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Architecture of the cytoplasmic face of the nuclear pore
Christopher J. Bley
1,#
,
Si Nie
1,#
,
George W. Mobbs
1,#
,
Stefan Petrovic
1,#
,
Anna T. Gres
1,#,†
,
Xiaoyu Liu
1,#,‡
,
Somnath Mukherjee
2
,
Sho Harvey
1
,
Ferdinand M. Huber
1,§
,
Daniel H.
Lin
1,¶
,
Bonnie Brown
1
,
Aaron W. Tang
1
,
Emily J. Rundlet
1,††
,
Ana R. Correia
1,‡‡
,
Shane
Chen
3
,
Saroj G. Regmi
3
,
Taylor A. Stevens
1
,
Claudia A. Jette
1
,
Mary Dasso
3
,
Alina Patke
4
,
Alexander F. Palazzo
5
,
Anthony A. Kossiakoff
2
,
André Hoelz
1,*
1
California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 East
California Boulevard, Pasadena, CA 91125, USA
2
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL
60637, USA
3
Division of Molecular and Cellular Biology, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD 20892, USA
4
California Institute of Technology, Division of Biology and Biological Engineering, 1200 East
California Boulevard, Pasadena, CA 91125, USA
5
Department of Biochemistry, University of Toronto, Toronto, ON M5G 1M1, Canada
Abstract
INTRODUCTION—
The subcellular compartmentalization of eukaryotic cells requires selective
transport of folded proteins and protein/nucleic acid complexes. Embedded in nuclear envelope
*
Corresponding author: hoelz@caltech.edu (A.H.).
Present address: Clinical Research Methodology, Scientific Solutions, Worldwide Clinical Trials, 600 Park Offices Drive Suite 200,
Research Triangle Park, NC 27709, USA.
Present address: Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, 609
Charles E. Young Drive East, Los Angeles, CA 90095, USA.
§
Present address: Odyssey Therapeutics, Inc., Industriepark Höchst G875, Frankfurt am Main, 65926, Germany.
Present address: Whitehead Institute for Biomedical Research, 455 Main Street Cambridge, MA 02142, USA.
††
Present address: Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis,
TN 38105, USA & Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medicine, 1300 York Avenue, New York, NY
10065, USA.
‡‡
Present address: Amgen Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
#
these authors contributed equally to this work
Author contributions:
AH conceived and coordinated the study. CJB, SN, GWM, SP, ATG, XL, SM, SH, FMH, DHL, BB, AWT,
EJR, ARC, SC, SGR, TAS, CAJ, MD, AP, AFP, AAK, and AH designed research. CJB, SN, GWM, SP, ATG, XL, SM, SH, FMH,
DHL, BB, AWT, EJR, ARC, SC, SGR, TAS, CAJ, and AH performed research. CJB, SN, GWM, SP, ATG, XL, SM, SH, FMH, DHL,
BB, AWT, EJR, ARC, SC, SGR, TAS, CAJ, MD, AP, AFP, AAK, and AH analyzed data. SM and AAK (synthetic antibodies), and
SC, SGR, and MD (auxin-degron cell lines) provided new reagents. AP and AFP provided reagents and experimental guidance. CJB,
SN, GWM, SP, ATG, XL, AP, and AH integrated and conceptualized the results. CJB, SN, GWM, SP, AP, and AH wrote and revised
the manuscript, with contributions from all authors.
Competing interests:
The authors declare no financial conflicts of interest.
Supplementary Materials:
Materials and Methods
Supplementary Text
Figs. S1 to S89
Tables S1 to S18
References
HHS Public Access
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Science
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pores, generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear
pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The
~110 MDa human NPC is an ~1,000 protein assembly comprising multiple copies of ~34 different
proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an
inner ring encircling the central transport channel, and outer rings formed by Y-shaped coat
nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings
are decorated with compartment-specific asymmetric nuclear basket and cytoplasmic filament
nucleoporins, which establish transport directionality and provide docking sites for transport
factors and the small GTPase Ran. The cytoplasmic filament nucleoporins also play an essential
role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit
the central transport channel. Unsurprisingly, the NPC’s cytoplasmic face represents a hotspot for
disease-associated mutations and is commonly targeted by viral virulence factors.
RATIONALE—
Previous studies established a near-atomic composite structure of the human
NPC’s symmetric core by combining biochemical reconstitution elucidating the interaction
network between symmetric nucleoporins, crystal and single particle cryo-EM structure
determination of nucleoporins and nucleoporin complexes revealing their three-dimensional shape
and the molecular details of their interactions, quantitative docking in cryo-ET maps of the
intact human NPC uncovering nucleoporin stoichiometry and positioning, and cell-based assays
validating the physiological relevance of the biochemical and structural findings. Here, we
extended our approach to the cytoplasmic filament nucleoporins to elucidate the near-atomic
architecture of the cytoplasmic face of the human NPC.
RESULTS—
Using biochemical reconstitution, we elucidated the protein-protein and protein-
RNA interaction networks of the human and
C. thermophilum
cytoplasmic filament nucleoporins,
establishing an evolutionarily conserved hetero-hexameric cytoplasmic filament nucleoporin
complex (CFNC) held together by a central hetero-trimeric coiled-coil hub that tethers two
separate mRNP-remodeling complexes. Further biochemical analysis and determination of a
series of crystal structures revealed that the metazoan-specific cytoplasmic filament nucleoporin
NUP358 is composed of 16 distinct domains, including an N-terminal S-shaped
α
-helical solenoid
followed by a coiled-coil oligomerization element, numerous Ran-interacting domains, an E3
ligase domain, and a C-terminal prolyl-isomerase domain. Physiologically validated quantitative
docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles,
conjoined by the oligomerization element, are anchored through their N-terminal domains to
the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600Å into
the cytoplasm. Using cell-based assays, we demonstrated that NUP358 is dispensable for the
architectural integrity of the assembled interphase NPC and RNA export but is required for
efficient translation. After NUP358 assignment, the remaining 4-shaped cryo-ET density matched
the dimensions of the CFNC coiled-coil hub, in close proximity to an outer ring NUP93. Whereas
the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled-coil
channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed the
NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human
NPC. In contrast, two
C. thermophilum
CFNCs are anchored by a divergent mechanism involving
assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned
cryo-ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of
the nuclear basket component ELYS established that the equivalent position on the cytoplasmic
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face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric
distribution of nucleoporins.
CONCLUSION—
We have substantially advanced the biochemical and structural characterization
of the asymmetric nucleoporins’ architecture and attachment at the cytoplasmic and nuclear
faces of the NPC. Our near-atomic composite structure of the human NPC’s cytoplasmic face
provides a biochemical and structural framework for elucidating the molecular basis of mRNP
remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms
of nucleoporin diseases at the cytoplasmic face of the NPC.
Abstract
The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic
transport. Despite recent progress in elucidating the NPC symmetric core architecture, the
asymmetrically decorated cytoplasmic face, essential for mRNA export and a hotspot for
nucleoporin-associated diseases, has remained elusive. Here, we report a composite structure of
the human cytoplasmic face obtained by combining biochemical reconstitution, crystal structure
determination, docking into cryo-electron tomographic reconstructions, and physiological
validation. Whereas species-specific motifs anchor an evolutionarily conserved ~540kDa hetero-
hexameric cytoplasmic filament nucleoporin complex above the central transport channel,
attachment of the NUP358 pentameric bundles depends on the double-ring arrangement of the
coat nucleoporin complex. Our composite structure and its predictive power provide a rich
foundation for elucidating the molecular basis of mRNA export and nucleoporin diseases.
One-Sentence Summary:
An interdisciplinary analysis established the near-atomic molecular architecture of the cytoplasmic
face of the human nuclear pore complex.
The sequestration of genetic material in the nucleus represents one of the great hallmarks of
evolution but creates the necessity for selective bidirectional transport across the nuclear
envelope (
1
4
). The nuclear pore complex (NPC) is the sole gateway through which
folded proteins and protein/nucleic acid complexes cross the nuclear envelope, making
this transport organelle an essential machine for all eukaryotic life. Besides its direct
role as a transport channel, the NPC serves as an organizer for nuclear and cytoplasmic
processes that are essential for the flow of genetic information from DNA to RNA to protein,
including transcription, spliceosome assembly, mRNA export, and ribosome assembly (
1
4
).
Dysfunction of the NPC or its components represents a major cause of human disease (
2
,
5
,
6
).
Architecturally, the NPC consists of a central core with an 8-fold rotational symmetry
across a nucleocytoplasmic axis and a two-fold rotational symmetry across the plane of the
nuclear envelope, which links to compartment-specific asymmetric “cytoplasmic filaments”
(CF) and a “nuclear basket” structure (Fig.1A) (
1
,
2
). The NPC is built from ~34 different
proteins, termed nucleoporins (nups) that are organized into distinct subcomplexes. Multiple
copies of each nup in the NPC add up to an assembly that reaches an extraordinary
molecular mass of ~110MDa in vertebrates. The symmetric core of the NPC is composed
of an inner ring and two spatially segregated outer rings. The inner ring is embedded in
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nuclear envelope pores generated by the circumscribed fusion of the double membrane of
the nuclear envelope. The diffusion barrier is formed by unstructured phenylalanine-glycine
(FG) repeats that fill the central transport channel, imposing a gradually increasing barrier
to passive diffusion of macromolecules >40kDa (
1
4
). Transport factors collectively termed
karyopherins overcome the diffusion barrier by binding to FG repeats, thereby transporting
cargo across the nuclear envelope (
7
9
). A significant fraction of the FG repeats in the
inner ring is contributed by a hetero-trimeric channel nup complex (CNT) which is anchored
by a single assembly sensor motif (
10
12
). The outer rings sit atop the nuclear envelope,
sandwiching the inner ring from both sides. They are primarily formed by the Y-shaped coat
nup complex (CNC, also referred to as Y-complex or Nup107–160 complex), and serve as a
platform for the asymmetric incorporation of the CF and nuclear basket nups.
Two decades ago, the atomic level characterization of the NPC began with individual nup
domains and progressed to nup complexes of increasing size and complexity, culminating
in the ~400kDa hetero-heptameric CNC (
11
,
13
28
). Simultaneously, advances in cryo-
electron tomographic (cryo-ET) data acquisition and processing gradually increased the
resolution of intact NPC 3D reconstructions (
29
). Docking of the CNC into a ~32Å cryo-ET
map of the intact human NPC demonstrated that two reticulated eight-membered CNC
rings, linked by head-to-tail interactions, are present on each side of the nuclear envelope
(
27
,
30
). Moreover, this advance established that the resolution gap between high- and
low-resolution structural methods can be overcome by combining biochemical reconstitution
and X-ray crystallographic characterization of nups with cryo-ET reconstruction of the
intact NPC. Expansion of this approach to the nine nups constituting the inner ring rapidly
led to the reconstitution of two distinct ~425kDa inner ring complexes (IRCs) and the
elucidation of their components’ structures (
10
12
,
20
,
31
38
). In turn, this advance enabled
the determination of the near-atomic composite structure of the entire ~56MDa symmetric
core of the human NPC, establishing the stoichiometry and placement of all 17 symmetric
nups within a ~23Å cryo-ET reconstruction (
38
,
39
). Subsequently, the architecture of
the
Saccharomyces cerevisiae
NPC was determined with a similar approach, utilizing high-
resolution nup crystal structures and ~25Å cryo-ET maps of either detergent purified or
in-situ
NPCs (
40
,
41
). Compared to the human NPC, the
S.cerevisiae
NPC lacks the distal
CNC ring and associated nups on both sides of the nuclear envelope, but the relative nup
arrangement within the rest of the symmetric core remains essentially identical (
38
,
39
,
42
).
Projecting from the cytoplasmic face of the NPC, the CF nups recruit cargo•transport factor
complexes for nucleocytoplasmic transport and orchestrate the export and remodeling of
messenger ribonucleoprotein particles (mRNPs) in preparation for translation (
2
,
43
). The
nine-component CF nup machinery represents a hotspot for human diseases ranging from
degenerative brain disorders and cardiac diseases to cancer (
2
,
5
,
6
). Although linked to the
human CF nups NUP358, NUP214, NUP62, NUP88, NUP98, GLE1, NUP42, RAE1, and
DDX19, the pathophysiology and optimal therapeutic strategies for these conditions remain
ill-defined.
Here, we present insight into the atomic and higher order architecture, function, and
mechanism of action of the CF nups in the human and thermophilic fungus
Chaetomium
thermophilum
NPCs. First, we uncover a conserved modular architecture within the hetero-
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