担当区分：最終著者,　責任著者   出版者・発行元：Oxford University Press ({OUP})  

DOI： 10.1093/pcp/pcz148

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nar/gky619

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

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

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

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

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担当区分：最終著者,　責任著者  

DOI： 10.1080/15592324.2015.1035853

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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

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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

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

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC 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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

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

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記述言語：英語   出版者・発行元：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

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DOI： 10.1073/pnas.0912234106

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

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

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

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

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

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

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

The 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

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

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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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

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

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/1471-2229-5-22

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記述言語：英語   出版者・発行元：JAPAN SOC CELL BIOLOGY  

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記述言語：英語   出版者・発行元：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

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

The 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

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

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

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

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

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

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

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

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

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記述言語：日本語   出版者・発行元：(一社)日本細胞生物学会  

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

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

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

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

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

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.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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

The 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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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記述言語：英語   出版者・発行元：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.

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

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

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

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.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

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

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.

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

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

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DOI： 10.14841/jspp.2011.0.0112.0

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DOI： 10.14841/jspp.2010.0.0266.0

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DOI： 10.14841/jspp.2010.0.S0003.0

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DOI： 10.14841/jspp.2010.0.0130.0

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DOI： 10.14841/jspp.2009.0.0113.0

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DOI： 10.14841/jspp.2009.0.0084.0

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DOI： 10.14841/jspp.2009.0.0810.0

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DOI： 10.14841/jspp.2008.0.0980.0

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DOI： 10.14841/jspp.2008.0.0313.0

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DOI： 10.14841/jspp.2008.0.0793.0

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DOI： 10.14841/jspp.2007.0.546.0

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DOI： 10.14841/jspp.2007.0.550.0

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DOI： 10.14841/jspp.2006.0.353.0

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DOI： 10.14841/jspp.2006.0.911.0

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DOI： 10.14841/jspp.2006.0.663.0

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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DOI： 10.14841/jspp.2005.0.805.0

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記述言語：日本語   出版者・発行元：秀潤社  

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DOI： 10.14841/jspp.2004.0.771.0

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC CELL BIOLOGY  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC CELL BIOLOGY  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   出版者・発行元：Japanese Society of Plant Physiologists  

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その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184557

記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC CELL BIOLOGY  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC CELL BIOLOGY  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-LISS  

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