Imamoto, N (Naoko)
Mol Cell Proteomics. 2012 Oct 19;: 23087160
Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and SILAC-based quantitative proteomics.
Makoto Kimura, Shingo Kose, Nobuaki Okumura, Kenichiro Imai, Maiko Furuta, Noriyuki Sakiyama, Kentaro Tomii, Paul Horton, Toshifumi Takao, Naoko Imamoto
The human importin-β family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acid in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. 1) cells are cultured in a medium containing a stable isotope. 2) cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-β family carrier, and then supplemented with a particular importin-β family carrier of interest. 3) proteins in the nuclei are extracted and analyzed quantitatively by LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-β family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes and also new potential cargoes, which we corroborated by in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possess at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin and Lys/Arg-rich segments capable of binding to both transportin and importin-β. Thus, our method will be useful to link a carrier to features shared among its cargoes and to specific nuclear localization signals.
Nucleus. 2012 Sep 1;3 (5): 22895094
Cellular Dynamics Laboratory; RIKEN Advanced Science Institute; Wako, Saitama, Japan.
Cellular stresses significantly affect nuclear transport systems. Nuclear transport pathways mediated by importin β-family members, which are active under normal conditions, are downregulated. During thermal stress, a nuclear import pathway mediated by a novel carrier, which we named Hikeshi, becomes active. Hikeshi is not a member of the importin β family and mediates the nuclear import of Hsp70s. Unlike importin β family-mediated nuclear transport, the Hikeshi-mediated nuclear import of Hsp70s is not coupled to the GTPase cycle of the small GTPase Ran but rather is coupled with the ATPase cycle of Hsp70s. Hikeshi-mediated nuclear import is essential for the attenuation and reversal of the thermal stress response in human cells. The mechanism and functions of this newly identified nuclear import pathway will be discussed.
Most cited papers:
Kerstin S Wendt, Keisuke Yoshida, Takehiko Itoh, Masashige Bando, Birgit Koch, Erika Schirghuber, Shuichi Tsutsumi, Genta Nagae, Ko Ishihara, Tsuyoshi Mishiro, Kazuhide Yahata, Fumio Imamoto, Hiroyuki Aburatani, Mitsuyoshi Nakao, Naoko Imamoto, Kazuhiro Maeshima, Katsuhiko Shirahige, Jan-Michael Peters
Research Institute of Molecular Pathology, Dr. Bohr Gasse 7, 1030 Vienna, Austria.
Cohesin complexes mediate sister-chromatid cohesion in dividing cells but may also contribute to gene regulation in postmitotic cells. How cohesin regulates gene expression is not known. Here we describe cohesin-binding sites in the human genome and show that most of these are associated with the CCCTC-binding factor (CTCF), a zinc-finger protein required for transcriptional insulation. CTCF is dispensable for cohesin loading onto DNA, but is needed to enrich cohesin at specific binding sites. Cohesin enables CTCF to insulate promoters from distant enhancers and controls transcription at the H19/IGF2 (insulin-like growth factor 2) locus. This role of cohesin seems to be independent of its role in cohesion. We propose that cohesin functions as a transcriptional insulator, and speculate that subtle deficiencies in this function contribute to 'cohesinopathies' such as Cornelia de Lange syndrome.
The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line.
Harukazu Suzuki, Alistair R R Forrest, Erik van Nimwegen, Carsten O Daub, Piotr J Balwierz, Katharine M Irvine, Timo Lassmann, Timothy Ravasi, Yuki Hasegawa, Michiel J L de Hoon, Shintaro Katayama, Kate Schroder, Piero Carninci, Yasuhiro Tomaru, Mutsumi Kanamori-Katayama, Atsutaka Kubosaki, Altuna Akalin, Yoshinari Ando, Erik Arner, Maki Asada, Hiroshi Asahara, Timothy Bailey, Vladimir B Bajic, Denis Bauer, Anthony G Beckhouse, Nicolas Bertin, Johan Björkegren, Frank Brombacher, Erika Bulger, Alistair M Chalk, Joe Chiba, Nicole Cloonan, Adam Dawe, Josee Dostie, Pär G Engström, Magbubah Essack, Geoffrey J Faulkner, J Lynn Fink, David Fredman, Ko Fujimori, Masaaki Furuno, Takashi Gojobori, Julian Gough, Sean M Grimmond, Mika Gustafsson, Megumi Hashimoto, Takehiro Hashimoto, Mariko Hatakeyama, Susanne Heinzel, Winston Hide, Oliver Hofmann, Michael Hörnquist, Lukasz Huminiecki, Kazuho Ikeo, Naoko Imamoto, Satoshi Inoue, Yusuke Inoue, Ryoko Ishihara, Takao Iwayanagi, Anders Jacobsen, Mandeep Kaur, Hideya Kawaji, Markus C Kerr, Ryuichiro Kimura, Syuhei Kimura, Yasumasa Kimura, Hiroaki Kitano, Hisashi Koga, Toshio Kojima, Shinji Kondo, Takeshi Konno, Anders Krogh, Adele Kruger, Ajit Kumar, Boris Lenhard, Andreas Lennartsson, Morten Lindow, Marina Lizio, Cameron Macpherson, Norihiro Maeda, Christopher A Maher, Monique Maqungo, Jessica Mar, Nicholas A Matigian, Hideo Matsuda, John S Mattick, Stuart Meier, Sei Miyamoto, Etsuko Miyamoto-Sato, Kazuhiko Nakabayashi, Yutaka Nakachi, Mika Nakano, Sanne Nygaard, Toshitsugu Okayama, Yasushi Okazaki, Haruka Okuda-Yabukami, Valerio Orlando, Jun Otomo, Mikhail Pachkov, Nikolai Petrovsky, Charles Plessy, John Quackenbush, Aleksandar Radovanovic, Michael Rehli, Rintaro Saito, Albin Sandelin, Sebastian Schmeier, Christian Schönbach, Ariel S Schwartz, Colin A Semple, Miho Sera, Jessica Severin, Katsuhiko Shirahige, Cas Simons, George St Laurent, Masanori Suzuki, Takahiro Suzuki, Matthew J Sweet, Ryan J Taft, Shizu Takeda, Yoichi Takenaka, Kai Tan, Martin S Taylor, Rohan D Teasdale, Jesper Tegnér, Sarah Teichmann, Eivind Valen, Claes Wahlestedt, Kazunori Waki, Andrew Waterhouse, Christine A Wells, Ole Winther, Linda Wu, Kazumi Yamaguchi, Hiroshi Yanagawa, Jun Yasuda, Mihaela Zavolan, David A Hume, Takahiro Arakawa, Shiro Fukuda, Kengo Imamura, Chikatoshi Kai, Ai Kaiho, Tsugumi Kawashima, Chika Kawazu, Yayoi Kitazume, Miki Kojima, Hisashi Miura, Kayoko Murakami, Mitsuyoshi Murata, Noriko Ninomiya, Hiromi Nishiyori, Shohei Noma, Chihiro Ogawa, Takuma Sano, Christophe Simon, Michihira Tagami, Yukari Takahashi, Jun Kawai, Yoshihide Hayashizaki
 RIKEN Omics Science Center, RIKEN Yokohama Institute, Kanagawa, Japan. These authors contributed equally to this work. These authors are the core writing group. These authors are affiliated with the FANTOM 4 headquarters.
Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.
Soo Jae Lee, Toshihiro Sekimoto, Eiki Yamashita, Emi Nagoshi, Atsushi Nakagawa, Naoko Imamoto, Masato Yoshimura, Hiroaki Sakai, Khoon Tee Chong, Tomitake Tsukihara, Yoshihiro Yoneda
Institute for Protein Research, Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan.
The sterol regulatory element-binding protein 2 (SREBP-2), a nuclear transcription factor that is essential for cholesterol metabolism, enters the nucleus through a direct interaction of its helix-loop-helix leucine zipper domain with importin-beta. We show the crystal structure of importin-beta complexed with the active form of SREBP-2. Importin-beta uses characteristic long helices like a pair of chopsticks to interact with an SREBP-2 dimer. Importin-beta changes its conformation to reveal a pseudo-twofold symmetry on its surface structure so that it can accommodate a symmetric dimer molecule. Importin-beta may use a similar strategy to recognize other dimeric cargoes.
Biological Macromolecules Laboratory, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan. firstname.lastname@example.org
We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.
Kazuhiro Maeshima, Kazuhide Yahata, Yoko Sasaki, Reiko Nakatomi, Taro Tachibana, Tsutomu Hashikawa, Fumio Imamoto, Naoko Imamoto
Cellular Dynamics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan. email@example.com
Nuclear pores are sophisticated gateways on the nuclear envelope that control macromolecular transport between the cytoplasm and nucleoplasm. So far the structural and functional aspects of nuclear pores have been extensively studied, but their distribution and density, which might reflect nuclear organization and function, remain unknown. Here, we report the cell-cycle-dependent dynamics of nuclear pores. Large distinct subdomains lacking nuclear pores are present on the nuclear surface of HeLaS3 cells in early cell-cycle stages. Such ;pore-free islands' gradually become dispersed in G1-S phase. Surprisingly, the islands are enriched with inner nuclear membrane proteins lamin A/C and emerin, but exclude lamin B. Lamin-A/C-enriched pore-free islands were also observed in human normal diploid fibroblasts and several cell lines, showing the generality of this phenomenon. Knockdown and ectopic expression analyses demonstrated that lamin A/C, but not emerin, plays an essential structural and regulatory role in the nuclear pore distribution and the formation of pore-free islands. These data thus provide strong evidence that the dynamics of nuclear pores are regulated by the reorganization of inner nuclear structures.
Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
mRNAs in eukaryotic cells are presumed to always associate with a set of proteins to form mRNPs. In Xenopus oocytes, a large pool of maternal mRNAs is masked from the translational apparatus as storage mRNPs. Here we identified Xenopus RAP55 (xRAP55) as a component of RNPs that associate with FRGY2, the principal component of maternal mRNPs. RAP55 is a member of the Scd6 or Lsm14 family. RAP55 localized to cytoplasmic foci in Xenopus oocytes and the processing bodies (P-bodies) in cultured human cells: in the latter cells, RAP55 is an essential constituent of the P-bodies. We isolated xRAP55-containing complexes from Xenopus oocytes and identified xRAP55-associated proteins, including a DEAD-box protein, Xp54, and a protein arginine methyltransferase, PRMT1. Recombinant xRAP55 repressed translation, together with Xp54, in an in vitro translation system. In addition, xRAP55 repressed translation in oocytes when tethered with a reporter mRNA. Domain analyses revealed that the N-terminal region of RAP55, including the Lsm domain, is important for the localization to P-bodies and translational repression. Taken together, our results suggest that xRAP55 is involved in translational repression of mRNA as a component of storage mRNPs.
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
Coherent x-ray diffraction microscopy is a lensless phase-contrast imaging technique with high image contrast. Although electron tomography allows intensive study of the three-dimensional structure of cellular organelles, it has inherent difficulty with thick objects. X rays have the unique benefit of allowing noninvasive analysis of thicker objects and high spatial resolution. We observed an unstained human chromosome using coherent x-ray diffraction. The reconstructed images in two or three dimensions show an axial structure, which has not been observed under unstained conditions.
Phosphoproteomics reveals new ERK MAP kinase targets and links ERK to nucleoporin-mediated nuclear transport.
Hidetaka Kosako, Nozomi Yamaguchi, Chizuru Aranami, Masato Ushiyama, Shingo Kose, Naoko Imamoto, Hisaaki Taniguchi, Eisuke Nishida, Seisuke Hattori
Division of Cellular Proteomics (BML), Institute of Medical Science, The University of Tokyo, Tokyo, Japan. firstname.lastname@example.org
Many extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, but the diversity of ERK-mediated processes suggests the existence of additional targets. Using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, we detected 38 proteins showing reproducible phosphorylation changes between ERK-activated and ERK-inhibited samples, including 24 new candidate ERK targets. ERK directly phosphorylated at least 13 proteins in vitro. Of these, Nup50 was verified as a bona fide ERK substrate. Notably, ERK phosphorylation of the FG repeat region of Nup50 reduced its affinity for importin-beta family proteins, importin-beta and transportin. Other FG nucleoporins showed a similar functional change after ERK-mediated phosphorylation. Nuclear migration of importin-beta and transportin was impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. Thus, we propose that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.
Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles.
Yuki Okada, Tadaki Suzuki, Yuji Sunden, Yasuko Orba, Shingo Kose, Naoko Imamoto, Hidehiro Takahashi, Shinya Tanaka, William W Hall, Kazuo Nagashima, Hirofumi Sawa
Laboratory of Molecular and Cellular Pathology, Graduate School of Hokkaido University, N15, W7, Kita-ku, Sapporo 060-8638, Japan.
The nuclear envelope is one of the chief obstacles to the translocation of macromolecules that are larger than the diameter of nuclear pores. Heterochromatin protein 1 (HP1) bound to the lamin B receptor (LBR) is thought to contribute to reassembly of the nuclear envelope after cell division. Human polyomavirus agnoprotein (Agno) has been shown to bind to HP1alpha and to induce its dissociation from LBR, resulting in destabilization of the nuclear envelope. Fluorescence recovery after photobleaching showed that Agno increased the lateral mobility of LBR in the inner nuclear membrane. Biochemical and immunofluorescence analyses showed that Agno is targeted to the nuclear envelope and facilitates the nuclear egress of polyomavirus-like particles. These results indicate that dissociation of HP1alpha from LBR and consequent perturbation of the nuclear envelope induced by polyomavirus Agno promote the translocation of virions out of the nucleus.
Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid.
Kiyoshi Tahara, Masatoshi Takagi, Miho Ohsugi, Takefumi Sone, Fumiko Nishiumi, Kazuhiro Maeshima, Yasuomi Horiuchi, Noriko Tokai-Nishizumi, Fumio Imamoto, Tadashi Yamamoto, Shingo Kose, Naoko Imamoto
Cellular Dynamics Laboratory, Discovery Research Institute, Institute of Physical and Chemical Research, Wako, Saitama, 351-0198, Japan.
Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-alpha/beta transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-alpha/beta was efficiently targeted to mitotic chromosomes. The addition of Ran-guanosine diphosphate and an energy source, which generates Ran-guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-beta-mediated chromosome loading of hKid. Our results indicate that the association of importin-beta and -alpha with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP-mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor-mediated mechanism for targeting proteins to mitotic chromosomes.