mediated transport of ATP release inhibitors such as ATP-binding cassette (ABC) proteins, hemichannels, maxi anion channels and P2X7 receptor. The ATP transport activity of proteoliposomes containing purified human VNUT was blocked by glibenclamide, carbenoxolone, 18 á-glycyrrhetinic acid, flufenamic acid, arachidonic acid and A438079 without the formation of Äø (positive inside) as a driving force being affected. Thus, inhibitors of ATP release may inhibit VNUT and subsequent ATP release, since the previous works proved that inhibitors of ATP release blocked VNUT-mediated ATP release at the cell level. © 2013 The Pharmaceutical Society of Japan.

DOI： 10.1248/bpb.b13-00544

Vesicular neurotransmitter transporters: An approach for studying transporters with purified proteins 査読

Hiroshi Omote, Yoshinori Moriyama

Physiology 28 ( 1 ) 39 - 50 2013年1月

記述言語：英語

Vesicular storage and subsequent release of neurotransmitters are the key processes of chemical signal transmission. In this process, vesicular neurotransmitter transporters are responsible for loading the signaling molecules. The use of a "clean biochemical" approach with purified, recombinant transporters has helped in the identification of novel vesicular neurotransmitter transporters and in the analysis of the control of signal transmission. © 2013 Int. Union Physiol. Sci./Am. Physiol. Soc.

DOI： 10.1152/physiol.00033.2012

Type 1 sodium-dependent phosphate transporter acts as a membrane potential-driven urate exporter 査読

Takaaki Miyaji, Tatsuya Kawasaki, Natsuko Togawa, Hiroshi Omote, Yoshinori Moriyama

Current Molecular Pharmacology 6 ( 2 ) 88 - 94 2013年

記述言語：英語掲載種別：研究論文（学術雑誌）

SLC17A1 protein (NPT1) was the first identified member of the SLC17 phosphate transporter family, and is known to mediate Na+/inorganic phosphate (Pi) co-transport when expressed in Xenopus oocytes. Although this protein was suggested to be a renal polyspecific anion exporter, its transport properties were not well characterized. The clean biochemical approach revealed that proteoliposomes comprising purified NPT1 as the only protein source transport various organic anions such as urate, p-aminohippuric acid (PAH), and acetylsalicylic acid (aspirin) in a membrane potential (δ̄)-driven and Cl- -dependent manner. Human NPT1 carrying an SNP mutation, Thr269Ile, known to increase the risk of gout, exhibited 32% lower urate transport activity compared to the wild type protein, leading to the conclusion that NPT1 is the long searched for transporter responsible for renal urate excretion. In the present article, we summarized the history of identification of the urate exporter and its possible involvement in the dynamism of urate under physiological and pathological conditions. © 2013 Bentham Science Publishers.

DOI： 10.2174/18744672113069990035

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter 査読

Natsuko Togawa, Takaaki Miyaji, Sho Izawa, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 302 ( 11 ) C1652 - C1660 2012年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Togawa N, Miyaji T, Izawa S, Omote H, Moriyama Y. A Na+ phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter. Am J Physiol Cell Physiol 302: C1652-C1660, 2012. First published March 28, 2012; doi:10.1152/ajpcell.00015.2012.-The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Delta psi)- and Cl- dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na+-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl--dependent manner at the expense of an electrochemical gradient of protons, especially Delta psi, across the membrane. The Delta psi- and Cl--dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.

DOI： 10.1152/ajpcell.00015.2012

Divalent Cation Transport by Vesicular Nucleotide Transporter 査読

Takaaki Miyaji, Keisuke Sawada, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 286 ( 50 ) 42881 - 42887 2011年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Background: The vesicular nucleotide transporter (VNUT) transports nucleotides in the presence of Mg2+.
Results: VNUT transports divalent cations in a membrane potential-and nucleotide-dependent manner.
Conclusion: VNUT transports divalent cations as a nucleotide complex.
Significance: VNUT functions as a divalent cation importer in secretory vesicles.

DOI： 10.1074/jbc.M111.277269

Functional characterization of vesicular excitatory amino acid transport by human sialin 査読

Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF NEUROCHEMISTRY 119 ( 1 ) 1 - 5 2011年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL

Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Delta psi)-driven aspartate and glutamate transport into synaptic vesicles in addition to H(+)/sialic acid cotransport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H(+)/sialic acid cotransport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Delta psi-driven aspartate and glutamate transport activity and H(+)/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H(+)/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Delta 268SSLRN272 still showed normal levels of Delta psi-driven aspartate and glutamate transport even though H(+)/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.

DOI： 10.1111/j.1471-4159.2011.07388.x

Characterization of the human MATE2 proton-coupled polyspecific organic cation exporter 査読

Toshinori Komatsu, Miki Hiasa, Takaaki Miyaji, Takuji Kanamoto, Takuya Matsumoto, Masato Otsuka, Yoshinori Moriyama, Hiroshi Omote

INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 43 ( 6 ) 913 - 918 2011年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

Human multidrug and toxic compound extrusion 2 (hMATE2) is a kidney-specific isoform of hMATE1, an exporter of toxic organic cations (OCs) of exogenous and endogenous origins at the final excretion step in the kidneys and liver (Otsuka et al., 2005), and contains a splicing variant, MATE2K, that has an exon of hMATE2 deleted (Masuda et al., 2006). In the present study, we characterized the degree of expression and the transport properties of hMATE2. Quantitative PCR analysis with probes specific for hMATE2 indicated the presence of hMATE2 mRNA in the kidneys, which corresponded to 39% of total mRNA encoding both hMATE2 and hMATE2K. hMATE2-specific antibodies immunostained the renal urinary tubules. Upon expression in HEK293 cells, hMATE2 was localized in intracellular vesicular structures, and thus transport activity of tetraethylammonium (TEA), a typical substrate for MATE transporters, by the cells was not detected. The hMATE2 protein was purified and reconstituted into liposomes. An artificially imposed pH gradient (Delta pH) across the proteoliposomal membrane drove the uptake of TEA. Dissipation of Delta pH by ammonium sulfate effectively inhibited the TEA uptake, while that of the membrane potential by valinomycin had little effect. The profiles of cis-inhibition of TEA transport by hMATE2 and hMATE2K are similar to each other. Thus, both hMATE2 and hMATE2K equally operate in the human kidneys to extrude OCs into the urine. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biocel.2011.03.005

Vesicular Neurotransmitter Transporter: Bioenergetics and Regulation of Glutamate Transport 査読

Hiroshi Omote, Takaaki Miyaji, Narinobu Juge, Yoshinori Moriyama

BIOCHEMISTRY 50 ( 25 ) 5558 - 5565 2011年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

Glutamate plays essential roles in chemical transmission as a major excitatory neurotransmitter. The accumulation of glutamate in Secretory vesicles is mediated by vesicular glutamate transporters (VGLUTs) that together with the driving electrochemical gradient of proteins influence the subsequent quantum release Of glutamate and the function of higher order neurons. The vesicular content of glutamate is Well correlated with membrane potential (Delta psi) which suggests that Delta psi determines the vesicular glutamate concentration The transport of glutamate into secretory vesicles is highly dependent on Cl(-). This anion stimulates glutamate transport but is inhibitory at higher concentrations. Accumulating evidence indicates that Cl(-) regulates glutamate transport through control of VGLUT activity and the H(+) electrochemical gradient. Recently, a comprehensive study demonstrated that Cl(-) regulation of VGLUT is competitively inhibited by metabolic intermediates such as ketone bodies. It also showed that ketone bodies are effective in controlling epilepsy. These results suggest a correlation between metabolic state and higher order brain function. We propose a novel function for Cl(-) as a fundamental regulator for signal transmission.

DOI： 10.1021/bi200567k

A Vesicular Transporter That Mediates Aspartate and Glutamate Neurotransmission 査読

Takaaki Miyaji, Hiroshi Omote, Yoshinori Moriyama

BIOLOGICAL & PHARMACEUTICAL BULLETIN 33 ( 11 ) 1783 - 1785 2010年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PHARMACEUTICAL SOC JAPAN

Aspartate, an excitatory amino acid, is known to be stored in synaptic vesicles and exocytosed from some neurons to perform aspartergic neurotransmission. Through in vitro reconstitution, we found that sialin, a lysosomal sialic acid exporter, is responsible for the vesicular storage of aspartate in hippocampal neurons and pinealocytes. Mutations found in Salla disease cause decreased aspartate transport activity without affecting sialic acid transport. Thus, sialin is a multifunctional transporter. It is possible that people with Salla disease lose the ability of aspartergic neurotransmission, and this could explain why Salla disease involves severe neurological defects.

DOI： 10.1248/bpb.33.1783

Metabolic Control of Vesicular Glutamate Transport and Release 査読

Narinobu Juge, John A. Gray, Hiroshi Omote, Takaaki Miyaji, Tsuyoshi Inoue, Chiaki Hara, Hisayuki Uneyama, Robert H. Edwards, Roger A. Nicoll, Yoshinori Moriyama

NEURON 68 ( 1 ) 99 - 112 2010年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CELL PRESS

Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl(-). Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl(-) acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity.

DOI： 10.1016/j.neuron.2010.09.002

Type 1 Sodium-dependent Phosphate Transporter (SLC17A1 Protein) Is a Cl--dependent Urate Exporter 査読

Masafumi Iharada, Takaaki Miyaji, Takahiro Fujimoto, Miki Hiasa, Naohiko Anzai, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 285 ( 34 ) 26107 - 26113 2010年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

SLC17A1 protein (NPT1) is the first identified member of the SLC17 phosphate transporter family and mediates the transmembrane cotransport of Na+/P-i in oocytes. Although this protein is believed to be a renal polyspecific anion exporter, its transport properties are not well characterized. Here, we show that proteoliposomes containing purified SLC17A1 transport various organic anions such as p-aminohippuric acid and acetylsalicylic acid (aspirin) in an inside positive membrane potential (Delta psi)-dependent manner. We found that NPT1 also transported urate. The uptake characteristics were similar to that of SLC17 members in its Cl- dependence and inhibitor sensitivity. When arginine 138, an essential amino acid residue for members of the SLC17 family such as the vesicular glutamate transporter, was specifically mutated to alanine, the resulting mutant protein was inactive in Delta psi-dependent anion transport. Heterologously expressed and purified human NPT1 carrying the single nucleotide polymorphism mutation that is associated with increased risk of gout in humans exhibited 32% lower urate transport activity compared with the wild type protein. These results strongly suggested that NPT1 is a Cl--dependent polyspecific anion exporter involved in urate excretion under physiological conditions.

DOI： 10.1074/jbc.M110.122721

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins 査読

Yo Sonoda, Alex Cameron, Simon Newstead, Hiroshi Omote, Yoshinori Moriyama, Michihiro Kasahara, So Iwata, David Drew

FEBS LETTERS 584 ( 12 ) 2539 - 2547 2010年6月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE BV

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called 'tricks-of-thetrade' and include case studies of several mammalian transporters. (C) 2010 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/j.febslet.2010.04.015

Vesicular Inhibitory Amino Acid Transporter Is a Cl-/gamma-Aminobutyrate Co-transporter 査読

Narinobu Juge, Akiko Muroyama, Miki Hiasa, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 284 ( 50 ) 35073 - 35078 2009年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of gamma-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Delta psi) (positive inside) but not Delta pH. VIAAT-mediated GABA uptake had an absolute requirement for Cl- and actually accompanied Cl- movement. Kinetic analysis indicated that one GABA molecule and two Cl- equivalents were transported during one transport cycle. VIAAT in which Glu(213) was specifically mutated to alanine completely lost the ability to take up both GABA and Cl-. Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl- transporter that co-transports Cl- with GABA or glycine in a Delta psi dependent manner. It is concluded that Cl- plays an essential role in vesicular storage of GABA and glycine.

DOI： 10.1074/jbc.M109.062414

[Localization mechanism of vesicular neurotransmitter transporters]. 査読

Omote, H., Moriyama, Y.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 16 Suppl ) 2220 - 2224 2008年12月

Vesicular glutamate transporter acts as a metabolic regulator 査読

Yoshinori Moriyama, Hiroshi Omote

BIOLOGICAL & PHARMACEUTICAL BULLETIN 31 ( 10 ) 1844 - 1846 2008年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PHARMACEUTICAL SOC JAPAN

Vesicular glutamate transporter (VGLUT) is responsible for the active transport of L-glutamate into synaptic vesicles and, thus, plays an essential role in the glutamatergic chemical transmission in the central and peripheral nervous systems. Recent studies indicated that VGLUT is also expressed and localized in various secretory vesicles in non-neuronal peripheral organelles such as hormone-containing secretory granules in endocrine cells. L-Glutamate is stored in VGLUT-containing organelles, secreted upon stimulation, and then acts as a paracrine and/or autocrine modulator to regulate cellular functions. Thus. VGLUT is a key molecule for glutamate signaling and is the core of a novel signaling system.

DOI： 10.1248/bpb.31.1844

Identification of a vesicular aspartate transporter 査読

Takaaki Miyaji, Noriko Echigo, Miki Hiasa, Shigenori Senoh, Hiroshi Omote, Yoshinori Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 ( 33 ) 11720 - 11724 2008年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes and is exocytosed and stimulates neighboring cells by binding to specific cell receptors. Although evidence increasingly supports the occurrence of aspartergic neurotransmission, this process is still debated because the mechanism for the vesicular storage of aspartate is unknown. Here, we show that sialin, a lysosomal H+/sialic acid cotransporter, is present in hippocampal synaptic vesicles and pineal SLMVs. RNA interference of sialin expression decreased exocytosis of aspartate and glutamate in pinealocytes. Proteoliposomes containing purified sialin actively accumulated aspartate and glutamate to a similar extent when inside positive membrane potential is imposed as the driving force. Sialin carrying a mutation found in people suffering from Salla disease (R39C) was completely devoid of aspartate and glutamate transport activity, although it retained appreciable H+/sialic acid cotransport activity. These results strongly suggest that sialin possesses dual physiological functions and acts as a vesicular aspartate/glutamate transporter. It is possible that people with Salla disease lose aspartergic (and also the associated glutamatergic) neurotransmission, and this could provide an explanation for why Salla disease causes severe neurological defects.

DOI： 10.1073/pnas.0804015105

Identification of a vesicular nucleotide transporter 査読

Keisuke Sawada, Noriko Echigo, Narinobu Juge, Takaaki Miyaji, Masato Otsuka, Hiroshi Omote, Akitsugu Yamamoto, Yoshinori Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 ( 15 ) 5683 - 5686 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATIP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.

DOI： 10.1073/pnas.0800141105

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter 査読

Takuya Matsumoto, Takuji Kanamoto, Masato Otsuka, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 294 ( 4 ) C1074 - C1078 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Human multidrug and toxic compound extrusion 1 ( hMATE1) is an electroneutral H+/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium ( TEA) and cimetidine as substrates after expression in human embryonic kidney 293 ( HEK- 293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK- 293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wildtype transporter with about a fourfold decrease in V-max value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/ or transport of TEA and cimetidine but that their individual roles are different.

DOI： 10.1152/ajpcell.00504.2007

A novel variant of mouse MATE-1 H+/organic cation antiporter with a long hydrophobic tall 査読

Ayumi Kobara, Miki Hiasa, Takuya Matsumoto, Masato Otsuka, Hiroshi Omote, Yoshinori Moriyama

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 469 ( 2 ) 195 - 199 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H+-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2007.10.010

[Identification, structure and function of a drug transporter at the final step of extrusion]. 査読

Hiasa M, Omote H, Moriyama Y

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 1 ) 52 - 58 2008年1月

Multidrug and toxic compound extrusion (MATE)-type proteins as anchor transporters for the excretion of metabolic waste products and xenobiotics 査読

Y. Moriyama, M. Hiasa, T. Matsumoto, H. Omote

XENOBIOTICA 38 ( 7-8 ) 1107 - 1118 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INFORMA HEALTHCARE

1. Multidrug and toxic compound extrusion (MATE)-type transporters, which were first identified as a bacterial drug transporter family, are present in almost all prokaryotes and eukaryotes, and are thus one of the mostly conserved transporter families in nature.
2. Recently, a mammalian MATE transporter was shown to be a long hypothesized electroneutral H(+)/organic cation exporter that is responsible for the excretion of metabolic waste products and xenobiotics at renal brush border membranes and bile canaliculi. Plant MATE-type transporters are involved in the detoxification of metals and secondary metabolites such as phenols through their vesicular storage or extrusion at the plasma membrane.
3. Thus, MATE transporters are involved in one of the basic mechanisms that maintain homeostasis through the excretion of metabolic waste products and xenobiotics in nature.

DOI： 10.1080/00498250701883753

Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H+/organic cation exporter 査読

Miki Hiasa, Takuya Matsumoto, Toshinori Komatsu, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 293 ( 5 ) C1437 - C1444 2007年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Mammalian multidrug and toxic compound extrusion ( MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H+-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney ( Otsuka et al. Proc Natl Acad Sci USA 102: 17923-17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587-593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H+-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K+. Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H+/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.

DOI： 10.1152/ajpcell.00280.2007

Molecular mechanism of vesicular glutamate transporter 査読

Hiroshi Omote, Narinobu Juge

SEIKAGAKU 79 ( 10 ) 956 - 960 2007年10月

Vesicular glutamate transporter contains two independent transport machineries 査読

Narinobu Juge, Yumi Yoshida, Shouki Yatsushiro, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 281 ( 51 ) 39499 - 39506 2006年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F0F1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (Delta psi)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for similar to 4 mM Cl-, was sensitive to Evans blue, but was insensitive to D, L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in L-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a Delta psi-dependent L-glutamate uptake and a Na+-dependent Pi uptake.

DOI： 10.1074/jbc.M607670200

The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations 査読

Hiroshi Omote, Miki Hiasa, Takuya Matsumoto, Masato Otsuka, Yoshinori Moriyama

TRENDS IN PHARMACOLOGICAL SCIENCES 27 ( 11 ) 587 - 593 2006年11月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE LONDON

Multidrug and toxic compound extrusion (MATE) proteins, comprising the most recently designated family of multidrug transporter proteins, are widely distributed in all kingdoms of living organisms, although their function is far from understood. The bacterial MATE-type transporters that have been characterized function as exporters of cationic drugs, such as norfloxacin and ethidium, through H+ or Na+ exchange. Plant MATE-type transporters are involved in the detoxification of secondary metabolites, including alkaloids. Mammalian MATE-type transporters are responsible for the final step in the excretion of metabolic waste and xenobiotic organic cations in the kidney and liver through electroneutral exchange of H+. Thus, we propose that members of the MATE family are organic cation exporters that excrete metabolic or xenobiotic organic cations from the body.

DOI： 10.1016/j.tips.2006.09.001

Secretion of L-glutamate from osteoclasts through transcytosis 査読

Riyo Morimoto, Shunsuke Uehara, Shouki Yatsushiro, Narinobu Juge, Zhaolin Hua, Shigenori Senoh, Noriko Echigo, Mitsuko Hayashi, Toshihide Mizoguchi, Tadashi Ninomiya, Nobuyuki Udagawa, Hiroshi Omote, Akitsugu Yamamoto, Robert H. Edwards, Yoshinori Moriyama

EMBO JOURNAL 25 ( 18 ) 4175 - 4186 2006年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl-and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1(-/-) knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1(-/-) mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

DOI： 10.1038/sj.emboj.7601317

破骨細胞によるトランスサイトーシスを介したグルタミン酸分泌とその生理的意義

上原俊介, 森本理代, 八代聖基, 樹下成信, 林美都子, 妹尾繁範, 溝口利英, 二宮禎, 宇田川信之, Hua Zhaolin, 表弘志, 山本章嗣, Edwards Robert H., 森山芳則

松本歯学 32 ( 2 ) 171 - 172 2006年8月

Interaction of transported drugs with the lipid bilayer and beta-glycoprotein through a solvation exchange mechanism 査読

H Omote, MK Al-Shawi

BIOPHYSICAL JOURNAL 90 ( 11 ) 4046 - 4059 2006年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOPHYSICAL SOCIETY

Broad substrate specificity of human P-glycoprotein (ABCB1) is an essential feature of multidrug resistance. Transport substrates of P-glycoprotein are mostly hydrophobic and many of them have net positive charge. These compounds partition into the membrane. Utilizing the energy of ATP hydrolysis, P-glycoprotein is thought to take up substrates from the cytoplasmic lea. et of the plasma membrane and to transport them to the outside of the cell. We examined this model by molecular dynamics simulation of the lipid bilayer, in the presence of transport substrates together with an atomic resolution structural model of P-glycoprotein. Taken together with previous electron paramagnetic resonance studies, the results suggest that most transported drugs are concentrated near the surface zone of the inner lea. et of the plasma membrane. Here the drugs can easily diffuse laterally into the drug-binding site of P-glycoprotein through an open cleft. It was concluded that the initial high-affinity drug-binding site was located in the interfacial surface area of P-glycoprotein in contact with the membrane interface. Based on these results and our recent kinetic studies, a "solvation exchange'' drug transport mechanism of P-glycoprotein is discussed. A molecular basis for the improved colchicine transport efficiency by the much-studied colchicine-resistance G185V mutant human P-glycoprotein is also provided.

DOI： 10.1529/biophysj.105.077743

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast 査読

A Ohashi-Kobayashi, K Ohashi, WB Du, H Omote, R Nakamoto, M Al-shawi, M Maeda

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 343 ( 2 ) 597 - 601 2006年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these Compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.03.002

Transmembrane topology of vesicular glutamate transporter 2 査読

SK Jung, R Morimoto, M Otsuka, H Omote

BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 ( 3 ) 547 - 549 2006年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PHARMACEUTICAL SOC JAPAN

Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.

DOI： 10.1248/bpb.29.547

A human transporter protein that mediates the final excretion step for toxic organic cations 査読

M Otsuka, T Matsumoto, R Morimoto, S Arioka, H Omote, Y Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 ( 50 ) 17923 - 17928 2005年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

In mammals, toxic electrolytes of endogenous and exogenous origin are excreted through the urine and bile. Before excretion, these compounds cross numerous cellular membranes in a transporter-mediated manner. However, the protein transporters involved in the final excretion step are poorly understood. Here, we show that MATE1, a human and mouse orthologue of the multidrug and toxin extrusion family conferring multidrug resistance on bacteria, is primarily expressed in the kidney and liver, where it is localized to the luminal membranes of the urinary tubules and bile canaliculi. When expressed in HEK293 cells, MATE1 mediates H+-coupled electroneutral exchange of tetraethylammonium and 1-methyl-4-phenylpyridinium. its substrate specificity is similar to those of renal and hepatic H+-coupled organic cations (OCs) export. Thus, MATE1 appears to be the long searched for polyspecific OC exporter that directly transports toxic OCs into urine and bile.

DOI： 10.1073/pnas.0506483102

The remarkable transport mechanism of P-glycoprotein: A multidrug transporter 査読

MK Al-Shawi, H Omote

JOURNAL OF BIOENERGETICS AND BIOMEMBRANES 37 ( 6 ) 489 - 496 2005年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER/PLENUM PUBLISHERS

Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that utilizes the energy of ATP hydrolysis to PUMP toxic xenobiotics out of cells. P-glycoprotein employs a Most unusual molecular mechanism to perform this drug transport function. Here we review Our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein. in the yeast Saccharomyces cerevisiae, has facilitated biophysical Studies in Purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results Of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling, and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised 10 place Our results within a proper structural context.

DOI： 10.1007/s10863-005-9497-5

Proteolipid of vacuolar H+-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant 査読

Shouki Yatsushiro, Shinya Taniguchi, Toshihide Mitamura, Hiroshi Omote, Yoshinori Moriyama

Biochimica et Biophysica Acta - Biomembranes 1717 ( 2 ) 89 - 96 2005年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Vacuolar H+-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamem.2005.08.011

Proteolipid of vacuolar H(+)-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant. 査読

Yatsushiro S, Taniguchi S, Mitamura T, Omote H, Moriyama Y

Biochimica et biophysica acta 1717 ( 2 ) 89 - 96 2005年11月

Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus 査読

M Otsuka, M Yasuda, Y Morita, C Otsuka, T Tsuchiya, H Omote, Y Moriyama

JOURNAL OF BACTERIOLOGY 187 ( 5 ) 1552 - 1558 2005年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

DOI： 10.1128/jb.187.1552-1558.2005

Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation 査読

H Omote, RA Figler, MK Polar, MK Al-Shawi

BIOCHEMISTRY 43 ( 13 ) 3917 - 3928 2004年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K,, for colchicine and a lower K,, for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.

DOI： 10.1021/bi0353651

3SD03 P-糖タンパク質の構造と薬剤排出の分子機構(構造から見た膜輸送)

表弘志, Al-Shawi Marwan K.

生物物理 44 S24 2004年

Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein 査読

MK Al-Shawi, MK Polar, H Omote, RA Figler

JOURNAL OF BIOLOGICAL CHEMISTRY 278 ( 52 ) 52629 - 52640 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degreesC, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (k(cat)), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.

DOI： 10.1074/jbc.M308175200

A novel electron paramagnetic resonance approach to determine the mechanism of drug transport by P-glycoprotein 査読

H Omote, MK Al-Shawi

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 47 ) 45688 - 45694 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

ATP-driven pumping of a variety of drugs out of cells by the human P-glycoprotein poses a serious problem to medical therapy. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Membrane permeability of transported drugs and consequent lack of an experimentally defined drug position have made resolution of the transport mechanism difficult by classical techniques. To overcome these obstacles we devised a novel EPR spin-labeled verapamil for use as a transport substrate. Spin-labeled verapamil was an excellent transport substrate with apparent turnover number, K-m and K-i values of 5.8 s(-1), 4 mum, and 210 muM, respectively, at pH 7.4 and 37 degreesC. The apparent affinities were similar to10-fold higher than for unlabeled verapamil. Spin-labeled verapamil stimulated ATPase activity similar to5-fold, was relatively hydrophilic, and had a very low flip-flop rate, making it an ideal transport substrate. The K-m for MgATP activation of transport was 0.8 mm. By measuring the mobility of spin-labeled verapamil during transport experiments, we were able to resolve the location of the drug in proteoliposome suspensions. Steady state gradients of spin-labeled verapamil within the range of K-i/K-m ratios were observed.

DOI： 10.1074/jbc.M206479200

Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: High-yield expression and purification of human P-glycoprotein 査読

RA Figler, H Omote, RK Nakamoto, MK Al-Shawi

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 376 ( 1 ) 34 - 46 2000年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae, Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0.4-0.7 mu mol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated, By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone, Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp, Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K-M(MgATP),V-max, and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies. (C) 2000 Academic Press.

Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation. 査読

Sambongi Y, Iko Y, Tanabe M, Omote H, Iwamoto-Kihara A, Ueda I, Yanagida T, Wada Y, Futai M

Science (New York, N.Y.) 286 ( 5445 ) 1722 - 1724 1999年11月

Stability of the Escherichia coli ATP synthase F0F1 complex is dependent on interactions between gamma Gln-269 and the beta subunit loop beta Asp-301-beta Asp-305 査読

H Omote, K Tainaka, K Fujie, A Iwamoto-Kihara, Y Wada, M Futai

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 358 ( 2 ) 277 - 282 1998年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

The role of the conserved sequence motif (DDLTDP306)-D-301 in the F0F1 ATP synthase beta subunit was assessed by mutagenic analysis in the Escherichia coli enzyme. Mutations gave variable effects on F-1 sector activity, stability, and membrane binding to the F-0 sector. Upon solubilization, F-1 sectors of the beta D302E and beta D305E mutants (beta Asp-302 and beta Asp-305 replaced by glutamate) dissociated into subunits, while mutants with other beta 305 substitutions failed to assemble. Membrane ATPase activities of beta 301 and 302 mutants were 20-70% of wild type. Replacements of the gamma subunit Gln-269 had similar effects. The membrane ATPase activities of the gamma Q269E or gamma Q269D mutants were significantly lower and their F-1 sectors dissociated into subunits upon solubilization. These results suggest that the beta 301-305 loop and the gamma subunit region around Gln-269 form a key region for the assembly of alpha(3)beta(3)gamma complex. These results are consistent with the X-ray crystallographic structure of bovine F-1 (J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1994) Nature 370, 621-628) where the beta(301)DDLTD(305) loop directly interacts with gamma Gln-269. (C) 1988 Academic Press.

Essential Cys-Pro-Cys Motif of Caenorhabditis elegans Copper Transport ATPase

Yoshimizu Takao, Omote Hiroshi, Wakabayashi Tokumitsu, SAMBONGI Yoshihiro, FUTAI Masamitsu

Bioscience, biotechnology, and biochemistry 62 ( 6 ) 1258 - 1260 1998年6月

記述言語：英語出版者・発行元：社団法人日本農芸化学会

Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast &lrtri;ccc2 mutant (copper ATPase gene disruptant). We found that CUA-1 with Cys-Pro-Cys to Cys-Pro-Ala mutation could not rescue the yeast &lrtri;ccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.

DOI： 10.1271/bbb.62.1258

その他リンク： https://jlc.jst.go.jp/DN/JALC/00085042551?from=CiNii

Mutational analysis of F₁F₀ ATPase: Catalysis and energy coupling

Omote, H., Futai, M.

Acta Physiologica Scandinavica, Supplement 163 ( 643 ) 1998年

Atomic force microscopy of Escherichia coli FoF1-ATPase in reconstituted membranes 査読

Takeyasu, K., Omote, H., Nettikadan, S., Tokumasu, F., Iwamoto-Kihara, A., Futai, M.

Annals of the New York Academy of Sciences 834 ( 1 Na/K-ATPase a ) 149 - 152 1997年11月

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Futai, M., Omote, H.

Journal of Bioenergetics and Biomembranes 28 ( 5 ) 1996年

Molecular imaging of Escherichia coli F₀F₁-ATPase in reconstituted membranes using atomic force microscopy

Takeyasu, K., Omote, H., Nettikadan, S., Tokumasu, F., Iwamoto-Kihara, A., Futai, M.

FEBS Letters 392 ( 2 ) 1996年

Chapter 3 F-type H⁺ ATPase (ATP synthase): Catalytic site and energy coupling

Futal, M., Omote, H.

Handbook of Biological Physics 2 ( C ) 1996年

Escherichia coli H+-ATPase (ATP synthase): Catalytic site and roles of subunit interactions in energy coupling 査読

M Futai, H Omote, M Maeda

BIOCHEMICAL SOCIETY TRANSACTIONS 23 ( 4 ) 785 - 789 1995年11月

BETA-SUBUNIT GLU-185 OF ESCHERICHIA-COLI H+-ATPASE (ATP SYNTHASE) IS AN ESSENTIAL RESIDUE FOR COOPERATIVE CATALYSIS 査読

H OMOTE, NP LE, MY PARK, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 270 ( 43 ) 25656 - 25660 1995年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Glu-beta 185 of the Escherichia coli H+-ATPase (ATP synthase) beta subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-beta 185 were less than 0.2% of the wild type one; the Asp-beta 185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-beta 185 F-1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F-1. The divalent cation dependences of the S-carboxymethyl-beta 185 and Asp-beta 185 ATPase activities were altered from that of the mild type, The Asp-beta 185, Cys-beta 185, S-carboxymethyl-beta 185, and Gln-beta 185 enzymes showed about 130, 60, 20, and 50% of the mild type unisite catalysis rates, respectively. The S-carboxymethyl-beta 185 and Asp-beta 185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-beta 185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg . ADP complex. These results suggest that Glu-beta 185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-beta 185 is located close to the catalytic site in the higher order structure (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).

BETA-GAMMA SUBUNIT INTERACTION IS REQUIRED FOR CATALYSIS BY H+-ATPASE (ATP SYNTHASE) - BETA-SUBUNIT AMINO-ACID REPLACEMENTS SUPPRESS A GAMMA-SUBUNIT MUTATION HAVING A LONG UNRELATED CARBOXYL-TERMINUS 査読

C JEANTEURDEBEUKELAER, H OMOTE, A IWAMOTOKIHARA, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 270 ( 39 ) 22850 - 22854 1995年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The mechanisms of energy coupling and catalytic cooperativity are not yet understood for H+-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M., (1990) J. Biol, Chem, 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52 --> Cys or Gly-beta 150 --> Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77 --> Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.

CONSERVED GLU-181 AND ARG-182 RESIDUES OF ESCHERICHIA-COLI H+-ATPASE (ATP SYNTHASE) BETA-SUBUNIT ARE ESSENTIAL FOR CATALYSIS - PROPERTIES OF 33 MUTANTS BETWEEN BETA-GLU-181 AND BETA-LYS-801 RESIDUES 査読

MY PARK, H OMOTE, M MAEDA, M FUTAI

JOURNAL OF BIOCHEMISTRY 116 ( 5 ) 1139 - 1145 1994年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPANESE BIOCHEMICAL SOC

Twenty-two mutants between beta Glu-161 and beta Lys-201 of Escherichia coli H+-ATPase beta subunit could grow by oxidative phosphorylation, but 11 other such mutants, beta Glu-181-->Gln, Asp, Asn, Thr, Ser, Ale, or Lys and beta Arg-182-->Lys, Ala, Glu, or Gin, could not. The beta Asp-181, beta Lys-182, and other defective mutants had 1.4, 1, and < 0.1%, respectively, of the wild-type membrane ATPase activity. Partially purified F-1-ATPases from all mutants at positions 181 and 182, except for the beta Asp-181 and beta Lys-182 mutants, showed very low unisite catalysis. Purified F-1-ATPases of the beta Gln-181 and beta Ala-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (less than or equal to 1% of that of the wild type): their defects could be attributed to decreased catalytic rates (low k(+2) and k(-2)). Changes of the k(+2) and k(-2) values in the beta Asp-181 enzyme, which showed detectable multi- and unisite catalysis, were less marked (27 and 21%, respectively, of wild-type rates). The beta Gln-182 enzyme showed defective catalysis (less than or equal to 0.1% of the multi- and similar to 1% of the unisite catalyses of the wild type), whereas the beta Lys-182 enzyme showed 1 and 85% of the wild-type multisite and unisite catalytic rates, respectively. beta Lys-182 had wild-type values of k(+2) and k(-2), but beta Gln-182 had k(+2) about 10-fold lower than that of wild type. The position 181 and 182 mutant enzymes had significantly increased K-d (k(-1)/k(+1)) values, reflecting decreased substrate binding. These results suggest that beta Glu-181 and beta Arg-182 are essential for substrate binding, although mutations with conservative substitutions at these positions do not have drastic effects. This study also indicates the importance of the conserved Gly-Glu-Arg (GER) sequence (beta 180-beta 182).

CATALYSIS AND ENERGY COUPLING OF H+-ATPASE (ATP SYNTHASE) - MOLECULAR BIOLOGICAL APPROACHES 査読

M FUTAI, MY PARK, A IWAMOTO, H OMOTE, M MAEDA

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1187 ( 2 ) 165 - 170 1994年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The molecular biological approach has provided important information for understanding the FoF1 H+-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174 --> Phe mutant was suppressed by the second mutation alpha Arg-296 --> Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23 --> Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

THE ALPHA/BETA SUBUNIT INTERACTION IN H+-ATPASE (ATP SYNTHASE) - AN ESCHERICHIA-COLI ALPHA-SUBUNIT MUTATION (ARG-ALPHA-296 -] CYS) RESTORES COUPLING EFFICIENCY TO THE DELETERIOUS BETA-SUBUNIT MUTANT (SER-BETA-174 -] PHE) 査読

H OMOTE, MY PARK, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 269 ( 14 ) 10265 - 10269 1994年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The Ser-beta 174 residue of the Escherichia coli H+-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (similar to 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296 --> Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H+-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits. About 1 mol of N-[C-14]ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.

Osmoenzyme-H(+)-ATP synthase: catalysis and H+ translocation

Futai, M., Omote, H., Maeda, M.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 39 ( 7 ) 1994年

ESCHERICHIA-COLI F0F1-ATPASE - RESIDUES INVOLVED IN CATALYSIS AND COUPLING 査読

RK NAKAMOTO, K SHIN, A IWAMOTO, H OMOTE, M MAEDA, M FUTAI

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 671 335 - 344 1992年11月

ESCHERICHIA-COLI ATP SYNTHASE (F-ATPASE) - CATALYTIC SITE AND REGULATION OF H+ TRANSLOCATION 査読

M FUTAI, A IWAMOTO, H OMOTE, Y ORITA, K SHIN, RK NAKAMOTO, M MAEDA

JOURNAL OF EXPERIMENTAL BIOLOGY 172 443 - 449 1992年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COMPANY OF BIOLOGISTS LTD

We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that betaLys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in betaLys-155 or betaThr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues betaSer-174, betaGlu-192 and betaVal-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.

EFFECTS OF MUTATIONS OF CONSERVED LYS-155 AND THR-156 RESIDUES IN THE PHOSPHATE-BINDING GLYCINE-RICH SEQUENCE OF THE F1-ATPASE-BETA SUBUNIT OF ESCHERICHIA-COLI 査読

H OMOTE, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 267 ( 29 ) 20571 - 20576 1992年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

BetaLys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of betaLys- 155 and betaThr-156, mutants (betaLys-155 --> Ala, Ser, or Thr; betaThr-156 --> Ala, Cys, Asp, or Ser; betaLys-155/betaThr-156 --> betaThr-155/betaLys-156; and betaThr-156/betaVal-157 --> betaAla-156/betaThr-157) were constructed, and their properties were studied extensively. The betaSer-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had <0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (betaAla-155, betaSer-155, betaAla-156, and betaCys-156) showed low rates of multisite (<0.02% of the wild type) and unisite (<1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the betaAla-156 and betaCys-156 enzymes and 10(2)-fold lower with the betaAla-155 and betaSer-155 enzymes. The betaThr-156 --> Ala or Cys enzyme showed an altered response to Mg2+, suggesting that betaThr-156 may be closely related to Mg2+ binding. These results suggest that betaLys-155 and betaThr-156 are essential for catalysis and are possibly located in the catalytic site, although betaThr-156 could be replaced by a serine residue.

ESCHERICHIA-COLI F0F1-ATPASE - RESIDUES INVOLVED IN CATALYSIS AND COUPLING 査読

RK NAKAMOTO, K SHIN, A IWAMOTO, H OMOTE, M MAEDA, M FUTAI

ION-MOTIVE ATPASES : STRUCTURE, FUNCTION, AND REGULATION 671 335 - 344 1992年

MUTATIONS IN SER174 AND THE GLYCINE-RICH SEQUENCE (GLY149, GLY150, AND THR156) IN THE BETA-SUBUNIT OF ESCHERICHIA-COLI H+-ATPASE 査読

A IWAMOTO, H OMOTE, H HANADA, N TOMIOKA, A ITAI, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 266 ( 25 ) 16350 - 16355 1991年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

A sequence motif in the beta-subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta-Gly149 by Ser suppressed the effect of the beta-Ser174 --> Phe mutation (defective H+-ATPase), but beta-Gly150 --> Ser substitution did not have this effect. A single mutation (beta-Gly149 --> Ser or beta-Gly150 --> Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta-Gly149 --> Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca2+-dependent activity, but had the wild-type level of Mg2+-dependent activity with active oxidative phosphorylation. Introduction of a beta-Gly149 --> Ser or beta-Gly150 --> Ser mutation with the beta-Ser174 --> Phe mutation also lowered the Ca2+-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta-Thr156 --> Ala or Cvs mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity.
The possible location of the catalytic site including divalent cation binding site(s) is discussed.

　前のページ - 次のページ　

1