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The nuclear pore complex (NPC) serves as the sole bidirectional gateway of macromolecules in and out of the nucleus. Owing to its size and complexity (∼1,000 protein subunits, ∼110 MDa in humans), the NPC has remained one of the foremost challenges for structure determination. Structural studies have now provided atomic-resolution crystal structures of most nucleoporins. The acquisition of these structures, combined with biochemical reconstitution experiments, cross-linking mass spectrometry, and cryo–electron tomography, has facilitated the determination of the near-atomic overall architecture of the symmetric core of the human, fungal, and algal NPCs. Here, we discuss the insights gained from these new advances and outstanding issues regarding NPC structure and function. The powerful combination of bottom-up and top-down approaches toward determining the structure of the NPC offers a paradigm for uncovering the architectures of other complex biological machines to near-atomic resolution.
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Supplemental Video 1: The NPC forms a diffusion barrier to separate the nucleus and cytoplasm. A movie depicting the architecture of the NPC, its interaction with the nuclear envelope, and the diffusion barrier in the central transport channel. The diffusion barrier of the NPC allows molecules smaller than ~40 kDa to pass, but not larger macromolecules.
Supplemental Video 2: Nucleocytoplasmic transport is facilitated by karyopherins and the Ran cycle. A movie depicting the basic steps involved in Ran-dependent, karyopherin-mediated macromolecular transport through the NPC. In canonical nuclear import, cargo macromolecules contain a linear nuclear localization signal (NLS) that is recognized by karyopherin-α, which serves as an adaptor for karyopherin-β. Import cargoes can also be recognized directly by karyopherin-β family members either through an NLS or through direct recognition of their three-dimensional fold. Karyopherin-β family members are able to cross the diffusion barrier and ferry bound cargoes with them. After import complexes arrive in the nucleus, they encounter RanGTP, which is maintained at a high concentration in the nucleus. RanGTP binding disassembles import complexes. During canonical nuclear export, cargo macromolecules contain a nuclear export signal (NES), which is recognized by the karyopherin-β family member Crm1. Unlike nuclear import complexes, RanGTP binding is required for nuclear export complex assembly. As for import complexes, because the karyopherins are able to cross the diffusion barrier they can ferry bound cargoes and RanGTP with them. At the cytoplasmic face, RanGAP, which is anchored to the NPC in humans, activates the GTPase activity of Ran, triggering GTP hydrolysis and export complex disassembly.