Dirk Görlich

Isolation of a protein that is essential for the first step of nuclear protein import

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.

Kinetic analysis of translocation through nuclear pore complexes

The mechanism of facilitated translocation through nuclear pore complexes (NPCs) is only poorly understood. Here, we present a kinetic analysis of the process using various model substrates. We find that the translocation capacity of NPCs is unexpectedly high, with a single NPC allowing a mass flow of nearly 100 MDa/s and rates in the order of 103 translocation events per second. Our data further indicate that high affinity interactions between the translocation substrate and NPC components are dispensable for translocation. We propose a selective phase model’ that could explain how NPCs function as a permeability barrier for inert molecules and yet become selectively permeable for nuclear transport receptors and receptor–cargo complexes.

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.

Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane

We have reproduced the process of protein transport across and of protein integration into the mammalian endoplasmic reticulum membrane by the use of proteoliposomes reconstituted from pure phospholipids and purified membrane proteins. The transport of some proteins requires only two membrane protein complexes: the signal recognition particle receptor, needed for targeting of a nascent chain to the membrane, and a novel complex, the Sec61p complex, that consists of Sec61p and two smaller polypeptides. The translocation of other proteins also needs the presence of the translocating chain-association membrane (TRAM) protein. The integration of two membrane proteins of different topologies into the membrane does not require additional components. These results indicate a surprising simplicity of the basic translocation machinery. They suggest that the Sec61p complex binds the ribosome during translocation and forms the postulated protein-conducting channel.

Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells.

The assembly of eukaryotic ribosomal subunits takes place in the nucleolus and requires nuclear import of ribosomal proteins. We have studied this import in a mammalian system and found that the classical nuclear import pathway using the importin α/β heterodimer apparently plays only a minor role. Instead, at least four importin β‐like transport receptors, namely importin β itself, transportin, RanBP5 and RanBP7, directly bind and import ribosomal proteins. We found that the ribosomal proteins L23a, S7 and L5 can each be imported alternatively by any of the four receptors. We have studied rpL23a in detail and identified a very basic region to which each of the four import receptors bind avidly. This domain might be considered as an archetypal import signal that evolved before import receptors diverged in evolution. The presence of distinct binding sites for rpL23a and the M9 import signal in transportin, and for rpL23a and importin α in importin β might explain how a single receptor can recognize very different import signals.

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope

BACKGROUND Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Distinct functions for the two importin subunits in nuclear protein import

THE import of nuclear proteins proceeds through the nuclear pore complex and requires nuclear localization signals (NLSs)1,2, energy3,4 and soluble factors5, namely importin-α(Mr 60K)6-12,28, importin-β (90K)8-11,13 and Ran14,15. Importin-α is primarily responsible for NLS recognition6-12,29 and is a member of a protein family that includes the essential yeast nuclear pore protein SRPlp (ref. 16). As the first event, the complex of importin-α and importin-β binds the import substrate in the cytosol8,9. Here we show that this nuclear pore targeting complex initially docks as a single entity to the nuclear pore via importin-β. Then the energy-dependent, Ran-mediated translocation through the pore results in the accumulation of import substrate and importin-α in the nucleus. In contrast, importin-β accumulates at the nuclear envelope, but not in the nucleoplasm. Immunoelectron microscopy detects importin-β on both sides of the nuclear pore. This suggests that the nuclear pore targeting complex might move as a single entity from its initial docking site through the central part of the nuclear pore before it disassembles on the nucleoplasmic side.

NTF2 mediates nuclear import of Ran

Importin β family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Rans small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2–RanGDP complex to the nuclear side of the NPCs. A direct NTF2–RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP–NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin β family transport receptor.