Comprehensive structure and functional adaptations of the yeast nuclear pore complex

Abstract

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Keywords: NPC evolution; Nuclear pore complex; cryo-electron microscopy; cryo-electron tomography; inner ring dilation; nuclear basket; nucleocytoplasmic transport; nucleoporins; structural isoforms.

Figure 1.. Overview of isolated and in situ yeast NPC structures

(A) (top) Cytoplasmic and cross-section views of a composite 3D map from the isolated NPC: double nuclear outer ring (gold), inner ring (IR, light blue), Nup82 complex and cytoplasmic outer ring (tan), Pom152 ring (light pink), membrane-detergent ring (white), central transporter (red), and spoke-transporter connections (marked “C”). (bottom) Three views of molecular models docked in a composite, 3D density map of the isolated NPC. Nup color coding is defined for spokes in Figures 2G and 2H and for Y-complexes in Figure 4D. Only half of the NPC model is shown in each view and the Pom152 ring is marked (*). (B) (top) Two views of the in situ yeast NPC: outer rings (OR, tan), Pom152 ring (pink), pore membrane (transparent white), and basket anchors (silver). (bottom) Three views of molecular models docked in the combined 3D density map of the in situ NPC. Positions of Nsp1 FG domain anchor points in the Nup82 complex are indicated (green dots). Scale bar 200 Å.

Figure 2.. Structure of the isolated yeast NPC

(A) Composite 3D map of the inner ring and double outer rings viewed from the nuclear side: nuclear, double outer ring (gold), inner ring (IR, light blue). B. The inner ring is shown with local 2-fold axes and membrane attachment complexes that contain Nup157 pairs, Pom152 and Pom34 (MAC157, black asterisks), and Nup170 pairs with Ndc1 (MAC170; blue asterisks). (C) A cross-section of the inner ring with ɑ-helical rods throughout. (D) A single spoke is segmented and viewed along the central 2-fold axis to reveal the three major layers. (E) A central slice through the spoke with the orientation of Figure 2H shows the gap between inner and outer spoke domains (ISD and OSD, respectively). Cavities are marked with asterisks. (F) A central section with three docked models for the spokes and double outer ring (oriented as in C). β-propellers in the Y-complexes are highlighted (light blue) and the disordered cytoplasmic outer ring is indicated (dashed lines). (G) Close-up of a modeled spoke viewed along the 2-fold axis. Connector helices are colored gold. (H) The spoke contains two domains, as indicated by dashed lines (gap), with distinctive functions. The Nup color palette reflects their distribution within the inner (green), adaptin-like (pink-red), and membrane-interacting (blue) layers. (I) A thin slab viewed along the central 2-fold axis reveals a vertical separation of the outer spoke domain (dashed lines; also see Figure 3). Relevant scale bars are indicated on the panels.

Figure 3.. Orphans and interaction motifs for Nic96 NTDs in the spoke

(A) Nsp1 complex three-helix bundles and Nic96 NTD connectors form two diagonal walls (green iso-surface). Figures 3A–3D are viewed along the central 2-fold axis (marked 2 or a black ellipse). (B) Nic96 connector ɑ-helices (gold and blue cylinders) form a bridge between opposing half walls (green); other connectors bind to 3-helix bundles from the Nsp1 complex. (C) Connector helices and strands with overlayed map density are superimposed on Nups 188 and 192 (white cylinders). Each Nic96 NTD forms a group of 3 elements, a group of 2 helices, and a pair of helices that bind to Nup188 or Nup192 CTDs. In addition, two orphan helices (pink) bind to Nup192 NTDs. (D) Connector interactions are shown with Nups displayed as ribbons. Outer and inner Nic96 CTDs (light blue, light yellow) form keystone supports behind Nup192 and Nup188. Nup192 CTDs form Karyopherin-like interactions with Nic96 connector helices. Nup192 and Nup188 NTDs are shown in white and gray, while their CTDs are color coded. (E) A pair of Nup192 molecules with IBB-like motifs (from panel 3D) are shown after a 90 degree rotation counterclockwise about a vertical axis. (F) Two views of the Nup192/IBB-like motif are superimposed with the 3D density map. (G) Left: the Nup192/IBB-like motif is shown with numbered helices (N- to C-, 1 to 12). Right: overlay of human Importin β co-crystal structures with IBBs of Importin-ɑ (tan, PDB 1QGK) and Snurportin 1 (gray, PDB 3LWW). Numerals of split helices are circled. (H) Two views of the Nup188 CTD with a pair of connector helices and the overlayed 3D density map. The curved Nup188 tail binds consecutive Nic96 NTD ɑ-helices in a long groove. Scale bars are indicated on the panels.

Figure 4.. A double outer ring in a subset of yeast NPCs

(A) The isolated yeast NPC with a double outer ring segmented into proximal (gold) and distal (tan) rings. Density for Nup188/192 (red) and two orphans (silver) are shown. A double ring protomer is outlined (dashed box). (B) Model of Y-complexes in the double outer ring with the overlaid density map. (C) Top: three pairs of Y-complexes from the 3D density map. A gap is visible between neighboring Y-complexes in the distal ring (tan). Bottom: molecular models are shown in the map with MBMs indicated (*). (D) Y-complexes from the distal (left) and proximal (center) outer rings are shown, along with a staggered pair (right). A torus-like bulge and Nup133 spur are present in the tails of distal and proximal Y-complexes, respectively. (E) The pair of Y-complexes outlined in Figure 4A is shown with density for a Nup188/192 molecule (red) and three orphans (numbered 1–3). Orphans, MBMs(*), and a tripod of β-propellers are labeled. Scale bars are indicated on the panels.

Figure 5.. Structure of the in situ NPC

(A) Panels 1–3: three views are shown for the in situ yeast NPC: inner ring (blue), cytoplasmic and nucleoplasmic outer rings (tan), Pom152 ring (pink), pore membrane (transparent white), and basket anchors (silver). Connections from Nsp1 complexes to FG domains are indicated with black dots. Cytoplasmic and nuclear outer rings differ in diameter (radial offset, dashed lines). Panels 4–6: the core scaffold with the Pom152 ring is separated into component rings. (B) Cross-section of the in situ NPC with molecular models for three protomers. (C) Side view of a nearly complete protomer. β-propellers with MBM contacts are indicated (*). (D and E) Model for the tilted cytoplasmic outer ring with the Nup82 complex (Nup82c) and Dyn2 dimers. β-propellers with MBMs (*) and N-terminal FG domains from Nsp1 and Nup159 are indicated (colored dots). (F) Tilted view of the flat nucleoplasmic outer ring with docked models. A gap is present between adjacent Y-complexes and MBM contact sites are indicated (*); the Nup133 spur contacts the membrane. Scale bars are indicated.

Figure 6.. Inner ring dilation, basket anchors, and FG domain connections

(A) Models for aligned inner rings from the isolated (left) and in situ NPC (right). Radial dilation increases the diameter of the central channel and creates a gap between adjacent spokes. Nup157 and Nup170 membrane attachment sites (red dots) and spoke layers are indicated. (B) The nuclear outer ring (tan) with basket anchor complexes (silver) is viewed from the nucleoplasm. Two anchor motifs are present for each Y-complex (dashed oval). (C) Basket anchor complexes have two similar motifs (red and blue); each motif contains a “bent T” and an extended SE that were mapped with 2-helix coiled coils. Contact sites with the Y-complex are indicated (red and blue asterisks). (D) Tilted view of a Y-complex in the nuclear outer ring with basket anchors. (E) Extended 3-helix bundles of the Nsp1 complex (green) form a ring that faces the central channel. Connections to the central transporter are mapped with rods (red and blue) and are aligned with N termini (dark blue) of Nsp1 heterotrimers at local 2-fold axes (2). (F) FG connections of the in situ NPC originate from three points on each spoke (*) that align with the N-termini of Nsp1 complexes (dark blue). Radial expansion creates a gap between adjacent spokes and separates adjacent Nsp1 complexes; a lateral contact is formed between adjacent spokes at the gap (arrow). Scale bars are indicated.

Figure 7.. Diagrammatic summary of major insights into the yeast NPC

(A) A wiring diagram for Nic96 connectors that extend from CTDs in the membrane interacting layer to the diagonal walls. Red stars indicate approximate positions of phosphorylation sites in human Nup93: membrane interacting layer (blue-purple), adaptin-like layer (red), and inner layer (green). (B) The IBB helix of Importin-ɑ/Karyopherin ɑ interacts with Importin-β/Karyopherin β1, and this motif is compared with Nic96 NTD helices bound to Nup192 and Nup188. (C) Three structural variants of the NPC are present in the same yeast cell. (D) Inner ring dilation forms gaps between adjacent spokes; this is accompanied by a radial displacement of the pore membrane and expansion of the Pom152 ring. (E) Duplication within major NPC sub-structures: related Nups are shown in red-brown and light blue. Basket anchor motifs (red and royal blue double lines) are present in two copies per Y-complex. This lateral duplication may encompass the basket itself (vertical dashed lines). (inset) Cartoon of a basket anchor motif with labelled structure elements.