DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites

Abstract

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-β2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-β2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-β2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. Here, the data lead to a model where high-affinity binding of karyopherin-β2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-β2 may act analogously to control condensates in diverse cellular contexts.

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); NIGMS; Howard Hughes Medical Institute HCIA; Welch Foundation
OSTI Identifier:
1548416
Alternate Identifier(s):
OSTI ID: 1440606
Grant/Contract Number:  
AC02-06CH11357; 1S10RR26461-1; 1S10OD018027-01; R01GM069909; U01GM98256-01; R01GM56322; R01GM118530; R01GM083960; P41GM109824; R01GM112108; R01GM114274; T32GM008203; T32GM008297
Resource Type:
Published Article
Journal Name:
Cell
Additional Journal Information:
Journal Name: Cell Journal Volume: 173 Journal Issue: 3; Journal ID: ISSN 0092-8674
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; karyopherin-β2; transportin-1; PY-NLS; FUS; amyotrophic lateral sclerosis; liquid-liquid phase separation; biomolecular condensate; low-complexity sequences; intrinsically disordered protein; RNA granule

Citation Formats

Yoshizawa, Takuya, Ali, Rustam, Jiou, Jenny, Fung, Ho Yee Joyce, Burke, Kathleen A., Kim, Seung Joong, Lin, Yuan, Peeples, William B., Saltzberg, Daniel, Soniat, Michael, Baumhardt, Jordan M., Oldenbourg, Rudolf, Sali, Andrej, Fawzi, Nicolas L., Rosen, Michael K., and Chook, Yuh Min. Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. United States: N. p., 2018. Web. doi:10.1016/j.cell.2018.03.003.
Yoshizawa, Takuya, Ali, Rustam, Jiou, Jenny, Fung, Ho Yee Joyce, Burke, Kathleen A., Kim, Seung Joong, Lin, Yuan, Peeples, William B., Saltzberg, Daniel, Soniat, Michael, Baumhardt, Jordan M., Oldenbourg, Rudolf, Sali, Andrej, Fawzi, Nicolas L., Rosen, Michael K., & Chook, Yuh Min. Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. United States. https://doi.org/10.1016/j.cell.2018.03.003
Yoshizawa, Takuya, Ali, Rustam, Jiou, Jenny, Fung, Ho Yee Joyce, Burke, Kathleen A., Kim, Seung Joong, Lin, Yuan, Peeples, William B., Saltzberg, Daniel, Soniat, Michael, Baumhardt, Jordan M., Oldenbourg, Rudolf, Sali, Andrej, Fawzi, Nicolas L., Rosen, Michael K., and Chook, Yuh Min. Sun . "Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites". United States. https://doi.org/10.1016/j.cell.2018.03.003.
@article{osti_1548416,
title = {Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites},
author = {Yoshizawa, Takuya and Ali, Rustam and Jiou, Jenny and Fung, Ho Yee Joyce and Burke, Kathleen A. and Kim, Seung Joong and Lin, Yuan and Peeples, William B. and Saltzberg, Daniel and Soniat, Michael and Baumhardt, Jordan M. and Oldenbourg, Rudolf and Sali, Andrej and Fawzi, Nicolas L. and Rosen, Michael K. and Chook, Yuh Min},
abstractNote = {Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-β2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-β2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-β2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. Here, the data lead to a model where high-affinity binding of karyopherin-β2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-β2 may act analogously to control condensates in diverse cellular contexts.},
doi = {10.1016/j.cell.2018.03.003},
journal = {Cell},
number = 3,
volume = 173,
place = {United States},
year = {2018},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1016/j.cell.2018.03.003

Citation Metrics:
Cited by: 69 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease
journal, June 2012


Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization
journal, September 2015


Nucleocytoplasmic transport enters the atomic age
journal, June 2001


A Yeast Model of FUS/TLS-Dependent Cytotoxicity
journal, April 2011


Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins
journal, October 2015


Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor
journal, November 2008

  • Mittag, Tanja; Orlicky, Stephen; Choy, Wing-Yiu
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 46
  • DOI: 10.1073/pnas.0809222105

Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective
journal, June 2009

  • Guex, Nicolas; Peitsch, Manuel C.; Schwede, Torsten
  • ELECTROPHORESIS, Vol. 30, Issue S1
  • DOI: 10.1002/elps.200900140

Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains
journal, February 2002

  • Jäkel, Stefan; Mingot, José-Manuel; Schwarzmaier, Petra
  • The EMBO Journal, Vol. 21, Issue 3
  • DOI: 10.1093/emboj/21.3.377

Structural Basis for Cell-Cycle-Dependent Nuclear Import Mediated by the Karyopherin Kap121p
journal, June 2013


CRM1 Mediates Nuclear Export of Nonstructural Protein 2 from Parvovirus Minute Virus of Mice
journal, October 1999

  • Ohshima, Takayuki; Nakajima, Toshihiro; Oishi, Takayuki
  • Biochemical and Biophysical Research Communications, Vol. 264, Issue 1
  • DOI: 10.1006/bbrc.1999.1478

Uncoupling Kapβ2 Substrate Dissociation and Ran Binding
journal, June 2002

  • Chook, Yuh Min; Jung, Astrid; Rosen, Michael K.
  • Biochemistry, Vol. 41, Issue 22
  • DOI: 10.1021/bi012122p

ATSAS 2.8 : a comprehensive data analysis suite for small-angle scattering from macromolecular solutions
journal, June 2017

  • Franke, D.; Petoukhov, M. V.; Konarev, P. V.
  • Journal of Applied Crystallography, Vol. 50, Issue 4
  • DOI: 10.1107/S1600576717007786

ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import
journal, July 2010

  • Dormann, Dorothee; Rodde, Ramona; Edbauer, Dieter
  • The EMBO Journal, Vol. 29, Issue 16
  • DOI: 10.1038/emboj.2010.143

Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012

  • Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
  • DOI: 10.1107/S0907444912001308

Weak Interactions Govern the Viscosity of Concentrated Antibody Solutions: High-Throughput Analysis Using the Diffusion Interaction Parameter
journal, July 2012


TDP-43 and FUS en route from the nucleus to the cytoplasm
journal, April 2017


SEDPHAT – A platform for global ITC analysis and global multi-method analysis of molecular interactions
journal, April 2015


Structural basis for leucine-rich nuclear export signal recognition by CRM1
journal, April 2009

  • Dong, Xiuhua; Biswas, Anindita; Süel, Katherine E.
  • Nature, Vol. 458, Issue 7242
  • DOI: 10.1038/nature07975

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity
journal, August 2017

  • Monahan, Zachary; Ryan, Veronica H.; Janke, Abigail M.
  • The EMBO Journal, Vol. 36, Issue 20
  • DOI: 10.15252/embj.201696394

Plasticity of an Ultrafast Interaction between Nucleoporins and Nuclear Transport Receptors
journal, October 2015


The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5' splice site-like sequences
journal, March 2009

  • Loughlin, F. E.; Mansfield, R. E.; Vaz, P. M.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 14
  • DOI: 10.1073/pnas.0802466106

Analysis of edge birefringence
journal, September 1991


Domain Architectures and Characterization of an RNA-binding Protein, TLS
journal, October 2004

  • Iko, Yuko; Kodama, Takashi S.; Kasai, Nobuyuki
  • Journal of Biological Chemistry, Vol. 279, Issue 43
  • DOI: 10.1074/jbc.M408552200

Nuclear import by karyopherin-βs: Recognition and inhibition
journal, September 2011

  • Chook, Yuh Min; Süel, Katherine E.
  • Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1813, Issue 9
  • DOI: 10.1016/j.bbamcr.2010.10.014

RNA-binding proteins with prion-like domains in health and disease
journal, April 2017

  • Harrison, Alice Ford; Shorter, James
  • Biochemical Journal, Vol. 474, Issue 8
  • DOI: 10.1042/BCJ20160499

Cross-saturation and transferred cross-saturation experiments
journal, April 2014

  • Ueda, Takumi; Takeuchi, Koh; Nishida, Noritaka
  • Quarterly Reviews of Biophysics, Vol. 47, Issue 2
  • DOI: 10.1017/S0033583514000043

Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels
journal, May 2012


Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma
journal, June 1993

  • Crozat, Anne; Åman, Pierre; Mandahl, Nils
  • Nature, Vol. 363, Issue 6430
  • DOI: 10.1038/363640a0

Conformational Heterogeneity of Karyopherinβ2 Is Segmental
journal, November 2007


Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding
journal, May 2017

  • Ozdilek, Bagdeser A.; Thompson, Valery F.; Ahmed, Nasiha S.
  • Nucleic Acids Research, Vol. 45, Issue 13
  • DOI: 10.1093/nar/gkx460

Karyopherin-β2 Recognition of a PY-NLS Variant that Lacks the Proline-Tyrosine Motif
journal, October 2016


RNA Seeds Higher-Order Assembly of FUS Protein
journal, November 2013


The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei
journal, November 2015


Structure of the nuclear transport complex karyopherin-β2–Ran˙GppNHp
journal, May 1999

  • Chook, Yuh Min; Blobel, Günter
  • Nature, Vol. 399, Issue 6733
  • DOI: 10.1038/20375

Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export
journal, October 2015


TDP-43 and FUS: a nuclear affair
journal, July 2011


The molecular mechanism of nuclear transport revealed by atomic-scale measurements
journal, September 2015


HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions
journal, April 2016

  • Brautigam, Chad A.; Zhao, Huaying; Vargas, Carolyn
  • Nature Protocols, Vol. 11, Issue 5
  • DOI: 10.1038/nprot.2016.044

MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009

  • Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042073

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

Polymer physics of intracellular phase transitions
journal, November 2015

  • Brangwynne, Clifford P.; Tompa, Peter; Pappu, Rohit V.
  • Nature Physics, Vol. 11, Issue 11
  • DOI: 10.1038/nphys3532

New developments in the ATSAS program package for small-angle scattering data analysis
journal, March 2012

  • Petoukhov, Maxim V.; Franke, Daniel; Shkumatov, Alexander V.
  • Journal of Applied Crystallography, Vol. 45, Issue 2
  • DOI: 10.1107/S0021889812007662

Biochemical Properties and Biological Functions of FET Proteins
journal, June 2015


Rules for Nuclear Localization Sequence Recognition by Karyopherinβ2
journal, August 2006


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Integrative Structure–Function Mapping of the Nucleoporin Nup133 Suggests a Conserved Mechanism for Membrane Anchoring of the Nuclear Pore Complex
journal, August 2014

  • Kim, Seung Joong; Fernandez-Martinez, Javier; Sampathkumar, Parthasarathy
  • Molecular & Cellular Proteomics, Vol. 13, Issue 11
  • DOI: 10.1074/mcp.M114.040915

Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale
journal, December 2009

  • Fischer, H.; de Oliveira Neto, M.; Napolitano, H. B.
  • Journal of Applied Crystallography, Vol. 43, Issue 1
  • DOI: 10.1107/S0021889809043076

Specific Binding of Modified RGG Domain in TLS/FUS to G-Quadruplex RNA: Tyrosines in RGG Domain Recognize 2′-OH of the Riboses of Loops in G-Quadruplex
journal, November 2013

  • Takahama, Kentaro; Oyoshi, Takanori
  • Journal of the American Chemical Society, Vol. 135, Issue 48
  • DOI: 10.1021/ja4086929

Stress granules as crucibles of ALS pathogenesis
journal, April 2013

  • Li, Yun R.; King, Oliver D.; Shorter, James
  • The Journal of Cell Biology, Vol. 201, Issue 3
  • DOI: 10.1083/jcb.201302044

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains
journal, April 2018


Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs
journal, September 2017

  • Lin, Yuan; Currie, Simon L.; Rosen, Michael K.
  • Journal of Biological Chemistry, Vol. 292, Issue 46
  • DOI: 10.1074/jbc.M117.800466

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation
journal, April 2018


Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains
journal, October 2017


NMRPipe: A multidimensional spectral processing system based on UNIX pipes
journal, November 1995

  • Delaglio, Frank; Grzesiek, Stephan; Vuister, GeertenW.
  • Journal of Biomolecular NMR, Vol. 6, Issue 3
  • DOI: 10.1007/BF00197809

A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins
journal, August 2015


Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)
journal, July 2012

  • Zhang, Zi Chao; Chook, Yuh Min
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 30
  • DOI: 10.1073/pnas.1207247109

Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS
journal, April 2011


The CCPN data model for NMR spectroscopy: Development of a software pipeline
journal, April 2005

  • Vranken, Wim F.; Boucher, Wayne; Stevens, Tim J.
  • Proteins: Structure, Function, and Bioinformatics, Vol. 59, Issue 4
  • DOI: 10.1002/prot.20449

Structure-based design of a pathway-specific nuclear import inhibitor
journal, April 2007

  • Cansizoglu, Ahmet E.; Lee, Brittany J.; Zhang, Zi Chao
  • Nature Structural & Molecular Biology, Vol. 14, Issue 5
  • DOI: 10.1038/nsmb1229

Atomic basis of CRM1-cargo recognition, release and inhibition
journal, August 2014


Biomolecular condensates: organizers of cellular biochemistry
journal, February 2017

  • Banani, Salman F.; Lee, Hyun O.; Hyman, Anthony A.
  • Nature Reviews Molecular Cell Biology, Vol. 18, Issue 5
  • DOI: 10.1038/nrm.2017.7

Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II
journal, October 2015


New polarized light microscope with precision universal compensator
journal, November 1995


Autophagy regulates amyotrophic lateral sclerosis-linked fused in sarcoma-positive stress granules in neurons
journal, December 2014


A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation
journal, August 2015