Updated on 2022/12/01

写真a

 
NISHIKAWA Shuh-ichi
 
Organization
Academic Assembly Institute of Science and Technology CHIKYU SEIBUTSU KAGAKU KEIRETU Professor
Graduate School of Science and Technology Life and Food Sciences Professor
Faculty of Science Department of Science Professor
Title
Professor
External link

Degree

  • 博士(理学) ( 1992.3   東京大学 )

Research Interests

  • cell nucleus

  • protein quality control

  • molecular chaperone

  • endoplasmic reticulum quality control

  • Arabidopsis thaliana

  • molecular chaperones

  • endoplasmic reticulum

  • Saccharomyces cerevisiae

  • reproduction

Research Areas

  • Life Science / Plant molecular biology and physiology

  • Life Science / Functional biochemistry

  • Life Science / Cell biology

Research History (researchmap)

  • Niigata University Faculty of Science   Professor

    2017.4

      More details

  • Niigata University Faculty of Science, Department of Biology   Professor

    2012.4 - 2017.3

      More details

  • Nagoya University Graduate School of Science, Division of Material Science, Graduate School of Science,School of Science Material Science   Associate Professor

    2001.7 - 2012.3

      More details

  • Nagoya Univeristy Graduate School of Science   Research Assistant

    1996.4 - 2001.7

      More details

  • Nagoya University Faculty of Science, Department of Chemistry   Research Assistant

    1993.4 - 1996.3

      More details

  • The University of Tokyo Faculty of Science   JSPS Research Fellow

    1992.4 - 1993.3

      More details

▶ display all

Research History

  • Niigata University   Faculty of Science Department of Science   Professor

    2017.4

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Professor

    2012.4

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Professor

    2012.4

  • Niigata University   Abolition organization Bioregulatory Science   Professor

    2012.4 - 2017.3

Professional Memberships

▶ display all

 

Papers

  • Expression of GEX1 Orthologs of Brassica rapa and Oryza sativa Rescued the Nuclear Fusion Defect of the Arabidopsis GEX1 Mutant Reviewed

    Ayaka Yabe, Shuh-ichi Nishikawa

    Plants   2022.7

     More details

    Authorship:Last author, Corresponding author   Publishing type:Research paper (scientific journal)  

    DOI: 10.3390/plants11141808

    researchmap

  • Arabidopsis GEX1 Is a Nuclear Membrane Protein of Gametes Required for Nuclear Fusion During Reproduction. Reviewed International journal

    Shuh-Ichi Nishikawa, Yuki Yamaguchi, Chiharu Suzuki, Ayaka Yabe, Yuzuru Sato, Daisuke Kurihara, Yoshikatsu Sato, Daichi Susaki, Tetsuya Higashiyama, Daisuke Maruyama

    Frontiers in plant science   11   548032 - 548032   2020.10

     More details

    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    During the life cycle of flowering plants, nuclear fusion, or karyogamy, occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. In Arabidopsis thaliana, nuclear fusion events during sexual reproduction proceed without the breakdown of the nuclear envelope, indicating that nuclear membrane fusion is essential for the completion of this process. Arabidopsis gamete expressed 1 (GEX1) is a membrane protein that is conserved among plant species. GEX1 shares homology with the yeast karyogamy protein Kar5, which is primarily expressed in the nuclear membrane. The GEX1 family represents a putative karyogamy factor. Herein, we show that GEX1 is required for the nuclear fusion events in Arabidopsis reproduction. GEX1-deficient mature female gametophytes were found to contain two unfused polar nuclei in close proximity within the central cell. Electron microscopy showed that the outer membrane of the polar nuclei was connected via the endoplasmic reticulum, whereas the inner membrane remained unfused. These results indicate that GEX1 is involved in polar nuclear membrane fusion following the fusion of the outer nuclear membrane. Furthermore, sperm nuclear fusion events were defective in the fertilized egg and central cell following plasmogamy in the fertilization of gex1-1 female gametophytes by gex1-1 pollen. An analysis of GEX1 localization in the female gametophyte using a transgenic line expressing GFP-tagged GEX1 driven by the GEX1 promoter showed that GEX1 is a nuclear membrane protein in the egg and central cell. Time-lapse live-cell imaging showed that in developing female gametophytes, the nuclear GFP-GEX1 signal was first detectable in the central cell shortly before the polar nuclei came in close contact, and then in the egg cell. Thus, we suggest that the GEX1-family proteins are nuclear membrane proteins involved in karyogamy in the reproduction of eukaryotes including flowering plants.

    DOI: 10.3389/fpls.2020.548032

    PubMed

    researchmap

  • ERdj3B-mediated quality control maintains anther development at high temperatures. Reviewed International journal

    Masaya Yamamoto, Shuhei Uji, Tomoyuki Sugiyama, Tomoaki Sakamoto, Seisuke Kimura, Toshiya Endo, Shuh-Ichi Nishikawa

    Plant physiology   2020.1

     More details

    Authorship:Last author, Corresponding author   Language:English  

    Pollen development is highly sensitive to heat stress, which impairs cellular proteostasis by causing misfolded proteins to accumulate. Therefore, each cellular compartment possesses a dedicated protein quality control system. An elaborate quality control system involving molecular chaperones, including immunoglobulin binding protein (BiP), heat shock protein 70, and regulatory J domain-containing co-chaperones (J proteins), allows the endoplasmic reticulum (ER) to withstand a large influx of proteins. Here, we found that Arabidopsis thaliana mutants of ER-localized DnaJ family 3B (ERdj3B), one of three ER-resident J proteins involved in ER quality control, produced few seeds at high temperatures (29{degree sign}C) due to defects in anther development. This temperature-sensitive fertility defect is specific to the defective interactions of BiP with ERdj3B, but not with the other two J proteins, indicating functional differences between ERdj3B and the other J proteins. RNA-seq analysis revealed that heat stress affects pollen development in both wild-type and mutant buds, but the erdj3b mutant is more susceptible, possibly due to defects in ER quality control. Our results highlight the importance of a specific ER quality control factor, ERdj3B, for plant reproduction, particularly anther development, at high temperatures.

    DOI: 10.1104/pp.19.01356

    PubMed

    researchmap

  • Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation Reviewed

    Daisuke Maruyama, Tetsuya Higashiyama, Toshiya Endo, Shuh-ichi Nishikawa

    Plant and Cell Physiology   61 ( 1 )   29 - 40   2020.1

     More details

    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press ({OUP})  

    Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58(IPK) (erdj3a p58(ipk)). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58(ipk) double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58(ip)(k), which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

    DOI: 10.1093/pcp/pcz158

    Web of Science

    researchmap

  • Development of a Heat-Inducible Gene Expression System Using Female Gametophytes of Arabidopsis thaliana Reviewed

    Dukhyun Hwang, Satomi Wada, Azusa Takahashi, Hiroko Urawa, Yasuhiro Kamei, Shuh-ichi Nishikawa

    Plant and Cell Physiology   60   2564 - 2572   2019.11

     More details

    Authorship:Last author, Corresponding author   Publisher:Oxford University Press ({OUP})  

    DOI: 10.1093/pcp/pcz148

    researchmap

  • The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1. Reviewed International journal

    Imai H, Abe T, Miyoshi T, Nishikawa SI, Ito K, Uchiumi T

    Nucleic acids research   46 ( 15 )   7820 - 7830   2018.7

     More details

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

    The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events. Biochemical and crystal structural data indicate that the conserved hydrophobic amino acid residues at the C-terminus of the archaeal stalk protein aP1 binds to the nucleotide-binding domain 1 (NBD1) of aABCE1, and that this binding is crucial for ATPase activation of aABCE1 on the ribosome. The functional role of the stalk•ABCE1 interaction in ATPase activation and the subunit dissociation is also investigated using mutagenesis in a yeast system. The data demonstrate that the ribosomal stalk protein likely participates in efficient actions of both archaeal and eukaryotic ABCE1 in ribosome recycling. The results also show that the stalk protein has a role in the function of ATPase as well as GTPase factors in translation.

    DOI: 10.1093/nar/gky619

    PubMed

    researchmap

  • Quality control of nonstop membrane proteins at the ER membrane and in the cytosol Reviewed

    Shunsuke Arakawa, Kaori Yunoki, Toshiaki Izawa, Yasushi Tamura, Shuh-ichi Nishikawa, Toshiya Endo

    SCIENTIFIC REPORTS   6   30795   2016.8

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen. Here we followed the fate of nonstop endoplasmic reticulum (ER) membrane proteins with different membrane topologies in yeast to evaluate the importance of the Ltn1-dependent cytosolic degradation and the Dom34-dependent release of the nonstop membrane proteins. Ltn1-dependent degradation differed for membrane proteins with different topologies and its failure did not affect ER protein import or cell growth. On the other hand, failure in the Dom34-dependent release of the nascent polypeptide from the ribosome led to the block of the Sec61 channel and resultant inhibition of other protein import into the ER caused cell growth defects. Therefore, the nascent chain release from the translation apparatus is more instrumental in clearance of the clogged ER translocon channel and thus maintenance of normal cellular functions.

    DOI: 10.1038/srep30795

    Web of Science

    PubMed

    researchmap

  • Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism Reviewed

    Daisuke Maruyama, Ronny Voelz, Hidenori Takeuchi, Toshiyuki Mori, Tomoko Igawa, Daisuke Kurihara, Tomokazu Kawashima, Minako Ueda, Masaki Ito, Masaaki Umeda, Shuh-ichi Nishikawa, Rita Gross-Hardt, Tetsuya Higashiyama

    CELL   161 ( 4 )   907 - 918   2015.5

     More details

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

    In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

    DOI: 10.1016/j.cell.2015.03.018

    Web of Science

    PubMed

    researchmap

  • BiP3 supports the early stages of female gametogenesis in the absence of BiP1 and BiP2 in Arabidopsis thaliana. Reviewed

    Maruyama D, Endo T, Nishikawa S

    Plant signaling & behavior   10 ( 7 )   e1035853   2015

     More details

    Authorship:Last author, Corresponding author  

    DOI: 10.1080/15592324.2015.1035853

    PubMed

    researchmap

  • Different Sets of ER-Resident J-Proteins Regulate Distinct Polar Nuclear-Membrane Fusion Events in Arabidopsis thaliana Reviewed

    Daisuke Maruyama, Masaya Yamamoto, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   55 ( 11 )   1937 - 1944   2014.11

     More details

    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect.

    DOI: 10.1093/pcp/pcu120

    Web of Science

    PubMed

    researchmap

  • Multiple BiP Genes of Arabidopsis thaliana are Required for Male Gametogenesis and Pollen Competitiveness Reviewed

    Daisuke Maruyama, Tomoyuki Sugiyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   55 ( 4 )   801 - 810   2014.4

     More details

    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.

    DOI: 10.1093/pcp/pcu018

    Web of Science

    PubMed

    researchmap

  • The Plant Organelles Database 3 (PODB3) Update 2014: Integrating Electron Micrographs and New Options for Plant Organelle Research Reviewed

    Shoji Mano, Takanori Nakamura, Maki Kondo, Tomoki Miwa, Shuh-ichi Nishikawa, Tetsuro Mimura, Akira Nagatani, Mikio Nishimura

    PLANT AND CELL PHYSIOLOGY   55 ( 1 )   e1   2014.1

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.

    DOI: 10.1093/pcp/pct140

    Web of Science

    PubMed

    researchmap

  • Tam41 Is a CDP-Diacylglycerol Synthase Required for Cardiolipin Biosynthesis in Mitochondria Reviewed

    Yasushi Tamura, Yoshihiro Harada, Shuh-ichi Nishikawa, Koji Yamano, Megumi Kamiya, Takuya Shiota, Takuya Kuroda, Osamu Kuge, Hiromi Sesaki, Kenichiro Imai, Kentaro Tomii, Toshiya Endo

    CELL METABOLISM   17 ( 5 )   709 - 718   2013.5

     More details

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

    CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.

    DOI: 10.1016/j.cmet.2013.03.018

    Web of Science

    PubMed

    researchmap

  • Analyses of protein-protein interactions by in vivo photocrosslinking in budding yeast Reviewed

    Takuya Shiota, Shuh-Ichi Nishikawa, Toshiya Endo

    Methods in Molecular Biology   1033   207 - 217   2013

     More details

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

    Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-l-phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the pBpa-incorporated proteins has been proven to be a powerful method for analyzing protein-protein interactions at the spatial resolution of amino-acid residues. Since photocrosslinking can be performed for pBpa-incorporated proteins that are properly assembled into a protein complex in living cells, this method will allow us to reveal protein-protein interactions of the target proteins at work. © 2013 Springer Science+Business Media, LLC.

    DOI: 10.1007/978-1-62703-487-6_14

    Scopus

    PubMed

    researchmap

  • Roles of Dom34:Hbs1 in Nonstop Protein Clearance from Translocators for Normal Organelle Protein Influx Reviewed

    Toshiaki Izawa, Tatsuhisa Tsuboi, Kazushige Kuroha, Toshifumi Inada, Shuh-ichi Nishikawa, Toshiya Endo

    CELL REPORTS   2 ( 3 )   447 - 453   2012.9

     More details

    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane. These stuck nonstop secretory proteins avoid proteasomal degradation in the cytosol, but are instead released into the ER lumen through stalled ribosome and translocator channels by Dom34:Hbs1. We also found that nonstop mitochondrial proteins are cleared from the mitochondrial translocator, the TOM40 complex, by Dom34:Hbs1. Clearance of stuck nonstop proteins from organellar translocator channels is crucial for normal protein influx into organelles and for normal cell growth, especially when nonstop mRNA decay does not function efficiently.

    DOI: 10.1016/j.celrep.2012.08.010

    Web of Science

    PubMed

    researchmap

  • Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation Reviewed

    Toshiaki Izawa, Hiroyuki Nagai, Toshiya Endo, Shuh-ichi Nishikawa

    MOLECULAR BIOLOGY OF THE CELL   23 ( 7 )   1283 - 1293   2012.4

     More details

    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC CELL BIOLOGY  

    The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.

    DOI: 10.1091/mbc.E11-08-0722

    Web of Science

    PubMed

    researchmap

  • Using image-based resources: Databases for plant organelle dynamics and applications based on image information Reviewed

    Mano, S, Y. Kimori, T. Takeda, T. Miwa, S. Nishikawa, T. Mimura, A. Nagatani, a, M. Nishimura

    "Image and Video Processing - An Introductory Guide" iConcept Press   pp.1 - 25   2012

     More details

  • The Plant Organelles Database 2 (PODB2): An Updated Resource Containing Movie Data of Plant Organelle Dynamics Reviewed

    Shoji Mano, Tomoki Miwa, Shuh-ichi Nishikawa, Tetsuro Mimura, Mikio Nishimura

    PLANT AND CELL PHYSIOLOGY   52 ( 2 )   244 - 253   2011.2

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    The Plant Organelles Database (PODB) was launched in 2006 and provides imaging data of plant organelles, protocols for plant organelle research and external links to relevant websites. To provide comprehensive information on plant organelle dynamics and accommodate movie files that contain time-lapse images and 3D structure rotations, PODB was updated to the next version, PODB2 (http://podb.nibb.ac.jp/Organellome). PODB2 contains movie data submitted directly by plant researchers and can be freely downloaded. Through this organelle movie database, users can examine the dynamics of organelles of interest, including their movement, division, subcellular positioning and behavior, in response to external stimuli. In addition, the user interface for access and submission has been enhanced. PODB2 contains all of the information included in PODB, and the volume of data and protocols deposited in the PODB2 continues to grow steadily. Moreover, a new website, Plant Organelles World (http://podb.nibb.ac.jp/Organellome/PODBworld/en/index.html),, which is based on PODB2, was recently launched as an educational tool to engage members of the non-scientific community such as students and school teachers. Plant Organelles World is written in layman's terms, and technical terms were avoided where possible. We would appreciate contributions of data from all plant researchers to enhance the usefulness of PODB2 and Plant Organelles World.

    DOI: 10.1093/pcp/pcq184

    Web of Science

    PubMed

    researchmap

  • LAP5 and LAP6 Encode Anther-Specific Proteins with Similarity to Chalcone Synthase Essential for Pollen Exine Development in Arabidopsis Reviewed

    Anna A. Dobritsa, Zhentian Lei, Shuh-ichi Nishikawa, Ewa Urbanczyk-Wochniak, David V. Huhman, Daphne Preuss, Lloyd W. Sumner

    PLANT PHYSIOLOGY   153 ( 3 )   937 - 955   2010.7

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC PLANT BIOLOGISTS  

    Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.

    DOI: 10.1104/pp.110.157446

    Web of Science

    PubMed

    researchmap

  • A vacuolar carboxypeptidase mutant of Arabidopsis thaliana is degraded by the ERAD pathway independently of its N-glycan Reviewed

    Masaya Yamamoto, Mitsuyoshi Kawanabe, Yoko Hayashi, Toshiya Endo, Shuh-ichi Nishikawa

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   393 ( 3 )   384 - 389   2010.3

     More details

    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Misfolded proteins produced in the endoplasmic reticulum (ER) are degraded by a mechanism, the ER-associated degradation (ERAD). Here we report establishment of the experimental system to analyze the ERAD in plant cells. Carboxypeptidase V (CPY) is a vacuolar enzyme and its mutant CPY* is degraded by the ERAD in yeast. Since Arabidopsis thaliana has AtCPY, an ortholog of yeast CPY, we constructed and expressed fusion proteins consisting of AtCPY and GFP and of AtCPY*, which carries a mutation homologous to yeast CPY* and GFP in A. thaliana cells. While AtCPY-GFP was efficiently transported to the vacuole, AtCPY* -GFP was retained in the ER to be degraded in proteasome- and Cdc48-dependent manners. We also found that AtCPY*-GFP was degraded by the ERAD in yeast cells, but that its single N-glycan did not function as a degradation signal in yeast or plant cells. Therefore, AtCPY*-GFP can be used as a marker protein to analyze the ERAD pathway, likely for nonglycosylated substrates, in plant cells. (C) 2010 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2010.02.001

    Web of Science

    PubMed

    researchmap

  • BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana

    Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   107 ( 4 )   1684 - 1689   2010.1

     More details

    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

    Nuclear fusion is an essential process in the sexual reproduction of animals and plants. In flowering plants, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse to produce the diploid central cell nucleus, and twice during double fertilization. The yeast Ig binding protein (BiP) is a molecular chaperone Hsp70 in the endoplasmic reticulum that regulates nuclear membrane fusion during mating. Here we report that in Arabidopsis thaliana, BiP is involved in the fusion of polar nuclei during female gametophyte development. BiP-deficient mature female gametophytes contain two unfused polar nuclei, in spite of their close contact. This indicates a surprising conservation of BiP function in nuclear fusion between plants and yeasts. We also found that endosperm nuclear division becomes aberrant after fertilization of the BiP-deficient female gametophytes with wild-type pollen. This is experimental evidence for the importance of fusion of the polar nuclei in the proliferation of endosperm nuclei.

    DOI: 10.1073/pnas.0905795107

    Web of Science

    J-GLOBAL

    researchmap

  • Seeing Is Believing: On the Use of Image Databases for Visually Exploring Plant Organelle Dynamics Reviewed

    Shoji Mano, Tomoki Miwa, Shuh-ichi Nishikawa, Tetsuro Mimura, Mikio Nishimura

    PLANT AND CELL PHYSIOLOGY   50 ( 12 )   2000 - 2014   2009.12

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Organelle dynamics vary dramatically depending on cell type, developmental stage and environmental stimuli, so that various parameters, such as size, number and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics. At present, imaging techniques are emerging as fundamental tools in plant organelle research, and the development of new methodologies to visualize organelles and the improvement of analytical tools and equipment have allowed the large-scale generation of image and movie data. Accordingly, image databases that accumulate information on organelle dynamics are an increasingly indispensable part of modern plant organelle research. In addition, image databases are potentially rich data sources for computational analyses, as image and movie data reposited in the databases contain valuable and significant information, such as size, number, length and velocity. Computational analytical tools support image-based data mining, such as segmentation, quantification and statistical analyses, to extract biologically meaningful information from each database and combine them to construct models. In this review, we outline the image databases that are dedicated to plant organelle research and present their potential as resources for image-based computational analyses.

    DOI: 10.1093/pcp/pcp128

    Web of Science

    PubMed

    researchmap

  • Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space (vol 106, pg 14403, 2009) Reviewed

    Kawano Shin, Yamano Koji, Naoe Mari, Momose Takaki, Terao Kayoko, Nishikawa Shuh-ichi, Watanabe Nobuhisa, Endo Toshiya

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   106 ( 47 )   20133   2009.11

  • Roles of Tom70 in Import of Presequence-containing Mitochondrial Proteins

    Hayashi Yamamoto, Kenji Fukui, Hisashi Takahashi, Shingo Kitamura, Takuya Shiota, Kayoko Terao, Mayumi Uchida, Masatoshi Esaki, Shuh-ichi Nishikawa, Tohru Yoshihisa, Koji Yamano, Toshiya Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 46 )   31635 - 31646   2009.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.

    DOI: 10.1074/jbc.M109.041756

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • LAP3, a novel plant protein required for pollen development, is essential for proper exine formation

    Anna A. Dobritsa, Shuh-Ichi Nishikawa, Daphne Preuss, Ewa Urbanczyk-Wochniak, Lloyd W. Sumner, Adam Hammond, Ann L. Carlson, Robert J. Swanson

    SEXUAL PLANT REPRODUCTION   22 ( 3 )   167 - 177   2009.9

     More details

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

    We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in beta-propeller enzymes. Insertion mutations in LAP3 lead to male sterility. To investigate possible roles for LAP3 in pollen development, we assayed the metabolite profile of anther tissues containing developing pollen grains and found that the lap3-2 defect leads to a broad range of metabolic changes. The largest changes were seen in levels of a straight-chain hydrocarbon nonacosane and in naringenin chalcone, an obligate compound in the flavonoid biosynthesis pathway.

    DOI: 10.1007/s00497-009-0101-8

    Web of Science

    J-GLOBAL

    researchmap

  • Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

    Shin Kawano, Koji Yamano, Mari Naoe, Takaki Momose, Kayoko Terao, Shuh-ichi Nishikawa, Nobuhisa Watanabe, Toshiya Endo

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   106 ( 34 )   14403 - 14407   2009.8

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

    The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 angstrom. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

    DOI: 10.1073/pnas.0901793106

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

    Machiko Sakoh-Nakatogawa, Shuh-ichi Nishikawa, Toshiya Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 18 )   11815 - 11825   2009.5

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.

    DOI: 10.1074/jbc.M900813200

    Web of Science

    CiNii Article

    J-GLOBAL

    researchmap

  • Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating

    Shuh-ichi Nishikawa, Aiko Hirata, Toshiya Endo

    GENES TO CELLS   13 ( 11 )   1185 - 1195   2008.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

    DOI: 10.1111/j.1365-2443.2008.01236.x

    Web of Science

    J-GLOBAL

    researchmap

  • Arabidopsis thaliana Has a Set of J Proteins in the Endoplasmic Reticulum that are Conserved from Yeast to Animals and Plants

    Masaya Yamamoto, Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   49 ( 10 )   1547 - 1562   2008.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    J domain-containing proteins (J proteins) are functional partners for heat shock protein 70 (Hsp70) molecular chaperones and mediate various cellular processes by regulating activities of Hsp70. Budding yeast has three J proteins in the endoplasmic reticulum (ER): Scj1p and Jem1p functioning in protein folding and quality control in the ER, and Sec63p functioning in protein translocation across the ER membrane as partners for BiP, an Hsp70 in the ER. Here we report that Arabidopsis thaliana has orthologs of these yeast ER J proteins, which we designated as AtERdj3A, AtERdj3B, AtP58(IPK), AtERdj2A and AtERdj2B. Tunicamycin treatment of Arabidopsis cells, which causes ER stress, led to up-regulation of AtERdj3A, AtERdj3B, AtP58(IPK) and AtERdj2B. Subcellular fractionation analyses showed their ER localization, indicating that the identified J proteins indeed function as partners for BiP in Arabidopsis cells. Since expression of AtERdj3A, AtERdj3B and AtP58(IPK) partially suppressed the growth defects of the yeast jem1 Delta scj1 Delta mutant, they have functions similar to those of Scj1p and Jem1p. T-DNA insertions of the AtERDJ2A gene resulted in pollen germination defects, probably reflecting its essential function in protein translocation. These results suggest that A. thaliana has a set of ER J proteins structurally and functionally conserved from yeast to plants.

    DOI: 10.1093/pcp/pcn119

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Identification and characterization of a Jem1p ortholog of Candida albicans: dissection of Jem1p functions in karyogamy and protein quality control in Saccharomyces cerevisiae

    Tadashi Makio, Shuh-ichi Nishikawa, Takeshi Nakayama, Hiroyuki Nagai, Toshiya Endo

    GENES TO CELLS   13 ( 10 )   1015 - 1026   2008.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Jem1p of yeast Saccharomyces cerevisiae is a J-domain containing co-chaperone (J protein) in the endoplasmic reticulum (ER) lumen. Jem1p is required for nuclear fusion during mating (karyogamy) and functions together with another J protein, Scj1p, in protein folding and quality control in the ER as a partner for the ER Hsp70 (BiP/Kar2p). Candida albicans has a gene encoding a homolog of S. cerevisiae Jem1p, CaJem1p. CaJem1p localized in the ER when expressed in S. cerevisiae, and expression of CaJem1p from a single-copy plasmid suppressed the temperature sensitive growth and the ER quality control defect of the jem1 Delta scj1 Delta mutant, indicating that CaJem1p is functional in S. cerevisiae. However, CaJem1p suppressed the karyogamy defect of the jem1 Delta mutant only when it was over-expressed from a multicopy plasmid. Domain-swapping experiments showed that this was due to the difference between the N-terminal domains of ScJem1p and CaJem1p. The N-terminal domain of ScJem1p is essential for its function and interacts with Nep98p, a component of the spindle pole body involved in karyogamy. Since the interaction of CaJem1p with Nep98p is weaker than that of ScJem1p, the Nep98p-ScJem1p interaction is likely important for promoting karyogamy in S. cerevisiae.

    DOI: 10.1111/j.1365-2443.2008.01223.x

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • The plant organelles database (PODB): a collection of visualized plant organelles and protocols for plant organelle research

    Shoji Mano, Tomoki Miwa, Shuh-ichi Nishikawa, Tetsuro Mimura, Mikio Nishimura

    NUCLEIC ACIDS RESEARCH   36   D929 - D937   2008.1

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:OXFORD UNIV PRESS  

    The plant organelles database (PODB; http://podb.nibb.ac.jp/Organellome) was built to promote a comprehensive understanding of organelle dynamics, including organelle function, biogenesis, differentiation, movement and interactions with other organelles. This database consists of three individual parts, the organellome database, the functional analysis database and external links to other databases and homepages. The organellome database provides images of various plant organelles that were visualized with fluorescent and nonfluorescent probes in various tissues of several plant species at different developmental stages. The functional analysis database is a collection of protocols for plant organelle research. External links give access primarily to other databases and Web pages with information on transcriptomes and proteomes. All the data and protocols in the organellome database and the functional analysis database are populated by direct submission of experimentally determined data from plant researchers and can be freely downloaded. Our database promotes the exchange of information between plant organelle researchers for the comprehensive study of the organelle dynamics that support integrated functions in higher plants. We would also appreciate contributions of data and protocols from all plant researchers to maximize the usefulness of the database.

    DOI: 10.1093/nar/gkm789

    Web of Science

    J-GLOBAL

    researchmap

  • Functional analysis of J proteins in the endoplasmic reticulum of Arabidopsis thaliana Reviewed

    Masaya Yamamoto, Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   48   S154 - S154   2007

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Web of Science

    researchmap

  • Reproduction defects of the T-DNA mutants of endoplasmic reticulum Hsp70 in Arabidopsis thaliana Reviewed

    Daisuke Maruyama, Toshiya Endo, Shuh-ichi Nishikawa

    PLANT AND CELL PHYSIOLOGY   48   S153 - S153   2007

     More details

    Language:English   Publisher:OXFORD UNIV PRESS  

    Web of Science

    researchmap

  • Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria Reviewed

    Yasushi Tamura, Yoshihiro Harada, Koji Yamano, Kazuaki Watanabe, Daigo Ishikawa, Chie Ohshima, Shuh-ichi Nishikawa, Hayashi Yamamoto, Toshiya Endo

    JOURNAL OF CELL BIOLOGY   174 ( 5 )   631 - 637   2006.8

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROCKEFELLER UNIV PRESS  

    Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and - 16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

    DOI: 10.1083/jcb.200603087

    Web of Science

    PubMed

    researchmap

  • Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth

    Shuh-ichi Nishikawa, Gregory M. Zinkl, Robert J. Swanson, Daisuke Maruyama, Daphne Preuss

    BMC PLANT BIOLOGY   5   2005.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:BIOMED CENTRAL LTD  

    DOI: 10.1186/1471-2229-5-22

    Web of Science

    J-GLOBAL

    researchmap

  • Identification of Tim40 that mediates protein sorting to mitochondrial intermembrane space Reviewed

    Mari Naoe, Yukimasa Ohwa, Daigo Ishikawa, Chie Olishima, Shuh-ichi Nishikawa, Hayashi Yamamoto, Toshiya Endo

    CELL STRUCTURE AND FUNCTION   30   101 - 101   2005.6

     More details

    Language:English   Publisher:JAPAN SOC CELL BIOLOGY  

    Web of Science

    researchmap

  • Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD)

    S Nishikawa, JL Brodsky, K Nakatsukasa

    JOURNAL OF BIOCHEMISTRY   137 ( 5 )   551 - 555   2005.5

     More details

    Language:English   Publisher:JAPANESE BIOCHEMICAL SOC  

    Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.

    DOI: 10.1093/jb/mvi068

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast

    K Nakatsukasa, S Okada, K Umebayashi, R Fukuda, S Nishikawa, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 48 )   49762 - 49772   2004.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The protein quality control system in the endoplasmic reticulum (ER) ensures that only properly folded proteins are deployed throughout the cells. When nonnative proteins accumulate in the ER, the unfolded protein response is triggered to limit further accumulation of nonnative proteins and the ER is cleared of accumulated nonnative proteins by the ER-associated degradation (ERAD). In the yeast ER, aberrant nonnative proteins are mainly directed for the ERAD, but a distinct fraction of them instead receive O-mannosylation. In order to test whether O-mannosylation might also be a mechanism to process aberrant proteins in the ER, here we analyzed the effect of O-mannosylation on two kinds of model aberrant proteins, a series of N-glycosylation site mutants of prepro-alpha-factor and a pro-region-deleted derivative of Rhizopus niveus aspartic proteinase-I (Deltapro) both in vitro and in vivo. O-Mannosylation increases solubilities of the aberrant proteins and renders them less dependent on the ER chaperone, BiP, for being soluble. The release from ER chaperones allows the aberrant proteins to exit out of the ER for the normal secretory pathway transport. When the gene for Pmt2p, responsible for the O-mannosylation of these aberrant proteins, and that for the ERAD were simultaneously deleted, the cell exhibited enhanced unfolded protein response. O-Mannosylation may therefore function as a fail-safe mechanism for the ERAD by solubilizing the aberrant proteins that overflowed from the ERAD pathway and reducing the load for ER chaperones.

    DOI: 10.1074/jbc.M403234200

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Identification of Tim40 that mediates protein sorting to the mitochondrial intermembrane space

    M Naoe, Y Ohwa, D Ishikawa, C Ohshima, S Nishikawa, H Yamamoto, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 46 )   47815 - 47821   2004.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four mitochondrial subcompartments. Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the intermembrane space. Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment. Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro. In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the intermembrane space in vitro. These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.

    DOI: 10.1074/jbc.M410272200

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Mitochondrial protein import - Requirement of presequence elements and TOM components for precursor binding to the TOM complex

    M Esaki, H Shimizu, T Ono, H Yamamoto, T Kanamori, S Nishikawa, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 44 )   45701 - 45707   2004.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F-0-ATPase are required for binding to the trans site. The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.

    DOI: 10.1074/jbc.M404591200

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import

    T Asai, T Takahashi, M Esaki, S Nishikawa, K Ohtsuka, M Nakai, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 19 )   19464 - 19470   2004.5

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequence-containing proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.

    DOI: 10.1074/jbc.M401291200

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Tom40 protein import channel binds to non-native proteins and prevents their aggregation

    M Esaki, T Kanamori, S Nishikawa, Shin, I, PG Schultz, T Endo

    NATURE STRUCTURAL BIOLOGY   10 ( 12 )   988 - 994   2003.12

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATURE PUBLISHING GROUP  

    Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation.

    DOI: 10.1038/nsb1008

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Nep98p is a component of the yeast spindle pole body and essential for nuclear division and fusion

    S Nishikawa, Y Terazawa, T Nakayama, A Hirata, T Makio, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 11 )   9938 - 9943   2003.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    During the mating of yeast Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. This process requires the functions of BiP/Kar2p, a member of the Hsp70 family in the endoplasmic reticulum, and its partner protein, Jem1p. To investigate further the role of BiP and Jem1p in nuclear fusion, we screened for partner proteins for Jem1p by the yeast two-hybrid system and identified Nep98p. Nep98p is an essential integral membrane protein of the nuclear envelope and is enriched in the spindle pole body (SPB), the sole microtubule-organizing center in yeast. Temperature-sensitive nep98 mutant cells contain abnormal SPBs lacking the half-bridge, suggesting the essential role of Nep98p in the organization of the normal SPB. Additionally, nep98 mutant cells show defects in mitotic nuclear division and nuclear fusion during mating. Because Jem1p is not required for nuclear division, Nep98p probably has dual functions in Jem1p-dependent karyogamy and in Jem1p-independent nuclear division.

    DOI: 10.1074/jbc.M210934200

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes

    H Yamamoto, M Esaki, T Kanamori, Y Tamura, S Nishikawa, T Endo

    CELL   111 ( 4 )   519 - 528   2002.11

     More details

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

    Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex.

    DOI: 10.1016/S0092-8674(02)01053-X

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • [Morphology of mitochondria]. Reviewed

    Kawai A, Nishikawa S, Endo T

    Nihon rinsho. Japanese journal of clinical medicine   60 Suppl 4   9 - 13   2002.4

     More details

  • Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells

    A Kawai, S Nishikawa, A Hirata, T Endo

    JOURNAL OF CELL SCIENCE   114 ( 19 )   3565 - 3574   2001.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:COMPANY OF BIOLOGISTS LTD  

    Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/ assembly or at elevated temperature. Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria. When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates. Inhibition of protein synthesis in the cytosol did not suppress the mitochondrial aggregation and functional impairment of Tim23, a subunit of mitochondrial protein translocator in the inner membrane, did not cause mitochondrial aggregation. Therefore defects of the Hsp70 function in protein import into mitochondria or resulting accumulation of precursor forms of mitochondrial proteins outside the mitochondria are not the causal reason for the aberrant mitochondrial morphology. By contrast, deletion of Mdj1p, a functional partner for mitochondrial Hsp70 in prevention of irreversible protein aggregation in the matrix, but not in protein import into mitochondria, caused aggregation of mitochondria, which was enhanced at elevated temperature (37 degreesC). The aggregation of mitochondria at 37 degreesC was reversed when the temperature was lowered to 23 degreesC unless protein synthesis was blocked. On the basis of these results, we propose that the mitochondrial matrix contains a protein that is responsible for the maintenance of mitochondrial morphology and requires mitochondrial Hsp70 for its function.

    Web of Science

    PubMed

    researchmap

  • Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation

    S Nishikawa, SW Fewell, Y Kato, JL Brodsky, T Endo

    JOURNAL OF CELL BIOLOGY   153 ( 5 )   1061 - 1069   2001.5

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROCKEFELLER UNIV PRESS  

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-cr-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the Lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and SCJ1 are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

    DOI: 10.1083/jcb.153.5.1061

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Mnl1p, an alpha-mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins

    K Nakatsukasa, S Nishikawa, N Hosokawa, K Nagata, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 12 )   8635 - 8638   2001.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha -mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha -mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.

    DOI: 10.1074/jbc.C100023200

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20

    Y Abe, T Shodai, T Muto, K Mihara, H Torii, S Nishikawa, T Endo, D Kohda

    CELL   100 ( 5 )   551 - 560   2000.3

     More details

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

    Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presenquence peptide derived from rat aldehyde dehydrogenase, The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

    DOI: 10.1016/S0092-8674(00)80691-1

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cytochrome b(2) import pathway

    M Esaki, T Kanamori, S Nishikawa, T Endo

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   96 ( 21 )   11770 - 11775   1999.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

    The import of cytochrome b(2) into mitochondria consists of two steps, The translocation of the first part of the presequence across the inner membrane is coupled with the translocation of the tightly folded heme-binding domain across the outer membrane and requires a membrane potential Delta Psi and the functions of mitochondrial Hsp70 (mHsp70) in the matrix, Once the heme-binding domain has passed the outer membrane, the translocation of the rest of the polypeptide chain across the outer membrane becomes independent of Delta Psi and mHsp70. Here we analyzed the late Delta Psi- and mHsp70-independent step in the transport of cytochrome bz fusion proteins into the intermembrane space (IMS), The import of the cytochrome b(2) fusion proteins containing two protein domains linked by a spacer segment into mitochondria was arrested at a stage at which one domain folded on each side of the outer membrane, along the pathway that is consistent with the stop-transfer model. The mature-size form of the translocation intermediate could move across the outer membrane in both directions, and the stabilization of the protein domain in the IMS promoted the forward translocation. On the other hand, the intermediate-size form of the translocation intermediate, which retains the anchorage to the inner membrane, was transported into the IMS independently of the stability of the protein domain in the IMS. These results suggest that two distinct mechanisms, the Brownian ratchet and the anchor diffusion mechanisms, can operate for the transmembrane movement of the mature-size form and the intermediate-size form, respectively, of cytochrome bz species.

    DOI: 10.1073/pnas.96.21.11770

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Appropriately spaced nuclear localizing signals are necessary for efficient nuclear import of nonnuclear proteins

    HJW Borgeld, K Naruse, S Nishikawa, J Zhang, A Kikuchi, K Furukawa, K Yagi, M Tanaka

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   256 ( 2 )   278 - 283   1999.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC  

    To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system. When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm. When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus. When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus. When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV49Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus. These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus. (C) 1999 Academic Press.

    DOI: 10.1006/bbrc.1999.0269

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

    T Kanamori, S Nishikawa, M Nakai, Shin, I, PG Schultz, T Endo

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   96 ( 7 )   3634 - 3639   1999.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

    Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

    DOI: 10.1073/pnas.96.7.3634

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Two distinct mechanisms operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdj1p/Yge1p chaperone system

    Y Kubo, T Tsunehiro, S Nishikawa, M Nakai, E Ikeda, A Toh-e, N Morishima, T Shibata, T Endo

    JOURNAL OF MOLECULAR BIOLOGY   286 ( 2 )   447 - 464   1999.2

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS LTD  

    The yeast mitochondrial Hsp70, Ssc1p, functions as a molecular chaperone with its partner proteins, Mdj1p (DnaJ homologue) and Yge1p (GrpE homologue). We have purified a mature form of Ssc1p from yeast mitochondria and those of Mdj1p and Yge1p from Escherichia coli overexpresser cells. With these purified components of the mitochondrial Hsp70 chaperone system, we have succeeded in reconstituting their chaperone functions in the protection of firefly luciferase against thermal damage in vitro. Heat-denatured luciferase is prevented from irreversible aggregation and is maintained in a refolding-competent state by Ssc1p and/or Mdj1p at 42 degrees C. Luciferase denatured at 42 degrees C is actively reactivated by Ssc1p, Mdj1p and/or Yge1p after lowering the temperature to 25 degrees C. The reactivation process of heat-denatured luciferase shows two-phase kinetics. The slow refolding process requires either Ssc1p or Mdj1p at 42 degrees C but the presence of Ssc1p, Mdj1p and Yge1p, and ATP hydrolysis, is essential at 25 degrees C. The slow refolding of luciferase involves multiple rounds of formation and dissociation of the complex between luciferase and Mdj1p/Ssc1p. On the other hand, the fast refolding process is most enhanced when luciferase is incubated with Ssc1p alone at 42 degrees C, and it requires neither the assistance of Mdj1p and Yge1p nor ATP hydrolysis. We have observed a similar two-pathway reactivation of heat-denatured luciferase by the bacterial Hsp70 and the yeast cytosolic Hsp70 systems. (C) 1999 Academic Press.

    DOI: 10.1006/jmbi.1998.2465

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis

    M Sato, K Sato, S Nishikawa, A Hirata, J Kato, A Nakano

    MOLECULAR AND CELLULAR BIOLOGY   19 ( 1 )   471 - 483   1999.1

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

    As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mf alpha 1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179-8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.

    Web of Science

    researchmap

  • Reinvestigation of the functions of the hydrophobic segment of Jem1p, a yeast endoplasmic reticulum membrane protein mediating nuclear fusion

    S Nishikawa, T Endo

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   244 ( 3 )   785 - 789   1998.3

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC  

    Jem1p is a DnaJ-like protein of the yeast ER membrane, which is required for nuclear membrane fusion during mating. We have determined the position of translation initiation codon for the JEM1 gene. Translation of Jem1p starts from the second ATG codon of the previously assumed JEM1 open reading frame, leading to the synthesis of a precursor protein of 645 amino acids long. The translated Jem1p precursor contains an N-terminal hydrophobic sequence that functions as a signal sequence and is removed upon import into the ER lumen. Jem1p is peripherally associated with the ER membrane probably through protein-protein interactions. (C) 1998 Academic Press.

    DOI: 10.1006/bbrc.1998.8342

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Analysis of the functional domain of the rat liver mitochondrial import receptor Tom20

    J Iwahashi, S Yamazaki, T Komiya, N Nomura, S Nishikawa, T Endo, K Mihara

    JOURNAL OF BIOLOGICAL CHEMISTRY   272 ( 29 )   18467 - 18472   1997.7

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the M-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Delta tom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.

    DOI: 10.1074/jbc.272.29.18467

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • The yeast JEM1p is a DnaJ-like protein of the endoplasmic reticulum membrane required for nuclear fusion

    S Nishikawa, T Endo

    JOURNAL OF BIOLOGICAL CHEMISTRY   272 ( 20 )   12889 - 12892   1997.5

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    DnaJ-like proteins are functional partners for Hsp70 molecular chaperones, Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family. The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment. An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen. We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p. The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature. The Delta jem1 mutant is defective in nuclear fusion, karyogamy, during mating. A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Delta jem1 Delta scj1 double mutant or defects in karyogamy of the Delta jem1 mutant. JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.

    DOI: 10.1074/jbc.272.20.12889

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking

    T Kanamori, SI Nishikawa, Shin, I, PG Schultz, T Endo

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   94 ( 2 )   485 - 490   1997.1

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

    Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway, Amino acids with benzophenone side chains of various lengths [DL-2-amino-3-(p-benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b(2)-dihydrofolate reductase fusion proteins, pb(2)(220)-DHFR and pb(2) Delta 19(220)DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively, In vitro import of pb(2)(220)-DHFR and pb(2) Delta 19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb(2) Delta 19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels, On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb(2)(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb(2)(220)-DHFR, but not pb(2) Delta 19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.

    DOI: 10.1073/pnas.94.2.485

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • MEMBRANE-PROTEIN RETRIEVAL FROM THE GOLGI-APPARATUS TO THE ENDOPLASMIC-RETICULUM (ER) - CHARACTERIZATION OF THE RER1 GENE-PRODUCT AS A COMPONENT INVOLVED IN ER LOCALIZATION OF SEC12P

    K SATO, S NISHIKAWA, A NAKANO

    MOLECULAR BIOLOGY OF THE CELL   6 ( 11 )   1459 - 1477   1995.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC CELL BIOL  

    Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyses have suggested that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early region of the Golgi apparatus. The rer1 mutant we isolated in a previous study mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene and determined its reading frame. RER1 encodes a hydrophobic protein of 188 amino acid residues containing four putative membrane spanning domains. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an epitope derived from the influenza hemagglutinin was added to the C-terminus of Rer1p and the cells expressing this tagged but functional protein were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern that is typical for Golgi proteins and clearly distinct from the ER staining. This punctate staining was in fact exaggerated in the sec7 mutant that accumulates the Golgi membranes at the restrictive temperature. Furthermore, double staining of Rer1p and Ypt1p, a GTPase that is known to reside in the Golgi apparatus, showed good colocalization. Subcellular fractionation experiments indicated that the fractionation pattern of Rer1p was similar to that of an early Golgi protein, Och1p. From these results, we suggest that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.

    Web of Science

    researchmap

  • INHIBITION OF ENDOPLASMIC-RETICULUM (ER)-TO-GOLGI TRANSPORT INDUCES RELOCALIZATION OF BINDING-PROTEIN (BIP) WITHIN THE ER TO FORM THE BIP BODIES

    S NISHIKAWA, A HIRATA, A NAKANO

    MOLECULAR BIOLOGY OF THE CELL   5 ( 10 )   1129 - 1143   1994.10

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC CELL BIOLOGY  

    Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the ''BIP body.'' This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BIP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.

    Web of Science

    researchmap

  • MUTATIONAL ANALYSIS OF THE SAR1 PROTEIN, A SMALL GTPASE WHICH IS ESSENTIAL FOR VESICULAR TRANSPORT FROM THE ENDOPLASMIC-RETICULUM

    A NAKANO, H OTSUKA, M YAMAGISHI, E YAMAMOTO, K KIMURA, S NISHIKAWA, T OKA

    JOURNAL OF BIOCHEMISTRY   116 ( 2 )   243 - 247   1994.8

     More details

    Language:English   Publisher:JAPANESE BIOCHEMICAL SOC  

    SAR1 encodes a 21-kDa GTPase, which is required for vesicle formation from the endoplasmic reticulum in yeast. Although it belongs to the expanding small GTPase superfamily, there are interesting structural features that are unique to the Sar1 protein. We performed a site-directed mutational study to identify the amino acid residues that are essential for the Sar1p function. Among seven mutants we constructed, four are functionless by themselves, while two confer temperature sensitivity to cells, When the mutant proteins are overproduced in wild-type cells, all of these six show a dominant negative effect on cell growth. The replacement by serine of the only cysteine residue present in Sar1p caused no significant change in the growth phenotype. These findings are not only important for analyzing the mechanism of the Sar1p action in yeast, but will also be very useful for studying the function of Sar1p counterparts in higher eukaryotes.

    Web of Science

    researchmap

  • IDENTIFICATION OF A GENE REQUIRED FOR MEMBRANE-PROTEIN RETENTION IN THE EARLY SECRETORY PATHWAY

    S NISHIKAWA, A NAKANO

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   90 ( 17 )   8179 - 8183   1993.9

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:NATL ACAD SCIENCES  

    The yeast SEC12 gene product (Sec12p) is an integral membrane protein required for the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Although this protein is almost exclusively localized in the ER, a significant fraction of Sec12p is modified by an enzyme that resides in the early compartment of the Golgi apparatus, suggesting that Sec12p is cycling between the ER and the early Golgi. We have taken a genetic approach to investigate the retention mechanism of Sec12p. Analysis of mutants that are defective in the retention of the Sec12-Mfalpha1 fusion protein in the early secretory compartments has identified a gene, RER1. A recessive mutation in RER1 causes mislocalization of the authentic Sec12p as well as two different Sec12 fusion proteins to the late Golgi apparatus and even to the cell surface. However, the rer1 mutant is not defective in the retention of an ER-resident soluble protein, BiP, suggesting that soluble and membrane proteins are retained in the ER by distinct mechanisms.

    DOI: 10.1073/pnas.90.17.8179

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • STRUCTURAL AND FUNCTIONAL DISSECTION OF A MEMBRANE GLYCOPROTEIN REQUIRED FOR VESICLE BUDDING FROM THE ENDOPLASMIC-RETICULUM

    C DENFERT, C BARLOWE, S NISHIKAWA, A NAKANO, R SCHEKMAN

    MOLECULAR AND CELLULAR BIOLOGY   11 ( 11 )   5727 - 5734   1991.11

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC MICROBIOLOGY  

    Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae. Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein. Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential. Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature. This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport. Sar1p membrane association was enhanced by elevated levels of Sec12p. These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.

    Web of Science

    researchmap

  • RECONSTITUTION OF GTP-BINDING SAR1 PROTEIN FUNCTION IN ER TO GOLGI TRANSPORT

    T OKA, S NISHIKAWA, A NAKANO

    JOURNAL OF CELL BIOLOGY   114 ( 4 )   671 - 679   1991.8

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROCKEFELLER UNIV PRESS  

    In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

    DOI: 10.1083/jcb.114.4.671

    Web of Science

    PubMed

    CiNii Article

    J-GLOBAL

    researchmap

  • THE GTP-BINDING SAR1 PROTEIN IS LOCALIZED TO THE EARLY COMPARTMENT OF THE YEAST SECRETORY PATHWAY

    S NISHIKAWA, A NAKANO

    BIOCHIMICA ET BIOPHYSICA ACTA   1093 ( 2-3 )   135 - 143   1991.7

     More details

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

    SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein. This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sar1p using this antibody. In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sar1p was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant. Membrane association of Sar1p was shown to be very tight. Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely. From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic.

    Web of Science

    researchmap

  • BIOGENESIS OF VACUOLAR MEMBRANE-GLYCOPROTEINS OF YEAST SACCHAROMYCES-CEREVISIAE

    S NISHIKAWA, N UMEMOTO, Y OHSUMI, A NAKANO, Y ANRAKU

    JOURNAL OF BIOLOGICAL CHEMISTRY   265 ( 13 )   7440 - 7448   1990.5

     More details

    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Web of Science

    researchmap

  • STUDIES ON THE TONOPLAST ACTION-POTENTIAL OF NITELLA-FLEXILIS

    T SHIMMEN, S NISHIKAWA

    JOURNAL OF MEMBRANE BIOLOGY   101 ( 2 )   133 - 140   1988

     More details

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

    Web of Science

    researchmap

▶ display all

Books

  • 第8章 タンパク質の一生:誕生から死まで in 医学のための細胞生物学(永田和宏,塩田浩平編)

    南山堂  2009 

     More details

  • 3.1.3 酵母を用いた翻訳 in RNA実験ノート上 RNAの基本的な取り扱いから解析手法まで(稲田利文,塩見晴彦編)

    羊土社  2008 

     More details

  • 3.8ミトコンドリア

    酵母のすべて-系統,細胞から分子まで 大隅良典・下田親 編 シュプリンガー・ジャパン  2007 

     More details

  • 第3章Short Topics 3GFPを用いた網羅的な可視化マーカーの構築

    新版 植物の細胞を観る実験プロトコール細胞工学別冊 植物細胞工学シリーズ22福田裕穂,西村幹夫,中野明彦 監修  2006 

     More details

  • 第11章(1)タンパク質の膜透過装置 タンパク質科学:構造,物性,機能 (後藤祐児,桑島邦博,谷澤克行編)

    化学同人  2005 

     More details

  • 無細胞蛋白質生合成系:蛋白質生産システム

    生物学実験法シリーズ: 遺伝子発現研究法 江尻慎一郎、平秀 編 学会出版センター  1999 

     More details

  • 6.2.5 酵母の細胞内構造体の調製法

    新版 微生物学実験法 大和田紘一,黒岩常祥,杉山純多,高橋秀夫,徳田元,渡辺信 編講談社サイエンティフィック  1999 

     More details

  • 6.2.5 酵母の細胞内構造体の調製法

    新版 微生物学実験法 大和田紘一,黒岩常祥,杉山純多,高橋秀夫,徳田元,渡辺信 編講談社サイエンティフィック  1999 

     More details

  • IV オルガネラの機能解析 4 ミトコンドリア

    酵母ラボマニュアル 酵母分子細胞生物学実験法 山本正幸・大矢禎一 編 シュプリンガー・フェアラーク東京  1998 

     More details

  • IV オルガネラの機能解析 4 ミトコンドリア

    酵母ラボマニュアル 酵母分子細胞生物学実験法 山本正幸・大矢禎一 編 シュプリンガー・フェアラーク東京  1998 

     More details

▶ display all

MISC

▶ display all

Presentations

▶ display all

Works

  • シロイヌナズナを用いた受精初期過程の分子機構の解析

    2001

     More details

Awards

  • 第9回井上研究奨励賞

    1993  

     More details

    Country:Japan

    researchmap

Research Projects

  • LINC複合体によるシロイヌナズナ有性生殖過程の核融合の制御機構

    Grant number:19K06704  2019.4 - 2022.3

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    西川 周一

      More details

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    researchmap

  • 陸上植物有性生殖における核膜融合の鍵メカニズム

    Grant number:19H04857  2019.4 - 2021.3

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    西川 周一

      More details

    Grant amount:\8580000 ( Direct Cost: \6600000 、 Indirect Cost:\1980000 )

    researchmap

  • 鍵と鍵穴に着目した有性生殖過程の核膜融合の分子機構と初期発生における意義の解明

    Grant number:17H05837  2017.4 - 2019.3

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    西川 周一

      More details

    Grant amount:\9360000 ( Direct Cost: \7200000 、 Indirect Cost:\2160000 )

    被子植物の有性生殖で観察される核融合過程では、核膜同士が融合することが必須である。本研究は、有性生殖過程の核膜融合で中心的な役割をはたす膜タンパク質であるGex1に着目した解析によって、シロイヌナズナ有性生殖過程の核膜融合の分子機構と、その初期発生における意義を明らかにすることを目的とする。
    本年度は、GFP融合タンパク質を用いた解析によって、Gex1が雌性配偶体では中央細胞と卵細胞に特異的に発現する核膜タンパク質であることを明らかにした。また、ライブイメージング解析によって、Gex1は中央細胞では極核の接近の時期に、卵細胞では極核の接触後に出現すること、Gex1は最初核膜全体に分布しているが、極核の融合過程で核膜上にドット様に局在することが示された。また、Brassica rapaとイネのGex1ホモログを用いた解析によって、Gex1機能に種特異性が存在することが示唆された。
    Gex1は内腔側に大きなドメインを有しており、ここが核膜融合で「鍵」として機能していると予想される。本研究では内腔側ドメインの中でもホモログ間での保存性の高いシステインリッチドメイン(Gex1-CRD)の構造解析を行った。その結果、Gex1-CRDは二量体を形成していること、二量体の間に疎水性の溝が存在することが示された。
    昨年度の研究で、核膜タンパク質であるSUNタンパク質が極核融合で機能することを示唆された。SUNタンパク質は核外膜のKASHタンパク質とLINC複合体を形成するが、これが極核融合に必要であることを示唆する結果を得た。核膜融合機構の解析の新たなアプローチとして、核膜融合を制御する低分子化合物のスクリーニングを東山班(伊丹グループ)との共同研究で開始し、スクリーニング条件を確立するとともに約5,000化合物のスクリーニングを進めている。

    researchmap

  • Mechanisms of nuclear membrane fusion during Arabidopsis reproduction

    Grant number:16K07394  2016.4 - 2019.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Nishikawa Shuh-ichi, HIGASHIYAMA Tetsuya, SATO Yoshikatsu, KURIHARA Daisuke, MARUYAMA Daisuke, KAMEI Yasuhiro, URAWA Hiroko, WADA Satomi, HWANG Dukhyum, SUZUKI Chiharu, TAKAHASHI Azusa, NAGAO Haruka

      More details

    Grant amount:\4810000 ( Direct Cost: \3700000 、 Indirect Cost:\1110000 )

    During the life cycle of angiosperms, nuclear fusion occurs three times. Two of these nuclear fusion events are sperm nuclear fusion events that occur during double fertilization. The third nuclear fusion is the polar nuclear fusion during female gametogenesis. These nuclear fusion events require the nuclear membrane fusion processes to complete.
    In this study, we analyzed mechanisms of nuclear membrane fusions during Arabidopsis reproduction using live-cell imaging and the newly-developed female gametophyte-specific gene induction system. We showed that the Hsp70 molecular chaperone system in the endoplasmic reticulum is required for nuclear membrane fusion in the sperm nuclear fusion process in addition to the polar nuclear fusion. We also showed that fertilization-coupled sperm nuclear fusion is essential for proper endosperm proliferation after fertilization. We identified two nuclear membrane proteins that are required for nuclear membrane fusion events during polar nuclear fusion.

    researchmap

  • Analyses of nuclear fusion during plant reproduction using gametophyte cell-specific gene induction systems

    Grant number:26650094  2014.4 - 2016.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research  Grant-in-Aid for Challenging Exploratory Research

    Nishikawa Shuh-ichi, KAMEI Yasuhiro, URAWA Hiroko, YAMAGUCHI Yuki, WADA Satomi

      More details

    Grant amount:\4030000 ( Direct Cost: \3100000 、 Indirect Cost:\930000 )

    In this study, we developed a new gene induction method, which allows cell-specific gene induction in Arabidopsis female gametophytes using heat shock-induced Cre-loxP recombination and female gametophyte-specific promoters. Female gametophyte-specific gene expressions were induced by heat treatment of flower buds of constructed transgenic lines. We also showed induction of cell-specific gene expression by irradiating isolated female gametophytes with the infrared laser. The developed method will be a good tool to analyze functions of proteins involved in the fusion of polar nuclei during female gametophyte development.

    researchmap

  • 細胞膜受容体の小胞体品質管理を介した花粉成熟過程のストレス耐性機構の解析

    Grant number:25120711  2013.4 - 2015.3

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    西川 周一

      More details

    Grant amount:\8580000 ( Direct Cost: \6600000 、 Indirect Cost:\1980000 )

    本研究では、細胞膜の受容体様キナーゼ(LRR-RLK)に着目した解析によって、小胞体品質管理因子であるAtERdj3BやUGGTといった小胞体分子シャペロンの花粉成熟過程における役割を明らかにするとともに、小胞体分子シャペロン・品質管理装置によるLRR-RLKの認識と機能発現の特異性の解明を目指している。
    本年度は、昨年度に引き続き領域内の共同研究を進め、花粉形成過程で機能するLRR-RLKの細胞外ドメインと小胞体Jタンパク質との相互作用の解析を行った。そして、昨年度見いだしたLRR-RLK細胞外ドメインとAtERdj3Bとの特異的な相互作用は、熱ストレスによって増加することを示す結果を得た。また、LRR-RLK細胞外ドメインに関する部分断片を用いた解析で、AtERdj3Bが特に認識する領域の推定を行った。
    本年度はまた、葯1個を用いた遺伝子発現解析系を用いた解析を進めた。そして、定量的解析に適した組織量の検討を行うとともに、定量PCR実験の標準化でこれまで利用されてきたハウスキーピング遺伝子でも、葯の発達ステージによって発現量が大きく異なるものがあることを示した。また、発達中の葯におけるUGGTなどの発現時期および組織の解析を行い、UGGTが花粉成熟期の葯で特異的に発現していること、タペート組織で特異的に発現するAtERdj3Bとは異なり、UGGTはタペート組織崩壊後の葯でも発現していることを示した。

    researchmap

  • 小胞体品質管理による花粉成熟過程のストレス耐性機構の解析

    Grant number:23120512  2011.4 - 2013.3

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    西川 周一

      More details

    Grant amount:\8450000 ( Direct Cost: \6500000 、 Indirect Cost:\1950000 )

    研究代表者らは、小胞体品質管理で中心的な役割をはたす小胞体分子シャペロンAtERdj3Bを欠損したシロイヌナズナ変異株が、高温ストレス下で花粉成熟の異常を示すことを見いだした。昨年度の解析によって、これはRpk2という花粉成熟過程における遺伝子発現に必要な細胞膜のLRR型キナーゼの、細胞膜輸送の欠損によることが示唆されている。研究代表者らはまた、糖タンパク質特異的な小胞体品質管理因子であるUGGTを欠損した変異株も高温ストレス下で花粉成熟の異常を示すことを見いだしている。
    本年度はまず、高温ストレス下で生育したuggt変異株の花芽におけるRpk2下流の遺伝子発現解析と、Rpk2の細胞膜輸送について検討した。この結果、uggt変異株で見られる高温ストレス下での花粉成熟異常は、Rpk2以外のLRR型キナーゼの欠損によることを示唆する結果を得た。本年度はaterdj3b uggt二重変異株の構築と解析も行った。得られた二重変異株は通常の生育温度でも不稔となり、これは花粉形成の欠損によることが示された。二重変異株はまた、生育異常を示した。この結果は、AtERdj3BとUGGTによる小胞体品質管理は、花粉形成過程以外でもLRR型キナーゼの機能維持に必要であることを示唆している。
    本年度は、LRR型キナーゼと小胞体分子シャペロン間の相互作用を解析する実験系の構築も行った。これまでに、小麦胚芽無細胞タンパク質合成系を用いて、Rpk2をはじめとする数種類のLRR型キナーゼの細胞外ドメインと、AtERdj3Bなどの小胞体分子シャペロンを可溶性の状態で合成することに成功している。今後は、合成したこれらタンパク質間の相互作用解析を進める計画である。

    researchmap

  • Analyses of ER-resident molecular chaperone-dependent nuclear membrane fusion during fertilization of Arabidopsis thaliana

    Grant number:23570051  2011 - 2013

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    NISHIKAWA Shuh-ichi

      More details

    Grant amount:\5460000 ( Direct Cost: \4200000 、 Indirect Cost:\1260000 )

    We showed that BiP, an Hsp70 molecular chaperone in the endoplasmic reticulum, function in the fusions of the outer and inner nuclear membranes of the polar nuclei with different sets of J-domain containing partner proteins during the female gametogenesis of A. thaliana. Live imaging analyses showed that the polar nuclei fusion defect can be recovered during the first endosperm nuclear division after fertilization and that the fusion of sperm nuclei at fertilization is required for proper endosperm proliferation. We also identified candidates of nuclear membrane proteins required for the polar nuclei fusion.

    researchmap

  • Organelle Differentiation as the Strategy for Environmental Adaptation in Plants

    Grant number:16085101  2004 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas  Grant-in-Aid for Scientific Research on Priority Areas

    NISHIMURA Mikio, MIMURA Tetsuro, NISHIKAWA Shuh-ichi, NISHIMURA Ikuko, MORITA Miyo

      More details

    Grant amount:\45800000 ( Direct Cost: \45800000 )

    researchmap

  • Regulation of plant cell function and development by endoplasmic reticulum quality control

    Grant number:16085202  2004 - 2008

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas  Grant-in-Aid for Scientific Research on Priority Areas

    SHUH-ICHI Nishikawa, TOSHIYA Endo, TOHRU Yoshihisa, TOSHIYA Endo, TOHRU Yoshihisa

      More details

    Grant amount:\95900000 ( Direct Cost: \95900000 )

    researchmap

  • Versatile molecular functions of the ribosomal stalk protein in translation cycle

    Grant number:19H03155  2019.4 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

      More details

    Grant amount:\17550000 ( Direct Cost: \13500000 、 Indirect Cost:\4050000 )

    researchmap

  • 真核生物停滞リボソーム上におけるぺプチジルtRNA分解のメカニズム解明

    Grant number:18K06080  2018.4 - 2021.3

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    伊東 孝祐, 西川 周一

      More details

    Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

    生体内では様々な要因で翻訳は異常停止し、合成途中の未成熟ペプチドがtRNAに結合したままのぺプチジルtRNAが停滞リボソーム内に産生される。このような状態は細胞にとって有害であり、翻訳の品質管理機構によって解消されなければならない。現在までの研究により、ペプチド部位が短鎖の場合、ぺプチジルtRNAはリボソームから細胞質に放出され、そしてぺプチジルtRNA加水分解酵素 (Pth) によりペプチドとtRNAに分解されて翻訳停滞が解消されることがわかっている。一方、ペプチド部位が長鎖の場合、ぺプチジルtRNAは停滞リボソーム上でペプチドとtRNAに分解されることが知られているが、この反応を担う因子は真核生物では未だ不明である。一方、我々は最近、Pthがユビキチン化タンパク質運搬因子と直接相互作用することを見い出している。本研究の目的は以下の通りである。
    1) 長鎖ぺプチジルtRNAを停滞リボソーム上で分解する因子を分子遺伝学的手法で探索する。
    2) Pth-ユビキチン化タンパク質運搬因子の相互作用様式をX線結晶構造解析で明らかにする。
    3) Pthとユビキチン化タンパク質運搬因子が共同でリボソーム上の長鎖ぺプチジルtRNAの分解に働いている可能性を追究する。
    研究成果であるが、1)については、長鎖ぺプチジルtRNAを発現するモデル酵母株を作製し終えた。2)については、ほぼ構造解析が終了した。3)については、2)の構造解析の結果をもとに行う実験であり、2)がほぼ終了しているので来年度予定通り実行できる状態である。

    researchmap

  • Functional role of the ribosomal stalk protein in translation recycling

    Grant number:16H04741  2016.4 - 2019.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Uchiumi Toshio, IMAI Hirotatsu

      More details

    Grant amount:\17420000 ( Direct Cost: \13400000 、 Indirect Cost:\4020000 )

    Translation of genetic information, i.e., protein synthesis, on a macromolecular complex called the ribosome occurs in four stages: initiation, elongation, termination, and recycling. So far in research, much less is known about mechanism of the recycling. In this project, we have focused on a ribosome recycling factor, ABCE1 (ATPase), and investigated molecular mechanism of the action of ABCE1 by using techniques of biochemistry and structural biology. As results, we have clarified that a ribosomal protein component, termed the “stalk”, directly binds to a specific site of ABCE1, recruits it to the functional site within the ribosome, and stimulates ATP hydrolysis. Thus, our results demonstrate that interaction between ABCE1 and the stalk protein leads to dissociation of ribosomes to subunits, or ribosome recycling.

    researchmap

  • Elucidation of the mechanism of the control of protein trafficking at mitochondrial membranes

    Grant number:22227003  2010.4 - 2016.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)  Grant-in-Aid for Scientific Research (S)

    ENDO Toshiya, TAMURA Yasushi, NISHIKAWA Shuh-ichi

      More details

    Grant amount:\210600000 ( Direct Cost: \162000000 、 Indirect Cost:\48600000 )

    The protein trafficking as well as lipid trafficking systems at mitochondrial membranes were analyzed to elucidate the mechanisms of their functions. By using site-specific photocrosslinking, the outer membrane translocator TOM complex was analyzed for its inter-subunit and subunit-precursor interactions and the dynamic assembly structures were revealed. Cellular mechanisms to clear the mitochondrial translocon clogged by non-stop proteins arising form mRNA lacking a stop codon were revealed. Tam41 at the mitochondrial inner membrane was found to be a key enzyme for the cardiolipin synthetic pathway and the mechanism of phosphatidic acid transport between the outer and inner membranes by Ups1-Mdm35 was elucidated by determination of its high-resolution structures with and without a phosphatidic acid.

    researchmap

  • Elucidation of the yeast systems that achieve and maintain functions and organizations of mitochondrial and ER proteins

    Grant number:19058005  2007 - 2011

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas  Grant-in-Aid for Scientific Research on Priority Areas

    ENDO Toshiya, NISHIKAWA Shuh-ichi, YOSHIHIA Tohru

      More details

    Grant amount:\198000000 ( Direct Cost: \198000000 )

    We have revealed the mechanisms of various aspects of protein transport into mitochondria, including cooperation of translocators, driving force for protein translocation, roles of multiple targeting signals in the presequence, mapping of subunit arrangements in the translocator complex, assembly of β-barrel proteins etc. We have also revealed the mechanisms of protein quality control in the ER, including roles of interactions between Mnl1 and PDI, ER retention of aberrant proteins by Yos9, roles of Hsp70 in plant sexual reproduction etc.

    researchmap

  • 酵母ミトコンドリアタンパク質配送における行き先シグナル解読機構の解明

    Grant number:19036007  2007 - 2008

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    遠藤 斗志也, 西川 周一

      More details

    Grant amount:\6300000 ( Direct Cost: \6300000 )

    酵母細胞内にamberサプレッサーtRNAとこれに非天然アミノ酸BPA(p-benzoyl-L-phenylalanine)をチャージできるアミノアシルtRNA合成酵素(BpaRS)を発現させると, 目的タンパク質内のamberコドンで指定した位置にBPAを組み込むことができる。この細胞または細胞抽出液に紫外光を照射するとBPAと近傍のタンパク質間に架橋が形成されるので, 架橋産物の相手を同定すれば, 目的タンパク質についてどの位置でどんなタンパク質と相互作用しているかをマッピングすることができる。
    この手法を酵母ミトコンドリア外膜のトランスロケータTOM40複合体構成因子Tom22に適用した。Tom22は一回膜貫通型タンパク質で, N端ドメインをサイトゾルに, C端ドメインを膜間部側に持つ。Tom22分子全体に5残基毎にBPAを導入し, 光架橋を行い, TOM40複合体構成因子および内膜のトランスロケータ構成因子の抗体を用いて, 架橋相手を同定した。Tom22のサイトゾルドメインはTOM40複合体の二種類の受容体, Tom70とTom20と相互作用していた。詳細なマッピングから, Tom22はTom20が認識する前駆体の行き先シグナルを別な角度からサンドイッチする形で認識している可能性が示唆された。Tom22の膜貫通ドメインはTOM40複合体の膜透過チャネルTom40と相互作用し, 複合体を安定に保っているらしいこと, 一分子のTom22は二分子のTom40と相互作用していることが分かった。さらに, Tom22の膜間部ドメインは内膜のトランスロケータTIM23複合体の受容体サブユニットTim50と相互作用していることが分かった。

    researchmap

  • Control and alteration of mitochondrial protein traffic

    Grant number:18107003  2006 - 2009

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S)  Grant-in-Aid for Scientific Research (S)

    ENDO Toshiya, YOSHIHISA Tohru, NISHIKAWA ShuhーIchi

      More details

    Grant amount:\111800000 ( Direct Cost: \86000000 、 Indirect Cost:\25800000 )

    To understand the mechanisms of the functions of mitochondrial protein translocators, which control the traffic of mitochondrial proteins, we have discovered a new maintenance factor Tam41, revealed the cooperation of receptors Tom20 and Tom22 in recognition of mitochondrial targeting signals, revealed the cooperation of translocators, the TOM40 complex and TIM23 complex, in transfer of mitochondrial precursor proteins, determined the high-resolution structure of the import motor assistant protein Tim15, analyzed the competition between the sorting signals for the outer membrane and matrix/inner membrane, achieved the interaction mapping of the outer membrane translocator subunit Tom22, and altered the signal recognition functions of the receptor Tom20.

    researchmap

  • Mechanisms and Physiological Roles of Endoplacmic Reticulum Quality Control Regulated by Protein Glycosylation

    Grant number:17370067  2005 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    NISHIKAWA Shuh-ichi, ENDO Toshiya

      More details

    Grant amount:\15480000 ( Direct Cost: \14400000 、 Indirect Cost:\1080000 )

    The endoplasmic reticulum (ER) is the entrance of the secretory pathway in eukaryotic cells. Secretory and membrane proteins undergo folding and various modification processes before they are exported to the Golgi apparatus. Quality control mechanisms in the ER monitor these processes to ensure that defective proteins that failed to acquire correct functional structures are not deployed throughout the cells. Misfolded proteins retained in the ER are degraded by a mechanism called endoplasmic reticulum-associated degradation (ERAD) . The aim of our research is to elucidate mechanisms of the ER quality control system mediated by carbohydrate modification of proteins using budding yeast and Arabidopsis thaliana.
    Yeast Mnllp is a mannosidase family protein in the ER and is proposed to function in the substrate recognition in the ERAD of misfolded glycoproteins. We found that Mnllp forms a complex with protein disulfide isomerase (PDI) through disulfide bonds. Analyses using mutants of Mnllp and PDI showed that the Mnll p-PDI interaction is important for ERAD. To analyze relationships between protein folding and carbohydrate modification in substrate recognition in ERAD, we developed new ERAD substrates using CPY*, a widely used ERAD substrate in yeast. Analyses using the newly developed ERAD substrates showed that a N-linked carbohydrate chain required for ERAD of CPY* is recognized as a ERAD signal only when it was in the misfolded domain.
    Analyses using mammalian and yeast cells showed that calnexin/calreticulin, EDEM (Mnllp in yeast) and OS9 function in carbohydrate chain-mediated ER quality control. Arabidopsis has orthologs of these ER quality control machineries. We constructed Arabidopsis knockout mutants of these quality control machineries and found that simultaneous deletion of AtCNX1 and AtCRT1 resulted in growth retardation. We also constructed AtCPY*-GFP, a novel ERAD substrate in plant cells. In contrast to yeast CPY*, we showed that ERAD of AtCPY*GFP does not require its N-linked carbohydrate modification

    researchmap

  • 品質管理による小胞体由来コンパートメント形成の分子機構と生理機能制御

    Grant number:16044219  2004 - 2005

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    西川 周一, 遠藤 斗志也, 吉久 徹

      More details

    Grant amount:\5400000 ( Direct Cost: \5400000 )

    小胞体内腔のhsp70であるBiPは,小胞体品質管理において中心的な役割をはたしている。本年度は,AtBiP1遺伝子に関する優性欠損変異体を,花粉特異的なLAT52プロモーターを用いて発現するシロイヌナズナ形質転換体を作製し,その解析を行った。得られた形質転換植物は,花粉形成は正常であったが,BiP変異遺伝子の次世代への伝達に欠損を示した。in vitroおよびsemi in vitro花粉伸長実験の結果,BiP変異体を発現しても花粉は発芽し花粉管を伸長するが,花粉管が雌しべ柱頭と花柱を通過する過程に欠損が生じることが明らかとなった。
    本研究では,BiPのパートナータンパク質として機能する,小胞体のJタンパク質の解析も行った。本年度は,出芽酵母の小胞体Jタンパク質であるJem1p, Scj1p, Sec63pのシロイヌナズナホモログ(AtJem1, AtScj1A, AtScj1B, AtSec63A, AtSec63B)に対する抗体を作製し,これを用いた解析を行った。シロイヌナズナ培養細胞の細胞分画によって,これらJタンパク質が小胞体に局在することが示唆された。また,AtJem1,AtScj1A, AtScj1Bは膜内腔側のタンパク質であることが示された。また,これら遺伝子に関する破壊株の作製を行い,AtJem1,AtScj1A, AtScj1BとAtSec63B各遺伝子の単独の破壊,AtScj1AとAtScj1Bの2重破壊は致死とはならないが,AtSec63Aの遺伝子破壊は致死もしくは雄性不稔となることを示した。

    researchmap

  • 酵母ミトコンドリアを巡るプロテインフラックス

    Grant number:16013218  2004

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    吉久 徹, 遠藤 斗志也, 西川 周一

      More details

    Grant amount:\6100000 ( Direct Cost: \6100000 )

    酵母ミトコンドリアへのタンパク質フラックスを解明する第1歩として、全ミトコンドリアタンパク質の2次元電気泳動カタログの作成を進め、現在、次元電気泳動し、解析可能な506のタンパク質スポットを得、その帰属を進めている。既知の結果については、酵母ミトコンドリアタンパク質のデータベースを構築した。現在、Web上での公開に向けて、ブラウジングインターフェースなどを構築している。
    次に、ミトコンドリア前駆体タンパク質が複数あるミトコンドリア外膜の局在化シグナル受容体の一つであるTom70に依存してミトコンドリアに取り込まれるプレ配列を持ったタンパク質について、Tom70依存性を決定づけている部分の解析を行った。プレ配列を含む各タンパク質のN-末端領域とDHFRとの融合タンパク質を用いたin vitroミトコンドリアインポート実験から、Tom70依存性がプレ配列部分で決定されるもの、成熟体部分で決定されるものなど様々な前駆体のタイプがあり、ミトコンドリアタンパク質受容体が前駆体の多様な部分と相互作用していることが明かとなった。
    他方、ミトコンドリアへのタンパク質フラックスに関わる既知遺伝子の多くが、酵母の生育に必須であることから、ミトコンドリアに局在する必須遺伝子を網羅的に解析し、タンパク質フラックスに関わる新たな遺伝子が4種類同定された。

    researchmap

  • Molecular mechanisms of the control of yeast mitochondrial protein fluxes.

    Grant number:15207009  2003 - 2005

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    ENDO Toshiya, YOSHIHISA Tohru, NISHIKAWA Shuh-ichi

      More details

    Grant amount:\50310000 ( Direct Cost: \38700000 、 Indirect Cost:\11610000 )

    The central process in the maintenance of functional integrity of mitochondria is the transport of 500-1000 different mitochondrial precursor proteins to mitochondria. Since mitochondria consist of four compartments, the outer and inner membranes, intermembrane space, and matrix, the flux of mitochondrial proteins from the cytosol should branch off in four different sub-fluxes that are directed for each of the four compartments. In the present study, we aimed at elucidation of the mechanisms for surveillance and control of the mitochondrial protein fluxes in yeast cells. We identified 5 new components, Tom38,Tom13,Tim40,Tim15,and Tim41,of the mitochondrial protein translocators. In the course of our efforts to assign roles in mitochondrial protein import to these new components, it has become evident that the pathways of mitochondrial protein transport are much more complex than previously envisaged and the processes of translocation across and assembly into mitochondrial membranes are controlled in a highly sophisticated manner by cooperation of the mitochondrial translocator complexes. Besides, it is important for the mitochondrial translocator systems to recognize destination signals of substrate proteins, at the branching points in the mitochondrial protein fluxes, and sort them to correct destination routes. In this connection, we found that the general import receptor, Tom20, in the outer membrane, plays important roles in increasing targeting specificity as well as import efficiency, that Tim50 in the inner membrane functions as a presequence receptor, and that substrate proteins for the TIM22 pathway in the inner membrane possess cryptic targeting signals for the TIM23 pathway. One of the important, but still unresolved questions is what drives the protein flux for mitochondria. Here we found that the translocation channel of the translocator itself has a chaperone-like activity, thereby promoting unfolding of the substrate proteins to be threaded into the narrow translocator channel.

    researchmap

  • Regulation of Arabidopsis life cycle by the hsp 70 system of the endoplasmic reticulum

    Grant number:15570034  2003 - 2004

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    NISHIKAWA Shuh-ichi

      More details

    Grant amount:\3700000 ( Direct Cost: \3700000 )

    The endoplasmic reticulum(ER) is the entrance of the secretory pathway in eukaryotic cells. Secretory and membrane proteins undergo folding and various modification processes before they are exported to the Golgi apparatus. Quality control mechanisms in the ER monitor these processes to ensure that defective proteins that failed to acquire correct functional structures are not deployed throughout the cells and play important roles in the maintenance of cell homeostasis. The aim of our research is to elucidate functions and mechanisms of the ER quality control system in plant development by analyzing functions of molecular chaperones in the ER, molecular machineries that control ER quality control. BiP, a Hsp 70 family molecular chaperone in the ER, plays a central role both in protein translocation across the ER membrane and ER quality control. In this study, we have developed a system to analyze BiP functions in plant cells using Arabidopsis thaliana.
    In order to compromise BiP functions in plant cells, we introduced a series of dominant negative mutations into the Arabidopsis BIP gene. When expressed one of the dominant negative mutants in the Arabidopsis culture cells using the 35S promoter, we observed change of subcellular distribution of 12S globulin-GFP fusion protein which can be attributed to its aggregation. We also constructed transgenic plants which express BiP mutants in pollen under the LAT52 promoter. Reciprocal cross experiments suggested that expression of BiP mutants caused pollen sterility. Analyses of yeast mutants identified three J-domain containing protein in the ER as partners for BiP during its function. We identified Arabidopsis homologues of yeast J-domain containing proteins in the ER and constructed their T-DNA insertion mutants.

    researchmap

  • 糖鎖修飾による小胞体品質管理の制御機構

    Grant number:15040210  2003 - 2004

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    西川 周一, 遠藤 斗志也

      More details

    Grant amount:\3100000 ( Direct Cost: \3100000 )

    本研究では,糖鎖修飾による小胞体品質管理制御のメカニズムを明らかにすることを目的としている.マンノース転移酵素Pmt2pによる異常タンパク質のO-マンノシル化は,小胞体におけるタンパク質の変性修復の機構のひとつであると考えられる.われわれはこれまでに,2種類の異常タンパク質をO-マンノシル化の基質として同定したが,これらはともに可溶性タンパク質の変異体であった.本年度は,Pmt2p依存にO-マンノシル化を受ける異常タンパク質として,出芽酵母Nep98pの温度感受性変異体を新たに同定した.Nep98pは膜貫通領域をひとつ持つ膜内在性の核膜タンパク質であり,温度感受性変異は内腔側領域に存在する.以上の結果は,高次構造に異常を示す領域が内腔側に存在することがO-マンノシル化を受けるためには必要であり,O-マンノシル化と基質の膜への結合性との間に関連がないことを示唆している.
    本研究では,マウスEDEMの酵母ホモログであり.ERADシグナルとなる糖鎖構造を認識する因子の有力な候補であるMnl1pの機能解析も目的としている.われわれは,ゲノム配列の解析から,シロイヌナズナのMnl1pホモログを見いだし,その機能が多細胞生物の個体レベルでどのような意義を持つかについても解析を行なっている.本年度は,シロイヌナズナのMnl1pホモログの機能について,酵母mnl1変異体のERAD欠損の相補能を指標として検討した.しかし,シロイヌナズナMnl1pホモログは,同時にその活性の検討を行ったマウスEDEMと共に,酵母mnl1変異体のERAD欠損の相補能を持たないことが明らかとなった.

    researchmap

  • 酵母ミトコンドリアを巡るプロテインフラックス

    Grant number:15013224  2003

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    吉久 徹, 西川 周一, 遠藤 斗志也

      More details

    Grant amount:\5800000 ( Direct Cost: \5800000 )

    酵母ミトコンドリアへのタンパク質フラックスを解明する第1歩として、全ミトコンドリアタンパク質の2次元電気泳動カタログの作成を進め、現在、次元電気泳動し、解析可能な506のタンパク質スポットを得、その帰属を進めている。既知の結果については、酵母ミトコンドリアタンパク質のデータベースを構築した。現在、Web上での公開に向けて、ブラウジングインターフェースなどを構築している。
    次に、ミトコンドリア前駆体タンパク質が複数あるミトコンドリア外膜の局在化シグナル受容体をどう使い分けているかを知る目的で、シグナル受容体の一つであるTom70を欠失したミトコンドリアへの網羅的な取り込み実験を行った。全酵母mRNA由来の翻訳産物を野生型またはTom70欠失型ミトコンドリアに取り込ませ、2次元電気泳動で展開して得られた114のスポット(うち37スポットが帰属済み)について定量的比較を行った。その結果、17のスポットでは、明らかにTom70欠失ミトコンドリアでの取り込みが低下していた。このうち帰属のできている5種類は、いずれもプレ配列を持つタイプの前駆体であることがわかった。他方、2種の未同定のスポットについては、取り込み効率が上昇した。前記5種に加え、Tom70欠失の影響が見られない10種のタンパク質について、各タンパク質のミトコンドリアへの取り込みを個別に解析することでその依存性を確認した。Tom70は主に内在性局在化シグナルを認識すると考えられてきたが、プレ配列を持つ前駆体にもTom70によって認識されるサブクラスがあることが示された。さらに、プレ配列を持つミトコンドリアタンパク質のTom70依存性は、all-or noneタイプの依存性ではなく、量的に影響されていることが明かとなった。
    他方、ミトコンドリアへのタンパク質フラックスに関わる既知遺伝子の多くが、酵母の生育に必須であることから、ミトコンドリアに局在する必須遺伝子を網羅的に解析し、タンパク質フラックスに関わる新たな遺伝子の探索も進行している。

    researchmap

  • Structures and functions of protein translocator systems

    Grant number:14037225  2002 - 2006

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas  Grant-in-Aid for Scientific Research on Priority Areas

    ENDO Toshiya, YOSHIHISA Tohru, NISHIKAWA Shuh-ichi

      More details

    Grant amount:\148600000 ( Direct Cost: \148600000 )

    The central process in the maintenance of functional integrity of mitochondria is the transport of 500-1000 different mitochondrial precursor proteins to mitochondria. Since mitochondria consist of four compartments, the outer and inner membranes, intermembrane space, and matrix, the flux of mitochondrial proteins from the cytosol should branch off in four different sub-fluxes that are directed for each of the four compartments. In the present study, we aimed at elucidation of the mechanisms for surveillance and control of the mitochondrial protein fluxes in yeast cells. We identified 5 new components, Tom38, Toml3, Tim4O, Timl5, and Tim4l, of the mitochondrial protein translocators. Besides, we determined the NMR structure of Timl5, and found that the ability of Tim15 to maintain solubility of mitochondrial Hsp70 represents one of the essential functions of Tim15 in yeast cell growth.
    Mitochondrial proteins have to become unfolded to move through the translocator channels. Here we found that the translocation channel of the translocator (Tom40) itself has a chaperone-like activity, thereby promoting unfolding of the substrate proteins to be threaded into the narrow translocator channel. We also analyzed the effects of various stabilization/destabilization mutations in the immunoglobulin-like module of the muscle protein titin on its import from the N-terminus or C-terminus into mitochondria. The effects of mutations on the import of the titin module from the N-terminus correlate well with those on forced mechanical unfolding in atomic force microscopy (AFM) measurements. On the other hand, import of the titin module from the N-terminus is sensitive to mutations in the N-terminal region, but not the ones in the C-terminal region that affect resistance to global unfolding in AFM experiments. We propose that the mitochondrial import system can catalyze precursor unfolding by reducing the stability of unfolding intermediates.

    researchmap

  • 酵母ミトコンドリアを巡るプロテインフラックス

    Grant number:14014214  2002

    日本学術振興会  科学研究費助成事業 特定領域研究  特定領域研究

    吉久 徹, 西川 周一, 遠藤 斗志也

      More details

    Grant amount:\5500000 ( Direct Cost: \5500000 )

    酵母ミトコンドリアへのタンパク質フラックスを解明する第1歩として、全ミトコンドリアタンパク質の2次元電気泳動カタログの作成を進めている。現在、精製したミトコンドリアを複数の条件下で2次元電気泳動し、解析可能な506のタンパク質スポットを得ている。このうち、既に166のスポットについて59種類のタンパク質に帰属することができた。また、2次元電気泳動では解析しにくい内在性膜タンパク質については、内在性膜タンパク質のみをSDS-PAGEで展開後、各バンドの同定を進めている。現在、これらの結果を元に、酵母ミトコンドリアタンパク質のデータベースを構築しており、将来的にはWeb上での公開を考えている。
    さらに、複数あることが知られているミトコンドリア外膜膜透過装置の局在化シグナル受容体が、ミトコンドリア前駆体タンパク質ごとにどのように使い分けられているかを知る目的で、シグナル受容体の一つであるTom70を欠失したミトコンドリアへの網羅的な取り込み実験を行った。全酵母mRNA由来の翻訳産物を野生型またはTom70欠失型ミトコンドリアに取り込ませ、2次元電気泳動で展開して得られた117のスポットについて定量的比較を行った。その結果、13のスポットでは、明らかにTom70欠失ミトコンドリアでの取り込みが低下していた。この内5種類については帰属ができ、いずれもプレ配列を持つタイプの前駆体であることが明らかとなった。さらに、個々のタンパク質のミトコンドリアへの取り込みを迅速に確認する為のシステムを構築し、これら5種のタンパク質がTom70に依存して取り込まれることを確認した。Tom70は主に内在性局在化シグナルを認識すると考えられてきたが、上記の結果は、プレ配列を持つ前駆体にもTom70によって認識されるサブクラスがあり、異なる受容体がサブクラス間で使い分けられていることを示唆している

    researchmap

  • Molecular mechanisms of the control and regulation of the mitochondrial protein flux.

    Grant number:13480207  2001 - 2002

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    ENDO Toshiya, NISHIKAWA Shuh-ichi, YOSHIHISA Tohru

      More details

    Grant amount:\14800000 ( Direct Cost: \14800000 )

    Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively. In the present study, we analyzed the mechanisms of the control and regulation of the protein flux into mitochondria.
    Based on the results of the site-specific photocrosslinking of the translocation intermediate, we have identified Tim50, a new component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex.
    We took a proteome-wide approach of mitochondrial protein import in vitro to find a set of substrate proteins for recognition by Tom70. Translation products of total yeast RNA were imported into isolated wild-type mitochondria and those without Tom70. Comparison of the imported proteins on 2D-electrophoresis gels between wild-type and Δtom70 mitochondria allowed us to identify proteins affected by deletion of Tom70 systematically.

    researchmap

  • 核分裂と核融合に必須な酵母核膜タンパク質Nep98pの機能解析

    Grant number:13043022  2001

    日本学術振興会  科学研究費助成事業 特定領域研究(A)  特定領域研究(A)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    Grant amount:\2400000 ( Direct Cost: \2400000 )

    申請者らは,酵母接合時の核内膜融合に必須な因子として,小胞体内腔の新規DnaJホモログJem1pを発見,さらにJem1pと相互作用する核膜タンパク質Nep98pを同定した。Nep98pは核膜融合に関与するが,増殖に必須であり,その変異株はG2/M期で細胞周期の進行が停止し,紡錘体形態に異常が見られた。そこでNep98pの機能を明らかにするために以下の実験を行った。
    (1)GFPとNep98pの融合タンパク質(NEP98遺伝子の破壊株の致死性を相補できる)は,過剰生産しない状態で核膜の特にSPB(スピンドル極体)に局在することが明らかになった。したがってNep98pがSPB形成および機能発現に関わっている可能性を支持する結果が得られた。(2)Nep98pの様々な部分欠失体を用いた解析から,Nep98pの内腔側は核膜局在に必要ないが,Nep98pのサイトゾル側の65〜145残基が核膜局在に必須であること,この領域が核膜融合の特に内膜融合よりも前のステップに必須であること,Nep98pの431〜650残基は酵母の増殖およびJem1pとの相互作用に必須であることを見いだした。また(3)Nep98pのJem1p相互作用領域をbaitとして酵母two-hybrird法を用いたスクリーニングを行い,Nep98pと相互作用する因子の検索を行った。その結果,Yfl042pおよびYlr072pという内在性膜タンパク質と思われる候補を得た。しかしこの二つのタンパク質の遺伝子を同時に破壊しても増殖や核融合に影響が見られら無かったので,それらがNep98pの真のパートナーであるかどうかは,さらに解析が必要である。

    researchmap

  • 酵母ミトコンドリアを巡るプロテインフラックス

    Grant number:13206028  2001

    日本学術振興会  科学研究費助成事業 特定領域研究(C)  特定領域研究(C)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    Grant amount:\8500000 ( Direct Cost: \8500000 )

    ミトコンドリアは外膜,膜間部,内膜,マトリクスの4つの区画から構成され,サイトゾルで合成されたタンパク質は外膜のTOM,内膜の少なくとも2種類のTIMとよばれるタンパク質複合体の働きによりミトコンドリアに移行し,適切なミトコンドリア内区画に配置される。取り込まれたタンパク質は,マトリクスの分子シャペロンHsp70,Hsp60の働きで機能化する。本研究では,以上のプロセスの全体像を,全ミトコンドリアタンパク質の流れ(プロテインフラックス)として網羅的に解析し,フラックスの大きさと分岐を制御する因子とメカニズムの解明の突破口を開くことをめざした.具体的にはまず500〜1000種類存在すると考えられるミトコンドリアタンパク質の同定のために,(1)酵母細胞からNycodenz密度勾配遠心を用いて高純度のミトコンドリアを単離精製し,マトリクス画分について二次元電気泳動を行い,CBB染色した各スポットを限定分解→質量スペクトルで同定した(2)単離ミトコンドリアの内在性膜タンパク質画分について,一次元電気泳動後,二次元目に展開せずに,限定分解とMS-MS測定によりタンパク質同定を行った。(3)酵母細胞から全mRNAを単離,in vitroで翻訳しRI標識タンパク質を合成した。これを基質として,in vitroで単離ミトコンドリアへの取り込み実験を行い,プロテアーゼでミトコンドリア内に取り込まれなかったタンパク質を消化した場合としない場合の各々について,ミトコンドリアを回収して二次元電気泳動を行い,標識タンパク質のスポットを検出した。(4)(3)の実験を受容体Tom70の欠損株について行うことにより,Tom70に依存してミトコンドリアに取り込まれるタンパク質を網羅的に解析するために,受容体への結合が律速段階となるようなin vitroインポートの実験条件を確立した。

    researchmap

  • 核膜融合に必須な分子シャペロンJemlpと相互作用する核膜タンパク質の機能解析

    Grant number:12680690  2000 - 2001

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    西川 周一

      More details

    Grant amount:\3400000 ( Direct Cost: \3400000 )

    本研究では,出芽酵母小胞体の分子シャペロンJem1pと相互作用する,出芽酵母の新規核膜タンパク質Nep98pの機能解析を目的として研究を行なっている.本年度は,Nep98pの機能および機能領域の解析を行なった.遺伝子破壊実験によって,Nep98pの機能が酵母の増殖に必須であることが示されている.そこでNep98pの機能する過程を明らかにするため,Nep98pに関する温度感受性変異株の作製を行なった.得られた温度感受性変異株を制限温度にシフトすると,G2/M期に対応する細胞の割合が増加した.また,そのような細胞中では核分裂の進行に欠損が観察され,抗チューブリン抗体を用いた蛍光抗体法によって,スピンドル形態に異常を示すことが明らかとなった.電子顕微鏡を用いた解析から,nep98温度感受性変異株ではスピンドル極体の構造に異常を持つことが明らかとなった.以上の結果から,Nep98pはスピンドル極体の機能発現に必須な役割をはたしていると考えられる.スピンドル極体の機能は,酵母接合時の核融合にも必要であり,nep98温度感受性変異株の接合子は2つの1倍体核の接近と核外膜の融合の過程に欠損を示すことが明らかとなった.
    Nep98pは膜貫通領域を1つ持つ膜内在性タンパク質であり,N末端をサイトゾル側に向けたトポロジーをとる.また,C末端側にはJem1pとの相互作用領域が存在する.これらの領域の機能を明らかにするため,様々なNep98pの部分欠失変異株を作製した.この結果,Jem1pとの相互作用領域は酵母の増殖に必須であり,ここがわずかでも欠失すると酵母は致死となることが明らかとなった.また,N末端のサイトゾル側ドメインは酵母の増殖に必須ではなかったが,接合時の核融合およびNep98pの核膜局在に必要であることが示された.

    researchmap

  • 酵母ミトコンドリアを巡るプロテインフラックス

    Grant number:12206039  2000

    日本学術振興会  科学研究費助成事業 特定領域研究(C)  特定領域研究(C)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    ミトコンドリアは外膜,膜間部,内膜,マトリクスの4つの区画から構成され,サイトゾルで合成されたタンパク質は外膜のTOM,内膜の少なくとも2種類のTIMとよばれるタンパク質複合体の働きによりミトコンドリアに移行し,適切なミトコンドリア内区画に配置される。取り込まれたタンパク質は,マトリクスの分子シャペロンHsp70,Hsp60の働きで機能化する。本研究では,以上のプロセスの全体像を,全ミトコンドリアタンパク質の流れ(プロテインフラックス)として網羅的に解析し,フラックスの大きさと分岐を制御する因子とメカニズムの解明の突破口を開くことをめざした。具体的にはまず500〜1000種類存在すると考えられるミトコンドリアタンパク質の同定のために,(1)酵母細胞からNycodenz密度勾配遠心を用いて高純度のミトコンドリアを単離精製し,さらに大量のミトコンドリアを可溶性膜間部,塩溶出性膜間部,マトリクス,膜画分に分画する方法を確立した。アフィニティタグをミトコンドリア表面に付け,磁気ビーズで高純度ミトコンドリアを単離・精製する方法も検討中である。(2)分画したミトコンドリアの各画分について二次元電気泳動を行い,CBB染色した各スポットを限定分解→質量スペクトルで同定することを試みた(現在データ解析中)。(3)酵母細胞から全mRNAを単離,in vitroで翻訳しRI標識タンパク質を合成した。これを基質として,in vitroで単離ミトコンドリアへの取り込み実験を行い,内膜の膜電位のある場合とない場合,プロテアーゼでミトコンドリア内に取り込まれなかったタンパク質を消化した場合としない場合の各々について,ミトコンドリアを回収して二次元電気泳動を行い,標識タンパク質のスポットを検出した。得られたスポットについて(2)のスポットとの対応付けを試みた。

    researchmap

  • ミトコンドリア行きターゲティングシグナル受容体による分子認識機構の解析

    Grant number:12019226  2000

    日本学術振興会  科学研究費助成事業 特定領域研究(A)  特定領域研究(A)

    遠藤 斗志也, 西川 周一

      More details

    Grant amount:\1900000 ( Direct Cost: \1900000 )

    近年,分子シャペロンによる基質認識やタンパク質のターゲティングシグナルの認識など,ブロードな基質スペクトルに特徴づけられる「あいまいな分子認識」の重要性が注目されている。本研究ではあいまいな認識の例として,ミトコンドリアタンパク質のプレシークエンスに書き込まれたミトコンドリアターゲティングシグナルを認識する受容体Tom20をとりあげた。様々な^<15>N標識した様々なプレ配列ペプチドについて,Tom20のサイトゾルドメイン(ΔTom20)を加えていったときにNMRシグナルが影響を受ける部位を調べることにより, 「Tom20結合セグメント」を同定した。またスピンラベルしたペプチドを用いて,ΔTom20結合時のプレ配列の配向も調べた。そして,プレ配列中のΔDTom20結合セグメントの位置はプレ配列によって異なる(フレキシブルである)が,ΔTom20結合時の配向は一定であることを見いだした。
    ΔTom20結合セグメントは,ΔTom20との結合によりNMRシグナルが影響を受ける残基であり,直接ΔTom20との結合に関わるのはその一部と考えられる。実際ΔTom20との複合体の立体構造を決定したALDHのプレ配列においてΔTom20との結合に直接関わるセグメントは,5残基である。ΔTom20との結合に直接関わるセグメントをΔTom20認識エレメントと呼ぶことにする。ALDH以外のプレ配列中のΔTom20認識エレメントを推測するために,各プレ配列中のΔTom20結合セグメントに共通するモチーフ,具体的にはAKDHのΔTom20認識エレメントと共通するモチーフを検索した。その結果,各プレ配列ペプチドにφχχφφというパターンが見いだされた。ただし,φは疎水性/芳香族残基(Leu,Met,Trp,Cys,Ala,Tyr,Phe,Ile),χは任意のアミノ酸残基である。

    researchmap

  • 核ゲノム及び葉緑体ゲノムにコードされたタンパク質の協調したチラコイド移行の解析

    Grant number:12025213  2000

    日本学術振興会  科学研究費助成事業 特定領域研究(A)  特定領域研究(A)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    Grant amount:\3500000 ( Direct Cost: \3500000 )

    核ゲノムにコードされたタンパク質は二枚の包膜を通過してストロマに達し,その後チラコイドに移行する。外包膜と内包膜にはTocとTic,チラコイドにはSecシステムとTATシステムという膜透過装置が存在する。安定な高次構造をとったタンパク質は,Secチャネルを介してチラコイド膜を通過できないが,葉緑体の包膜および,TATシステムを介してチラコイド膜を通過することができる。このことは(1)包膜のToc/Ticシステムやチラコイド膜のTATシステムには強力なunfolding活性があり,安定な高次構造であってもこれをほどくことができる,(2)チャネルが自在に広がることにより,タンパク質が安定な高次構造をとったまま膜透過できる,という二つの可能性を意味する。そこで二つの可能性を区別するために,包膜やチラコイド膜の膜透過チャネルの大きさを推定することを試みた。
    チラコイド内腔タンパク質である33Kタンパク質前駆体をin vitroで合成し,C末端に様々な大きさのケイ光発色団や金クラスターを付加した。そして修飾前駆体のin vitroでの葉緑体への取り込み実験において,付加したケイ光発色団や金クラスターが膜を通過できるかどうかを調べた。13×10Åのfluoresceinを付加した前駆体及び16×13ÅのTexas Redを付加した前駆体は包膜を通過,さらにチラコイド膜を通過して内腔に達した。一方20Åの金クラスターを付加した前駆体は包膜透過効率が40〜60%に低下し,チラコイド膜は通過できなかった。したがって包膜のToc/Ticチャネルは直径が20Å前後,チラコイド膜のSecチャネルは直径が15A前後と考えられる。このことは,Toc/Ticシステムには強力なunfolding活性があり,安定な高次構造をとった前駆体は高次構造がほどかれることによってToc/Ticチャネルを通過することを意味している。

    researchmap

  • 核ゲノム及び葉緑体ゲノムにコードされたタンパク質の協調したチラコイド移行の解析

    Grant number:11151214  1999

    日本学術振興会  科学研究費助成事業 特定領域研究(A)  特定領域研究(A)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    Grant amount:\3500000 ( Direct Cost: \3500000 )

    葉緑体の光合成機能発現の中心的プロセスは,核あるいは葉緑体ゲノムにコードされた光合成に関わるタンパク質の協調したチラコイド移行である。われわれは最近,葉緑体内部に大腸菌型Sec依存性のチラコイド移行経路があること,この経路と△pH依存の経路は共通の膜透過チャネルを利用していないことを見出した。そこで,下記のように,部位特異的光架橋法を前駆体の包膜通過に適用し,前駆体と包膜の膜透過装置の間の相互作用をマッピングした。また△pH依存のチラコイド膜透過経路が高次構造を保ったまま基質タンパク質を膜透過させるメカニズムを解明するために,この経路の新規膜透過チャネルの同定を行った。
    1.エンドウ葉緑体における△pH依存のチラコイド移行経路を解析する足がかりとして,シロイヌナズナやトウモロコシでTha4と共にこの経路に関与していることが明らかにされているHcf106遺伝子のクローニングを試みた。トウモロコシHcf106タンパク質において,他のHcf106やTha4と高いホモロジーが見られるドメインを含む50〜156アミノ酸残基領域をコードする遺伝子断片をプローブとして,エンドウcDNAのλライブラリーをスクリーニングした。この結果一つのクローンが得られ,塩基配列決定から248アミノ酸残基をコードするORFが同定された。
    2.プラストシアニン前駆体を用いて単離葉緑体への輸送実験を行い,ATP濃度を下げることにより,包膜透過反応の中間体が生成することを確認した。次に光反応性のアミノ酸であるBPAをサプレッサーtRNA法により部位特異的に導入したプラストシアニン前駆体を調製した。この前駆体と単離葉緑体を用いて包膜透過反応中間体を形成させ,UV照射により架橋反応を行った。その結果,Toc/Tic構成因子との架橋産物が,各部位で低濃度ATP、UV照射特異的に検出された。

    researchmap

  • ミトコンドリア行きターゲティングシグナル受容体による分子認識機構の解析

    Grant number:11132223  1999

    日本学術振興会  科学研究費助成事業 特定領域研究(A)  特定領域研究(A)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    Grant amount:\2000000 ( Direct Cost: \2000000 )

    タンパク質による分子認識の中でも,酵素による基質認識や抗体による抗原認識は,鍵と鍵穴の関係に例えられるように,厳密でシャープな基質スペクトルに特徴がある。一方近年,分子シャペロンによる基質認識やタンパク質のターゲティングシグナルの認識など,ブロードな基質スペクトルに特徴づけられる「あいまいな分子認識」の重要性が注目されている。本研究ではあいまいな認識の例として,ミトコンドリアタンパク質のプレシークエンスに書き込まれたミトコンドリアターゲティングシグナルを認識する受容体Tom20をとりあげた。Tom20の受容体ドメイン(△50Tom20)とプレシークエンスペプチド(pALDH(12-22))との複合体の立体構造をNMRで決定した。
    △50Tom20は,5本のαヘリックスから成り,そのうち4本が疎水的コアを作る。プレシークエンスpALDH(12-22)は1-3番目のヘリックスがつくる疎水性の溝に結合している。結合したペプチドは水-脂質界面がないにもかかわらず両親媒性のヘリックス構造をとり,ヘリックスの片側に並んだ疎水性残基が△50Tom20上の疎水性の溝にはまりこんでいる。したがって,プレシークエンスのTom20への結合は主として疎水性相互作用によって担われていることがわかる。一方ヘリックスの反対側に並んだ親水性残基は溶媒に露出する形になる。水-脂質界面がないのにプレシークエンスに両親媒性ヘリックス構造が誘起されたことは,プレシークエンスの受容体への結合においては,プレシークエンスがいったんミトコンドリア外膜に結合してから受容体に移行する必要はないことが示された。また,プレシークエンスの変異体を用いた実験から,Tom20のプレシークエンス認識においては,両親媒性ヘリックスを形成したプレシークエンスの疎水性残基とTom20との間の疎水性相互作用が重要であり,静電的相互作用は重要でないことが明らかになった。

    researchmap

  • Analyses of mechanisms of protein import into mitochondria by using unnatural amino acids.

    Grant number:10480156  1998 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    ENDO Toshiya, NISHIKAWA Shun-ichi, YOSHIHISA Tohru

      More details

    Grant amount:\13600000 ( Direct Cost: \13600000 )

    Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively. In the present study, artificially aminoacylated suppressor tRNAs were used to introduce photoreactive unnatural amino acids into model mitochondrial precursor proteins to map interactions between precursor proteins and translocation machineries along the import pathway.
    A model precursor protein, pSu9-DHFR, was arrested at two distinct stages, stage A (accumulated at 0℃) and stage B (accumulated at 30℃), in the translocation across the outer membrane and interactions between the arrested precursor protein and TOM proteins were analyzed at high resolution not achieved previously. Although the stage-A and the stage-B intermediates were previously assigned to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer lembrane. The mature domain is unfolded and bound to Tom40 at stage B while it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage-B intermediate, but not of the stage-A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. These results indicate that translocation of the presequence and unfolding of the mature domain are not necessarily coupled.
    We have also applied the approach of site-specific photocrosslinking to trie processes of carrier proteins transport to the mitochondrial inner membrane. Photoreactive unnatural amino acids were introduced at various positions of ADP/ATP carrier (AAC), which was arrested at various stages along its import pathway to the mitochondrial inner membrane. Subsequent photcrosslinking rexperiments evealed that AAC interacts with Tom70 and Tom40 in the outer membrane, Tim9 and Tim 10 in the intermembrane space, and Tim22 in the inner membrane in a stage-dependent manner.

    researchmap

  • 分子シャペロンJemlpによる核膜融合の制御機構

    Grant number:10780439  1998 - 1999

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    西川 周一

      More details

    Grant amount:\2000000 ( Direct Cost: \2000000 )

    DnaJファミリーのタンパク質はhsp70のパートナータンパク質として,hsp70の活性の制御を行っている.申請者らは酵母小胞体膜の内腔側に存在するDnaJファミリーのタンパク質としてJemlpを同定し,これが小胞体内腔のhsp70であるBiPのパートナータンパク質として,酵母接合時の核膜融合の過程に必須な役割をはたしていることを明らかにしてきた.Jemlpは表在性膜タンパク質であり,膜内在性タンパク質と相互作用することによって核膜融合を制御している可能性が考えられた.そこで昨年度に,Jemlpと相互作用するタンパク質を酵母two hybrid法を用いて検索し,新奇の核膜タンパク質をコードするNEP98遺伝子を分離した.
    NEP98遺伝子産物NEp98pは,膜貫通領域を1つ持つ,98kDaの核膜の膜内在性タンパク質である.Nep98pは,N末端がサイトゾル側に突出し,C末端部のJemlpとの相互作用領域が小胞体内腔側を向いた膜トポロジーを示す.また,酵母細胞内でNep98pがJemlpと相互作用していることが架橋実験によって示された.NEP98遺伝子の破壊を行ったところ酵母は致死となり,Nep98pは酵母の増殖に必須であることが明らかとなった.そこで,nep98温度感受性変異株の作製を行なったところ,得られた変異株は核の分裂に異常を示すことが明らかとなった.以上の結果はNep98pが核の融合・分裂に関与している可能性を示唆している.

    researchmap

  • 核外膜・内膜融合過程のアッセイ系の確立と新規シャベロンJemlpの機能解析

    Grant number:10878104  1998

    日本学術振興会  科学研究費助成事業 萌芽的研究  萌芽的研究

    遠藤 斗志也, 西川 周一

      More details

    Grant amount:\2100000 ( Direct Cost: \2100000 )

    分子遺伝学的解析が容易な出芽酵母は,細胞分裂に伴う核のベシクル化は見られないものの,接合時に核の融合が起こるので,核膜融合の機構を研究する良い実験系を提供する。われわれは最近,hsp70のパートナータンパク質であり分子シャペロンでもあるDnaJホモログ(JemIpと命名)が小胞体膜に存在し,その内腔側ドメインが核膜融合に関与していることを見いだした。本研究では,核質,核内膜,核膜(小胞体)内腔のいずれかをGFP(green fluorescent protein)でラベルした細胞とBFP(blue fluorescent protein)でラベルした細胞について接合反応を行い,核膜融合における外膜の融合と内膜の融合のステップを,ケイ光顕微鏡を用いて核融合をGFPとBFPの染色像が重なっていく過程としてモニターするアッセイ系を新しく開発することを目標とした。
    核膜(小胞体膜)をGFPで標識するため,小胞体膜タンパク質であるSec63pとGFPの融合タンパク質を構築し,GAL1プロモーターを用いて酵母内で発現した。Sec63p-GFP融合タンパク質を発現している細胞では小胞体の染色像が観察され,ラベルが成功したことが示された。次に核膜(小胞体)内腔をラベルするために,小胞体内腔局在型GFPとして,N末端に酵母α接合因子のシグナル配列,C末端に小胞体局在化シグナルであるHDEL配列を融合したタンパク質(ER-GFP)を遺伝子レベルで作製した。GAL1プロモーターを用いてER-GFPを酵母細胞内で発現させたところ,期待された核膜および小胞体の染色像は得られず,液胞の染色像が得られた。現在,HDEL配列とGFPの間にスペーサーの導入を行なうなどの改善を試みている。

    researchmap

  • 核ゲノム及び葉緑体ゲノムにコードされたタンパク質の協調したチラコイド移行の解析

    Grant number:10170212  1998

    日本学術振興会  科学研究費助成事業 特定領域研究(A)  特定領域研究(A)

    遠藤 斗志也, 西川 周一, 吉久 徹

      More details

    Grant amount:\2000000 ( Direct Cost: \2000000 )

    葉緑体の光合成機能発現の中心的プロセスは,核あるいは葉緑体ゲノムにコードされた光合成に関わるタンパク質の協調したチラコイド移行である。本研究では,まずタンパク質のチラコイド移行経路のうち,Sec依存型経路とΔpH依存型経路が,共通の膜透過チャネルを利用している可能性について検討した。チラコイド膜をスパンする形で膜透過反応が停止した中間体を生成することによりΔpH依存型経路を飽和したところ,ΔpH依存型経路の基質であるi23Kのチラコイドへの取り込みは阻害されたが,Sec依存型経路の基質であるi33Kのチラコイドへの取り込みは,全く阻害されなかった。したがってSec依存型経路とΔpH依存型経路は,異なる膜透過チャネルを使用しているものと考えられる。チラコイドへのタンパク質移行に関わるSec依存型経路とΔpH依存型経路は完全に独立しているのか,あるいは膜透過チャネルは共通して利用しているのかが,大きな問題であったが,今回の結果はこの問題に決着をつけるものである。
    大腸菌のSecYの場合と同様,葉緑体SecYもSecAやSecEなどとチラコイド膜上で膜透過装置複合体を形成していることが期待されるが,この仮想的SecY複合体の実体は明らかでない。そこで,シロイヌナズナSecE遺伝子をクローニングし,塩基配列を決定した。in vitroで合成したSecE前駆体は,単離葉緑体に効率よく取り込まれ、葉緑体内でプロセシングを受けて成熟体となった。オルガネラ分画等の結果,成熟体SecEはチラコイド膜に内在性膜タンパク質として組込まれたことが分かった。シロイヌナズナSecEに対する抗体を用いた抗体染色により、エンドウ葉緑体のチラコイドにもSecEが存在することが明らかになった。

    researchmap

  • Analyses of molecular mechanisms of mitochondrial protein transport by using unnatural amino acids

    Grant number:09044070  1997 - 1998

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for international Scientific Research  Grant-in-Aid for international Scientific Research

    ENDO Toshiya, SCHULTZ Peter G., NISHIKAWA Shun-ichi, YOSHIHISA Toru

      More details

    Grant amount:\5500000 ( Direct Cost: \5500000 )

    Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively. In the present study, artificially aminoacylated suppressor tRNAs were used to introduce photoreactive unnatural amino acids into model mitochondrial precursor proteins to map interactions between precursor proteins and translocation machineries along the import pathway.
    A model precursor protein, pSu9-DHFR, was arrested at two distinct stages, stage A (accumulated at 0℃) and stage B (accumulated at 30℃), in the translocation across the outer membranes and interactions between the arrested precursor protein and TOM proteins were analyzed at high resolution not achieved previously. Although the stage-A and the stage-B intermediates were previously assigned to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B while remains folded at stage A. After dissociation from the TOM complex, translocation of the stage-B intermediate, but not of the stage-A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. These results indicate that translocation of the presequence and unfolding of the mature domain are not necessarily coupled. We have also applied the approach of site-specific photocrosslinking to the process of carrier proteins transport to the mitochondrial inner membrane and of protein translocation across the chloroplast envelope membranes.

    researchmap

  • 遺伝学的アプローチによる酵母ミトコンドリアの形態制御に関する研究

    Grant number:09878127  1997

    日本学術振興会  科学研究費助成事業 萌芽的研究  萌芽的研究

    遠藤 斗志也, 西川 周一

      More details

    Grant amount:\2100000 ( Direct Cost: \2100000 )

    ミトコンドリアは細胞の増殖に伴って複製され,その位置,形,数は細胞の状態や機能と関連してダイナミックに変動する。こうしたミトコンドリア形態の制御をつかさどる遺伝子を同定するために,遺伝学的解析の容易な出芽酵母を用いて,ミトコンドリア形態に異常の有る変異株を単離することを計画した。
    ミトコンドリアの形態は,ミトコンドリアに特異的に結合するケイ光色素による染色法またはミトコンドリアタンパク質に対する抗体を用いるケイ光抗体法によって,直接顕微鏡で観察することができる。しかし,前者には,呼吸欠損株のミトコンドリアを観察できないという問題(ミトコンドリア形態に異常がある変異株は呼吸欠損株になる可能性が高い)が,後者には,試料調製の手間がかかるため,10^3のオーダー以上の株を吸う突然変異株の選別には向いていないという問題がある。そこで本研究では、クラゲ由来のケイ光性タンパク質GFPに遺伝子レベルでミトコンドリア行きシグナルをつないだ融合タンパク質を酵母細胞内で発現させ,ミトコンドリア内にGFPを送り込むことにより,ミトコンドリアの形態をケイ光顕微鏡により簡便に観察できるシステムを構築した。この方法は手間がかからず,呼吸欠損になる可能性が高いミトコンドリア形態異常株を,10^3のオーダーの温度感受性変異株や呼吸欠損株の集団から選別するのに適している。この方法を用いて,温度感受性変異株91株の中から19,呼吸欠損株33株の中から39のミトコンドリア形態異常株を選別した。この中から単一変異により温度感受性とミトコンドリア形態異常の表現系を示す株を4,単一変異により呼吸欠損とミトコンドリア形態異常の表現系を示す株を10得た。さらに相補性テストを行うことにより,温度感受性とミトコンドリア形態異常の表現系を示す株を2つの相補性群に,呼吸欠損とミトコンドリア形態異常の表現系を示す株は6つの相補性群に分けることができた。前者の変異株のうち1つについて変異遺伝子をクローニングすることに成功した。現在,この変異遺伝子の塩基配列の解析を行うとともに,残りの変異株についても変異遺伝子の同定を進めている。

    researchmap

  • Molecular anatomy of protein translocation machineries in yeast mitochondria

    Grant number:08458180  1996 - 1997

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    ENDO Toshiya, NISHIKAWA Shuh-ichi

      More details

    Grant amount:\7500000 ( Direct Cost: \7500000 )

    Most of mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively, In the present study, we analyzed subunit arrangements, structures, and precursor interactions of the mitochondrial translocation machineries.
    The TOM complex consists of receptor proteins, Tom7O, Tom37, Tom2O and Tom22, a channel protein, Tom4O, and small Tom proteins, Tom5, Tom6 and Tom7, By using differential solubilization methods, we found that Tom7OiTom37 and Tom2O/Tom22/Tom4O form distinct subcomplex structures in the TOM complex. We prepared cytosolic domains of Tom7O and Tom22 in large amounts and analyzed Tom7O-Tom2O interactions by glycerol gradient centrifugation. The results showed that a positively charged segment just downstream of the N-terminal transmembrane segment of Tom22 is important for interactions of Tom2O with possibly a negatively charged domain of Tom7O.Next, a tertiary structure of the cytosolic domain of Tom2O was determined by NMR spectroscopy. A binding site for presequences is also mapped on the NMR structure. In the last, we introduced a photoreactive unnatural amino acids into various positions along mitochondrial precursor protein by suppressor *RNA method, By using these labeled precursor proteins, interactions of the translocating polypeptide chains with components of the mitochondrial translocation machineries were analyzed by photocrosslinking at a high resolution that had not been obtained so far.

    researchmap

  • 酵母ミトコンドリアhsp70のシャペロン反応制御の分子機構

    Grant number:08780674  1996

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    西川 周一

      More details

    Grant amount:\1000000 ( Direct Cost: \1000000 )

    本研究では,酵母ミトコンドリアマトリクスの分子シャペロンhsp70であるSsclpと,そのパートナータンパク質Mdjlp,YgelpおよびTim44の精製を行ない,これらのシャペロン機能のin vitroでの解析を行なった.
    精製したSsclpは弱いATPase活性を有しており,これはMdjlp存在下で1.8倍,MdjlpとYgelpが共に存在すると7倍に促進された.以上の結果は大腸菌のDnaK-DnaJ-GrpEの系で得られた結果と良い一致を示しており,MdjlpとYgelpがDnaJ,GrpEと同様の機構でSsclpの機能制御を行なっていると示唆されるので,その詳細な機構について解析を行なっている.また,Mdjlpは酵母サイトゾルのhsp70であるSsalpのATPase活性も促進した.一方,Tim44のSsclpのATPase活性促進効果は現在のところ観察されていない.
    変性ルシフェラーゼのrefolding促進活性を指標にSsclpのシャペロン活性について検討したところ,DnaK-DnaJ-Grp,Ssalp-Ydjlpの場合とは異なり,Ssclp-Mdjlp-Ygelpはグアニジン塩酸によって変性したルシフェラーゼのrefoldingを促進する活性は持っていなかった.しかし,熱処理(42℃,10分)によってルシフェラーゼを変性する際にSsclpとATPが存在するとルシフェラーゼの変性が部分的に抑制された.また,熱処理後にMdjlp,Ygelpを更に加えると,変性したルシフェラーゼはrefoldし,活性の回復が観察された.現在,このシャペロン活性の違いについて更に解析を進めている.

    researchmap

  • High-level expression of recombinant proteins by controling the formation of the inclusion body

    Grant number:07558222  1995 - 1996

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)  Grant-in-Aid for Scientific Research (A)

    ENDO Toshiya, NISHIKAWA Shuh-ichi, NAKAI Masato

      More details

    Grant amount:\3900000 ( Direct Cost: \3900000 )

    Recent progress in recombinant DNA technology has allowed us to introduce genes for foreign useful proteins into microorganisms such as E.coli and to obtain their gene products in large amounts. In the present project, we aimed at, by controlling the properties of the expressed recombinant proteins to form inclusion bodies, increasing the expression efficiency of the recombinant proteins, suppressing their possible cell toxicity, and making their purification more feasible.
    We used yeast mitochondrial MIF4p as a model recombinant protein with low expression efficiency in E.coli cells. We replaced the presequence of MIF4p with various peptides including SynA1, SynA2, SynB1, SynB2, SynC (derivatives of the preseuqnece of yeast cytochrome oxidase subunit IV) and T7TAG,and made corresponding fusion genes. We then introduced the fusion genes into E.coli cells and induced their overexpression. The expression levels of the fusion proteins were assessed by *mmunoblotting and compared with that of the mature, presequence-less MIF4p. MIF4p tagged with T7TAG and with SynB2 showed higher expression levels than the mature MIF4p. The Syn B2-MIF4p fusion protein was found in an inclusion body while the T7TAG-MIF4p fusion protein was recovered in a soluble fraction. These peptides (SynB2 and T7TAG) will offer a useful starting point to further optimize their abilities to increase expression levels of the attached proteins in E.coli cells.

    researchmap

  • 酵母無細胞タンパク質合成系の改変によるSsaタンパク質のシャペロン機能解析

    Grant number:07780621  1995

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    西川 周一

      More details

    Grant amount:\900000 ( Direct Cost: \900000 )

    本研究では,酵母無細胞タンパク質合成系を元にしてサイトゾル中のSsaタンパク質の量を制御できる実験系の開発を行なった.まず,Ssa1タンパク質のC末端部分に(His)_6タグを導入した組み替えタンパク質(Ssa1-Hisp)のみをSsaタンパク質として発現している酵母株より,Moldaveらの方法に従って無細胞タンパク質合成用の酵母ライセ-トを調製した.次に,(His)_6タグに対するアフィニティーカラムであるニッケルレジンカラムを用いて,この酵母ライセ-トからSsaタンパク質を除去する条件の検討を行なった.その結果,タンパク質合成能を保持した状態でライセ-ト中のSsaタンパク質の85%を除去することができた.
    Ssaタンパク質を除去した酵母ライセ-トを用いてミトコンドリアタンパク質前駆体(pCOXIV-DHFR,F1β前駆体)を合成し,単離ミトコンドリアへの輸送実験を行なったところ,これらは共に,Ssaタンパク質除去前のライセ-トを用いて合成した場合と同程度の輸送効率でミトコンドリアに取り込まれた.抗Ssaタンパク質抗体はこれら前駆体の単離ミトコンドリアへの輸送を阻害したので,Ssaタンパク質は前駆体のミトコンドリアへの輸送に必要であることが示唆される.そこでこれはSsaタンパク質の除去が不十分であったためではないかと考えられる.現在,誘導可能なプロモーターであるGAL1プロモーターによるSSA1-His遺伝子の発現制御と組み合わせて,Ssaタンパク質の除去効率の改善を行なっている.
    Ssaタンパク質の活性はパートナータンパク質であるYdj1pによって制御されている.パートナータンパク質の側からSsaタンパク質の活性制御を行なうため,YDJ1遺伝子破壊株から酵母ライセ-トを調製した.このライセ-トを用いて合成した前駆体タンパク質の性質についても,現在解析を進めている.

    researchmap

  • Quality control in the endoplasmic reticulum

    1995

      More details

    Grant type:Competitive

    researchmap

  • Transmembrane Traffic of Proteins with Unnatural Amino Acids

    Grant number:06044091  1994 - 1995

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for international Scientific Research  Grant-in-Aid for international Scientific Research

    ENDO Toshiya, SCHULTZ Peter, KANAMORI Takashi, NISHIKAWA Shun-ichi, NAKAI Masato

      More details

    Grant amount:\6800000 ( Direct Cost: \6800000 )

    Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and are subsequently important into mitochondria. Translocation of precursor proteins across the mitochondrial outer and inner membranes requires coordinated functions of the translocation machineries in the two membranes. In order to analyze the interactions between components of the translocation machineries and translocating precursor proteins in details, we site-specifically incorporated an unnatural amino acid with a photoreactive cross-linking group, pBzPhe.
    We used pSu9 (1-69)-DHFR consisting of the presequence of the F_0-ATPase subunit 9 fused to mouse DHFR as a model precursor protein. Six mutant genes carrying the amber stop codon at specific positions along the polypeptide were constructed by in vitro mutagenesis. The mutant pSu9 (1-69)-DHFR fusion proteins (T4, A17, A29, T44, A56, and Y67) were synthesized in the presence of suppressor tRNA,which was charged with pBz-Phe, and RI-labeled Met by coupled transcription/translation with rabbit reticulocyte lysate. All the proteins except for T44 were synthesized efficiently. Besides, mutant fusion proteins containing pBz-Phe could be imported into mitochondria efficiently.
    Since translocation across the mitochondrial membranes require unfolding of the precursor protein, stabilization of the tertiary structure of the DHFR domain of the fusion proteins by methotrexate blocks their import into mitochondria, resulting in the formation of translocation intermediates. These translocation intermediates in transit through the translocation channels should be in close contact with the components of the translocation machineries. Therefore irradiation of the intermediates will lead to cross-linking of the fusion proteins with the components of the translocation machineries. Indeed, we could observe cross-linked products which are dependent on the positions of introduced pBz-Phe. These forms may represent cross-linked products between the fusion proteins and the components of the translocation machineries due to their specific interactions.

    researchmap

  • タンパク質のミトコンドリア内仕分け機構に関与する新規酵母突然変異株の分離

    Grant number:06858077  1994

    日本学術振興会  科学研究費助成事業 奨励研究(A)  奨励研究(A)

    西川 周一

      More details

    Grant amount:\900000 ( Direct Cost: \900000 )

    ミトコンドリア膜間部タンパク質であるシトクロムb_2は,その局在化の過程で最後に膜間部においてプロセシングをうけるが,このステップの変異株(imp変異株)が温度感受性の呼吸欠損株として分離されている.そこで,膜間部タンパク質の仕分けの変異株を,まず温度感受性呼吸欠損株として分離した.
    酵母野性株をエチルメタンスルホン酸を用いて変異源処理し,約19,000個のコロニーをスクリーニングした結果,215株の温度感受性呼吸欠損株を得た.これらの株を制限温度下で培養後,細胞抽出液を調製し,SDSポリアクリルアミドゲル電気泳動,抗シトクロムb_2抗体を用いたウェスタンブロットを行なった.この結果,制限温度下でシトクロムb_2の中間体が蓄積している株を1株,シトクロムb_2の前駆体が蓄積している株を4株得た.まず,制限温度下でシトクロムb_2の中間体を蓄積する1株について遺伝解析を行なった結果,この株はIMP1遺伝子に変異が生じており,膜間部タンパク質の仕分け機構の変異株ではないことが明らかとなった.一方,制限温度下でシトクロムb_2の前駆体を蓄積する株では,制限温度下においてF_1βタンパク質のような他のミトコンドリアタンパク質の前駆体の蓄積は観察はされなかった.このことは,これらの株はシトクロムb_2の局在化に特異的に欠損を持っていることを示唆している.そこで現在,これら4株についてさらに遺伝解析を進めている.これらの株の解析によって,シトクロムb_2の局在化機構に関与する因子が同定されることが期待される.

    researchmap

▶ display all

 

Teaching Experience

  • 基礎生物科学実習II

    2022
    Institution name:新潟大学

  • 生物学特論 VI

    2022
    Institution name:新潟大学

  • 植物生理学特論I

    2022
    Institution name:新潟大学

  • 生物学実習I

    2022
    Institution name:新潟大学

  • 先端科学技術総論

    2022
    Institution name:新潟大学

  • 生物学実験

    2021
    Institution name:新潟大学

  • 植物機能制御論I

    2021
    Institution name:新潟大学

  • 理学スタディ・スキルズ

    2021
    Institution name:新潟大学

  • 自然科学総論Ⅳ

    2020
    Institution name:新潟大学

  • 日本事情自然系A

    2019
    Institution name:新潟大学

  • 課題研究II(生物学)

    2018
    Institution name:新潟大学

  • 理学スタディ・スキルズ

    2018
    -
    2021
    Institution name:新潟大学

  • 自然科学基礎実験

    2017
    Institution name:新潟大学

  • 生物学総合演習

    2017
    Institution name:新潟大学

  • 課題研究I(生物学)

    2017
    Institution name:新潟大学

  • 生物学基礎実習b

    2017
    Institution name:新潟大学

  • 細胞・遺伝学実習

    2017
    Institution name:新潟大学

  • 生物学基礎演習

    2017
    Institution name:新潟大学

  • 植物機能制御論Ⅰ

    2015
    -
    2017
    Institution name:新潟大学

  • 実践型食づくりプロジェクト

    2015
    Institution name:新潟大学

  • 生命・食料科学博士特定研究Ⅱ

    2015
    Institution name:新潟大学

  • 研究発表(博士)演習(学会発表含む)

    2015
    Institution name:新潟大学

  • 基礎生命科学(博士)演習(中間発表)

    2015
    Institution name:新潟大学

  • 生命・食料科学博士セミナーⅡ

    2015
    Institution name:新潟大学

  • 生物学実習

    2015
    Institution name:新潟大学

  • 生命科学への招待(生物学学習法)

    2015
    Institution name:新潟大学

  • 外国語論文解説・討論Ⅱ

    2015
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅡ

    2014
    -
    2015
    Institution name:新潟大学

  • 研究発表演習(中間発表)

    2014
    -
    2015
    Institution name:新潟大学

  • 文献詳読Ⅱ

    2014
    -
    2015
    Institution name:新潟大学

  • 外国語論文解説・討論Ⅰ

    2014
    -
    2015
    Institution name:新潟大学

  • 研究発表

    2014
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学博士セミナーⅠ

    2014
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学博士特定研究Ⅰ

    2014
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅡ

    2014
    -
    2015
    Institution name:新潟大学

  • Biology Today

    2014
    Institution name:新潟大学

  • 植物生理学演習

    2013
    Institution name:新潟大学

  • 植物生理学I

    2013
    Institution name:新潟大学

  • 生物学基礎A

    2013
    Institution name:新潟大学

  • 植物生理学III

    2013
    Institution name:新潟大学

  • 植物生理学特論Ⅰ

    2013
    -
    2020
    Institution name:新潟大学

  • 生物学実験 I

    2013
    -
    2019
    Institution name:新潟大学

  • 基礎生物科学実習II

    2013
    -
    2017
    Institution name:新潟大学

  • 課題研究I(生物学科)

    2013
    -
    2016
    Institution name:新潟大学

  • 課題研究II

    2013
    -
    2016
    Institution name:新潟大学

  • 植物分子生理学実習

    2013
    -
    2016
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅠ

    2013
    -
    2015
    Institution name:新潟大学

  • 文献詳読Ⅰ

    2013
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅠ

    2013
    -
    2015
    Institution name:新潟大学

  • 植物機能制御論Ⅱ

    2013
    Institution name:新潟大学

▶ display all