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      Structural and biochemical analyses of the nuclear pore complex component ELYS identify residues responsible for nucleosome binding

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          Abstract

          The nuclear pore complex embedded within the nuclear envelope is the essential architecture for trafficking macromolecules, such as proteins and RNAs, between the cytoplasm and nucleus. The nuclear pore complex assembly occurs on chromatin in the post-mitotic phase of the cell cycle. ELYS (MEL-28/AHCTF1) binds to the nucleosome, which is the basic chromatin unit, and promotes assembly of the complex around the chromosomes in cells. Here we show that the Arg-Arg-Lys (RRK) stretch of the C-terminal ELYS region plays an essential role in the nucleosome binding. The cryo-EM structure and the crosslinking mass spectrometry reveal that the ELYS C-terminal region directly binds to the acidic patch of the nucleosome. These results provide mechanistic insight into the ELYS-nucleosome interaction, which promotes the post-mitotic nuclear pore complex formation around chromosomes in cells.

          Abstract

          Wataru Kobayashi et al. use cryo-EM to study the C-terminal ELYS subunit of the nuclear pore complex. They show that a three-residue stretch binds to the acidic patch of the nucleosome, providing new mechanistic insights into ELYS nucleosome interactions.

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          Most cited references29

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          Transport into and out of the nucleus.

          I Macara (2001)
          A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.
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            Orchestrating nuclear envelope disassembly and reassembly during mitosis.

            Cell division in eukaryotes requires extensive architectural changes of the nuclear envelope (NE) to ensure that segregated DNA is finally enclosed in a single cell nucleus in each daughter cell. Higher eukaryotic cells have evolved 'open' mitosis, the most extreme mechanism to solve the problem of nuclear division, in which the NE is initially completely disassembled and then reassembled in coordination with DNA segregation. Recent progress in the field has now started to uncover mechanistic and molecular details that underlie the changes in NE reorganization during open mitosis. These studies reveal a tight interplay between NE components and the mitotic machinery.
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              Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography*

              Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.
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                Author and article information

                Contributors
                kurumizaka@iam.u-tokyo.ac.jp
                Journal
                Commun Biol
                Commun Biol
                Communications Biology
                Nature Publishing Group UK (London )
                2399-3642
                3 May 2019
                3 May 2019
                2019
                : 2
                : 163
                Affiliations
                [1 ]ISNI 0000 0001 2151 536X, GRID grid.26999.3d, Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, , The University of Tokyo, ; 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan
                [2 ]ISNI 0000 0004 1936 9975, GRID grid.5290.e, Graduate School of Advanced Science and Engineering, , Waseda University, ; 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480 Japan
                Author information
                http://orcid.org/0000-0001-7412-3722
                Article
                385
                10.1038/s42003-019-0385-7
                6499780
                d9679e5f-7a1e-484f-ab01-104d348a59d8
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 19 December 2018
                : 8 March 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001691, MEXT | Japan Society for the Promotion of Science (JSPS);
                Award ID: JP18H05534
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100003382, MEXT | JST | Core Research for Evolutional Science and Technology (CREST);
                Award ID: JPMJCR16G1
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100009619, Japan Agency for Medical Research and Development (AMED);
                Award ID: JP18am0101076
                Award Recipient :
                Categories
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                Custom metadata
                © The Author(s) 2018

                nucleosomes,cryoelectron microscopy
                nucleosomes, cryoelectron microscopy

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