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Molecular mechanism of the nuclear protein import cycle

Nature Reviews Molecular Cell Biology volume 8, pages 195–208 (2007)Cite this article

Key Points

  • The classic nuclear protein import cycle functions as a biological molecular ratchet and is powered by the Ran GTPase that modulates interactions between carrier molecules and their cargoes. In the cytoplasm, cargo molecules carrying a classic nuclear localization signal (NLS) sequence are attached to the carrier importin-β by the importin-α adaptor. Cargoes are released in the nucleus following RanGTP binding to importin-β, after which the importins are recycled.

  • Nuclear pores facilitate the equilibration of cargo:carrier complexes between the nucleus and the cytoplasm. Transport directionality is imposed by import-complex dissociation by RanGTP in the nucleus and by RanGTP hydrolysis in the cytoplasm. Energy is used to orchestrate the binding and release of cargoes in the appropriate compartments rather than to move material directly through the pores.

  • Movement of material backwards and forwards through nuclear pores is facilitated by interactions with nuclear pore proteins (nucleoporins) that contain Phe-Gly (FG) sequence repeats. These proteins also obstruct the passage of proteins that lack an NLS.

  • Nucleoporins on the nuclear face of the NPC accelerate import-complex disassembly and provide a molecular ratchet to prevent futile cycles in which cargo is returned to the cytoplasm.

  • Molecular flexibility is important in modulating interactions between importins and their partners.

Abstract

The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the nuclear protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.

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Figure 1: Overview of nuclear protein import.
Figure 2: Formation of the importin-α:cargo complex in the cytoplasm.
Figure 3: Import-complex disassembly.
Figure 4: Nucleoporin catalysis of import-complex disassembly.
Figure 5: Nuclear export of importin-α.

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    Murray Stewart

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Supplementary information

Supplementary information S1 (movie)

Structure of importin-a bound to the nucleoplasmin nuclear localization signal (NLS). Importin-α (green) is constructed from a tandem series of Armadillo (ARM) repeats that stack to form a gently curving banana-like molecule. NLSs (blue) bind to the inner concave surface. See also Figure 2b. (PDB accession number 1EYJ). (MOV 4209 kb)

Supplementary information S2 (movie)

Structure of importin-β complexed with the importin-α IBB (importin-β binding) domain. 'Importin-β' (cyan) is constructed from 19 tandem HEAT repeats, each of which contains two α-helices (See BOX 2). The HEAT repeats stack to form a helicoidal molecule that coils around the IBB domain α-helix (red) like a snake around its prey. (PDB accession number 1QGK). (MOV 8513 kb)

Supplementary information S3 (movie)

Structure of Ran showing the structural changes between the GDP- and GTP-bound states. The larger switch I and smaller switch II loops change conformation dramatically depending on the state of the bound nucleotide (GTPbound is red; GDP-bound is blue) and these changes control the way in which Ran interacts with its partners in the nuclear protein import pathway. The remainder of the Ran chain (shown as cyan for GTP-bound and yellow for GDP-bound) is virtually unchanged by the state of the bound nucleotide. (PDB accession files 1BYU (RanGDP) and 1RRP (RanGTP). Only residues 8-177 are shown. (MOV 6825 kb)

Supplementary information S4 (movie)

Conformational changes in importin-β between different functional states. Comparison of the structures of importin-β when bound to either the IBB domain (cyan) or RanGTP (yellow) shows the large conformational change that occurs between different functional states. When bound to RanGTP, the importin-β helicoid pitch increases dramatically so that it no longer matches the IBB domain's α-helix, thus leading to import complex disassembly (see also Figure 3c). IBB domain is red. (PDB accession codes 1QGK and 2BKU). (MOV 13233 kb)

Supplementary information S5 (movie)

Structure of the complex between yeast importin-β (Kap95p) and RanGTP. Importin-β (yellow) coils around RanGTP (cyan) interacting with it at three sites (see also Figure 3 a,b). GTP is shown in space-filling format. (PDB accession code 2BKU). (MOV 8743 kb)

Supplementary information S6 (movie)

Structure of the complex between Nup50 and importin-α. The Nup50 Nterminus (blue) binds to two sites on importin-α (green): a higher affinity site at the importin-α C-terminus (at the top of the molecule in this movie) and a lower affinity site that overlaps the NLS binding area (in the central region of the importin-α chain). Nup50 binding actively displaces nuclear localization signals (NLSs) from importin-α. (PDB accession code 2C1M). (MOV 3736 kb)

Supplementary information S7 (movie)

Structure of the CAS:importin-α:RanGTP nuclear export complex. CAS (yeast Cse1, yellow) coils around both RanGTP (cyan) and importin-α (green) in the complex. The IBB domain (blue) is sandwiched between CAS and importin-α. The binding of the IBB domain to the NLS binding sites is crucial for the formation of this complex and ensures that only cargo-free importin-α is exported to the nucleus, thus preventing futile transport cycles. (PDB accession code 1WA5). (MOV 9573 kb)

Supplementary information S8 (movie)

Structure of isolated CAS. Isolated CAS (cyan), corresponding to the species present in the cytoplasm after the export complex dissociates following RanGTP hydrolysis, assumes a "closed" conformation in which its N-terminus binds to a region near the centre of the molecule. (PDB accession code 1Z3H). (MOV 7422 kb)

Supplementary information S9 (movie)

Comparison between the open and closed forms of CAS. CAS undergoes a considerable conformational change between the open form assumed in the CAS:importin-α:RanGTP export complex (yellow) and the closed form (cyan) that results from the disassembly of this complex in the cytoplasm following GTP hydrolysis on Ran. The helicoidal pitch has changed substantially between the two forms (compare with S4, movie), consistent with the molecule being flexible. (PDB accession codes 1Z3H and 1WA5). (MOV 10126 kb)

Supplementary information S10 (movie)

Illustration of the conformational change in CAS between the open and closed states. The MolMov package (http://bioinfo.mbb.yale.edu/MolMovDB) was used to simulate the transition between the "open" and "closed" forms of CAS. (PDB accession codes 1Z3H and 1WA5). (MOV 3809 kb)

Supplementary information S11 (movie)

Structure of nuclear transport factor 2 (NTF2). NTF2 is a dimer constructed from two chains (cyan and yellow). Each chain generates a hydrophobic cavity to which RanGDP binds (see S12, movie), whereas a hydrophobic patch formed between the two NTF2 chains binds the FxFG repeats from nucleoporins. (PDB accession number 1GY6). (MOV 8015 kb)

Supplementary information S12 (movie)

Complex between nuclear transport factor 2 (NTF2) and RanGDP. Two RanGDP chains (red and yellow) bind to the hyrodophobic cavities in the NTF2 dimer (blue and cyan) to facilitate its transport into the nucleus for nucleotide exchange using RanGEF. GDP is shown as space-filling format. (PDB accession number 1A2K). (MOV 7023 kb)

Supplementary information S13 (movie)

Structure of the metazoan RanGEF, RCC1. RCC1 (cyan) is based on a seven-bladed propeller structure. It accelerates the rate of nucleotide exchange on Ran by stabilizing the nucleotide-free state. (PDB accession code 1A12). (MOV 3546 kb)

Supplementary information S14 (movie)

Structure of the yeast RanGAP, Rna1. RanGAP (green) is constructed from leucine-rich repeats (LLRs each based on an α-helix and a β-strand) and accelerates the rate of the Ran GTPase by ∼105. (PDB accession code 2CA6). (MOV 4027 kb)

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Glossary

Coiled coil

A protein fold in which two α-helices coil around one another.

WD propeller

A protein fold formed by a series of repeating WD sequence motifs that structurally resemble the blades of a propeller.

α-helical solenoid

A protein fold formed by successive repeats, each of which contains a number of α-helices that form a loop that is like a coil of a spring or solenoid.

Adaptor proteins

Proteins that augment cellular responses by recruiting other proteins to a complex. They usually contain several protein:protein interaction domains.

Scanning Ala mutagenesis

A technique in which successive residues in a region of a sequence of a protein are mutated to Ala to define those that influence an activity, such as binding to another protein.

Random walk

The path followed by taking successive steps, each in a random direction relative to the previous step.

Entropy

The component of free energy due to the disorder or randomness of the system. Increases in entropy (disorder) lower the free energy, whereas increases in order (lower entropy) increase energy.

Enthalpy

The heat component of free energy that, in biological systems, is derived primarily from chemical bonds.

Le Chatelier's principle

Le Chatelier's principle states that if a dynamic equilibrium is disturbed by changing conditions, the position of equilibrium moves to counteract the change.

Thermal ratchet

A molecular mechanism by which the thermal (Brownian) motion of a particle is biased (or rectified) so that there is net movement in a particular direction. A thermal ratchet requires an input of energy in order not to violate the second law of thermodynamics.

Helicoidal pitch

The distance along the axis of a helicoid corresponding to a rotation of 360°.

Helicoid

A spiral that is shaped like the shell of a snail such that its radius decreases progressively along its axis.

Sensitivity analysis

A procedure to determine the sensitivity of the outcomes of a model to changes in its parameters.

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Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 8, 195–208 (2007). https://doi.org/10.1038/nrm2114

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