U. Kutay

Identification of different roles for RanGDP and RanGTP in nuclear protein import.

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.

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Rans 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.

Export of Importin α from the Nucleus Is Mediated by a Specific Nuclear Transport Factor

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

Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin‐β mediates translocation via direct interaction with NPC components and carries importin‐α with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin‐β which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin‐β for its multiple partners. Ran and importin‐α respectively require residues 1–364 and 331–876 of importin‐β for binding. Thus, RanGTP‐mediated release of importin‐α from importin‐β is likely to be an active displacement rather than due to simple competition between Ran and importin‐α for a common binding site. Importin‐β has at least two non‐overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin‐β from the NPC. Importin‐β fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin‐β mutants are very efficient inhibitors of NLS‐dependent protein import. Surprisingly, however, they also inhibit M9 signal‐mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES‐containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.

Identification of a tRNA-Specific Nuclear Export Receptor

In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.

Structural View of the Ran–Importin β Interaction at 2.3 Å Resolution

Abstract Transport receptors of the Importin β family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin β. The structure of Importin β shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin β family that use direct RanGTP‐binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin β (Impβ) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impβ/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impβ together with Imp7. Import by the Imp7/Impβ heterodimer is strictly Ran dependent, the Ran‐requiring step most likely being the disassembly of the cargo–receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impβ and Imp7, whereby the two Ran‐binding sites act synergistically. However, whereas an Impβ/RanGTP interaction appears essential for H1 import, Ran‐binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran‐binding site is essential when operating as an autonomous import receptor, i.e. independently of Impβ. Within the Impβ/Imp7 heterodimer, however, Imp7 plays a more passive role than Impβ and resembles an import adapter.

Yrb4p, a yeast Ran–GTP‐binding protein involved in import of ribosomal protein L25 into the nucleus

Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin‐β, the mediator of nuclear localization signal (NLS)‐dependent import into the nucleus, and to Pse1p. Like importin‐β, Yrb4p and Pse1p specifically bind to Gsp1p–GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p‐binding is dominant‐lethal and blocks bidirectional traffic across the NPC in wild‐type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p–GTP. Consistent with its putative role as an import receptor for L25‐like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC.

The translocation of transportin–cargo complexes through nuclear pores is independent of both Ran and energy

Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1] [2] [3] [4]. Import receptors such as importin beta or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin alpha/beta heterodimer, coupled directly to importin beta release from the NPCs. If the importin beta -RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5] [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin-substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin-substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin-substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin beta independently of Ran and energy.