Characterization of the membrane-coating Nup84 complex: paradigm for the nuclear pore complex structure - PubMed (original) (raw)

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Characterization of the membrane-coating Nup84 complex: paradigm for the nuclear pore complex structure

Erik W Debler et al. Nucleus. 2010 Mar-Apr.

Abstract

Nuclear pore complexes (NPCs) function as selective gates for nucleocytoplasmic transport. Although the NPC was discovered more than half a century ago, our knowledge of NPC components in atomic detail has exploded only over the past few years. Recent structural, biochemical, and in vivo studies of NPC components, in particular the membrane-coating heptameric Nup84 complex, have shed light onto the NPC architecture as well as onto its dynamic nature. Striking similarities were revealed between the components of the NPC and of coat protein complexes in the endocytic and secretory pathways, supporting their common evolutionary origin in a progenitor protocoatomer. Here, we summarize these findings and discuss emerging concepts that underlie the molecular architecture and the dynamics of the NPC. We conclude that the uncovered principles are not limited to the NPC, but are likely to extend to other macromolecular assemblies.

Keywords: binding promiscuity; coat protein complex; coatomer; crystallography; dynamic interaction; electron microscopy; macromolecular assembly; nucleocytoplasmic transport; nucleoporin; vesicle coats.

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Figures

Figure 1

Figure 1

Transport functions of the NPC. (A) Overall, the NPC consists of a cylindrical, symmetric core, which is asymmetrically decorated with filaments and a nuclear basket structure on the cytoplasmic and nucleoplasmic sides, respectively. Molecules smaller than ∼40 kDa freely diffuse through the NPC. (B) Active import and export of cargoes are facilitated by nuclear localization and nuclear export sequences (NLS and NES, respectively) that are recognized by transport factors, collectively termed karyopherins (kaps). The NLS of import cargoes is recognized either directly by an import karyopherin-β (kap-β) or via an adapter karyopherin (kap-α). Ran•GTP binding inside the nucleus leads to dissociation of the import complex (left). By contrast, the assembly of an NES-cargo kap-β export complex requires Ran•GTP binding. In the cytosol, this export complex is dissociated by GTP hydrolysis, which is catalyzed by Ran GTPase-activating protein (RanGAP) or Ran binding protein 1 (RanBP1). (C) The transport of large cargoes is thought to require the dilation of the central channel of the NPC (inset). (D) Inner nuclear membrane (INM) proteins are co-translationally integrated into the endoplasmic reticulum membrane, which is continuous with the outer nuclear membrane (ONM), and then imported to the INM. Similar to (B), the transport of INM proteins is also dependent on the Ran-cycle and karyopherins that likely travel through the central channel, while the cargo protein is anchored in the membrane and somehow pulled through. Substantial structural changes within the NPC would be necessary to facilitate this transport event.

Figure 2

Figure 2

Structural analysis of the heptameric Nup84 complex. Approximately 85% of the protein mass of the heptamer has been determined by x-ray crystallography. High-resolution crystal structures of the yeast nucleoporins Seh1•Nup85, Nup120, Sec13•Nup145C•Nup84, and the human nucleoporins Nup107 (homolog of Nup84) and Nup133 were fitted into a three-dimensional electron microscopy (EM) reconstruction. The crystallized N-terminal and C-terminal domains (NTD and CTD, respectively) are indicated. Two hinge regions are indicated at which the heptamer displayed marked flexibility. A 90°-rotated view is shown on the right, with the dimension of the heptamer indicated.

Figure 3

Figure 3

A schematic model of the NPC. The model includes four concentric cylinders composed of integral pore membrane proteins (POMs), coat nups, adaptor nups and channel nups. Natively unfolded FG-repeats of a number of nups make up the transport barrier in the central channel and are indicated by a transparent plug. Note that the surface topology of the pore membrane involves both convex and concave curvatures.

Figure 4

Figure 4

Flexibility and dynamics of NPC components. Spheres represent β-propeller domains, while cylinders represent α-helical solenoid domains (A) Crystal structures of the Seh1•Nup85 hetero-octamer (yellow and green, respectively) in three different conformational states revealed a rotation and a hinge motion. (B) Sec13•Nup145C (orange and blue, respectively) forms a hetero-octamer. (C) Sec13•Nup145C•Nup84 (colored as in panel B, Nup84 in yellow) forms a subcomplex that matches a hinge observed in one conformation of the heptameric Nup84 complex as determined by EM. A straight arrangement of this trimer would correspond to the second conformation of this subcomplex as revealed by EM. Notably, the homodimerization and heterodimerization surfaces of Nup145C are overlapping and therefore promiscuous. (D) Nup133 binds to Nup120 via a short N-terminal segment at the end of an unstructured region. Since the two proteins are localized at opposite ends of the Nup84 complex, this finding suggests a head-to-tail arrangement of the heptamers. Contraction and expansion of the unstructured region may allow for the flexible tethering of Nup120 and Nup133. (E) Four different states of Nup58/Nup45 tetramers have been crystallographically observed. The sliding of two Nup58/Nup45 dimers (purple) alters the overall dimensions of the tetramer. Nup58/Nup45 refers to two alternatively spliced variants (Nup58 and Nup45) and the determined structure is common to both proteins.

Figure 5

Figure 5

The architecture of the COPII coat. The vesicle-coating COPII complex consists of only five proteins. Sec13 and Sec31 assemble the outer shell, while Sec23, Sec24 and the membrane bound Sar1•GTP assemble the inner shell of COPII. In contrast to the pore membrane domain, the curvature of membrane-bound vesicles is convex in all directions.

Figure 6

Figure 6

The COPII coat is dynamic. (A) The Sec13•Sec31 hetero-octamer (red) is the repeating unit of the outer shell of the COPII complex. A cuboctahedral arrangement made of 12 modules is depicted, but other larger geometries of COPII cages were also observed by EM. Sec13 and Sec31 are shown in orange and grey, respectively. The inner shell of the COPII complex has been omitted for clarity. (B) With increasing cage size, the decreasing curvature of the vesicle is accounted for by a hinge motion that leads to more extended conformations of the Sec13•Sec31 hetero-octamer. Spheres represent β-propeller domains, while cylinders represent α-helical solenoid domains. Although Sec13 interacts with Sec31 and Nup145C in an analogous fashion, the higher-order organization differs and is facilitated by an additional β-propeller domain in Sec31 that is absent in Nup145C. The Sec13 binding site in Sec31 is located between the N-terminal β-propeller domain and the C-terminal α-helical solenoid domain.

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