The compartmentation of eukaryotic cells requires all nuclear proteins to be imported from the cytoplasm, whereas, for example, transfer RNAs, messenger RNAs, and ribosomes are made in the nucleus and need to be exported to the cytoplasm. Nuclear import and export proceed through nuclear pore complexes and can occur along a great number of distinct pathways, many of which are mediated by importin beta-related nuclear transport receptors. These receptors shuttle between nucleus and cytoplasm, and they bind transport substrates either directly or via adapter molecules. They all cooperate with the RanGTPase system to regulate the interactions with their cargoes. Another focus of our review is nuclear export of messenger RNA, which apparently largely relies on export mediators distinct from importin beta-related factors. We discuss mechanistic aspects and the energetics of transport receptor function and describe a number of pathways in detail.

MicroRNAs (miRNAs), which function as regulators of gene expression in eukaryotes, are processed from larger transcripts by sequential action of nuclear and cytoplasmic ribonuclease III-like endonucleases. We show that Exportin-5 (Exp5) mediates efficient nuclear export of short miRNA precursors (pre-miRNAs) and that its depletion by RNA interference results in reduced miRNA levels. Exp5 binds correctly processed pre-miRNAs directly and specifically, in a Ran guanosine triphosphate-dependent manner, but interacts only weakly with extended pre-miRNAs that yield incorrect miRNAs when processed by Dicer in vitro. Thus, Exp5 is key to miRNA biogenesis and may help coordinate nuclear and cytoplasmic processing steps.

The importin‐alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP‐bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC‐bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin‐beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin‐beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin‐beta probably triggers termination, i.e. the dissociation of importin‐alpha from importin‐beta and the subsequent release of the import substrate into the nucleoplasm.

NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-β has previously been shown to release importin-α from -β during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-α and -β, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.

SUMMARY Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled-coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

In eukaryotic cells, the enclosure of the genetic information in the nucleus allows the spatial and temporal separation of DNA replication and transcription from cytoplasmic protein synthesis. This compartmentalization not only permits a high level of regulation of these processes but at the same time necessitates a system of selective macromolecular transport between the nucleus and the cytoplasm. Transfer of macromolecules between both compartments is mediated by soluble receptors that interact with components of nuclear pore complexes (NPCs) to move their specific cargos. Transport occurs by way of a great variety of different pathways defined by individual receptors and accessory factors. Often, processes in substrate biogenesis that precede transport concurrently recruit transport factors to substrates, thus making transport responsive to correct and orderly synthesis of substrates. Some current challenges are to understand how transport factor-substrate interactions are controlled and integrated with sequential steps in substrate biogenesis, how large macromolecular complexes are restructured to fit through the NPC channel and to understand how transport factor-NPC interactions lead to actual translocation through the NPC.

The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.