Transport into and out of the nucleus

Abstract

A defining characteristic of eukaryotic cells is the possession of a nuclear envelope. Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope. The molecular basis for transport has been revealed only within the last few years. The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes. The carriers recognize localization signals on the cargo and can bind to pore proteins. They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear. Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo. The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process. Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions. Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport. This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.

FIG. 1

Structures of carrier complexes with Ran (A), the N-terminal domain of importinα (IBB domain) (B), and a bipartite NLS (C). H1 to H18 identify the HEAT motifs of transportin1 and importinβ. L7 is the loop within HEAT motif7. Ran (bound to GppNHp, a nonhydrolyzable analogue of GTP), the IBB domain, and the NLS are shown in white. Note how the HEAT motifs H11 to H18 are twisted up and around the IBB domain, compared to their positions in panel A (39, 263). A1 to A10 identify the ARM motifs, which are closely related to HEAT motifs. Note that the superhelical twist is in the same direction in all three structures and that the ligands all interact with the concave inner surface of the carriers.

FIG. 2

Mechanism of cargo import by direct interaction with an importin carrier protein. RanGTP is present at high concentrations only in the nucleus, where it disassembles the cargo-importin complex. The importin-RanGTP complex returns to the cytoplasm, where the GTP is hydrolyzed, releasing the RanGDP from the importin.

FIG. 3

Mechanism of cargo import by the importinα-importinβ pathway. In this mechanism, the cargo binds to an adapter, importinα, rather than directly to the carrier. The cargo-importinα-importinβ complex is disassembled in the nucleus by RanGTP. Importinβ returns to the cytosol, as shown in Fig. 2. The importinα requires a carrier for export, called CAS (CseI in budding yeast). Assembly of the export complex requires RanGTP. The ternary importinα-CAS-RanGTP complex is disassembled in the cytoplasm by hydrolysis of the GTP.

FIG. 4

Comparison of the Ran-GTP interacting surfaces between importin-β and transportin1. (A) Alignment of the Ran binding domains of importinβ and transportin1. Residues in importinβ that bind Ran are shown in yellow. Residues in transportin1 that bind Ran are shown in blue. Loop7 is indicated in pink. (B) Amino acid sequence of Ran showing the Switch 1 and Switch 2 regions that undergo a conformational switch between the GTP and GDP binding states, and the basic patch. Residues that bind importinβ are shown in yellow; residues that bind transportin1 are shown in blue (39, 263).

FIG. 4

Comparison of the Ran-GTP interacting surfaces between importin-β and transportin1. (A) Alignment of the Ran binding domains of importinβ and transportin1. Residues in importinβ that bind Ran are shown in yellow. Residues in transportin1 that bind Ran are shown in blue. Loop7 is indicated in pink. (B) Amino acid sequence of Ran showing the Switch 1 and Switch 2 regions that undergo a conformational switch between the GTP and GDP binding states, and the basic patch. Residues that bind importinβ are shown in yellow; residues that bind transportin1 are shown in blue (39, 263).

FIG. 5

Mechanism of cargo export. Cargo binds weakly to the export carrier in the absence of RanGTP but tightly in its presence. The ternary cargo-exportin-RanGTP complex is disassembled in the cytoplasm by GTP hydrolysis on Ran. The exportin is presumed to return empty to the nucleus.

FIG. 6

Structures of the RanGEF RCC1 (A) and of RanGAP (B). (A) The first 20 amino acids, containing the NLS, are absent. (B) The arginine finger residue is indicated. This residue is required for catalytic activity (93, 202).

FIG. 7

Structures of RanGDP-NTF2 (A) and RanGTP-RanBD (B). NTF2 forms a dimer, and each subunit associates with one molecule of RanGDP. Ran is shown in green, the nucleotide is shown in blue, and the NTF2 subunits are shown in red and yellow. Note the location of the Ran C-terminal arm (blue-green) in close juxtaposition to the basic patch on the RanGDP. When bound to a RanBD, the C-terminal arm (blue-green) of RanGTP is extended away from the body of the Ran (green) and embraces the RanBD (red). The N terminus of the RanBD (orange) loops around the Ran and contacts the basic patch (246, 264).

FIG. 8

Mechanism of Ran import. The carrier NTF2 binds specifically to RanGDP, present in the cytoplasm. In the nucleus, RanGEF catalyzes the exchange of GDP for GTP on Ran, which releases the NTF2. NTF2 then returns empty to the cytoplasm to pick up another molecule of RanGDP. The RanGTP associates with carriers (importins or exportins) which move to the cytosol, where the GTP is hydrolyzed, releasing RanGDP.

FIG. 9

The oily-spaghetti model for translocation through the nuclear pores. The approximate dimensions of the pore are shown. Nucleoporins containing FxFG repeats are shown as randomly oriented lines. Molecules with a diameter of <10 nm can diffuse freely through the pore. Larger molecules are hindered by the nucleoporins. Carrier proteins bind weakly and transiently to the nucleoporins. It is assumed that the free energy changes associated with conformational motions of the nucleoporins are very low, so that the carrier can diffuse from one binding site to another relatively unhindered. The time between binding events is on the order of a few microseconds, and the carrier is assumed to diffuse randomly back and forth across the pore. Vectoriality is imparted by the assembly and disassembly reactions driven by RanGTP and is not a property of the pore. Docking sites, which are situated at some distance outside of the pore, are not shown.