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.

Bidirectional transport of macromolecules between the nucleus and the cytoplasm occurs through the nuclear pore complexes (NPCs) by a signal-mediated mechanism that is directed by targeting signals (NLSs) residing on the transported molecules or "cargoes." Nuclear transport starts after interaction of the targeting signal with soluble cellular receptors. After the formation of the cargo-receptor complex in the cytosol, this complex crosses the NPC. Herein, we use gold particles of various sizes coated with cargo-receptor complexes to determine precisely how large macromolecules crossing the NPC by the signal-mediated transport mechanism could be. We found that cargo-receptor-gold complexes with diameter close to 39 nm could be translocated by the NPC. This implies that macromolecules much larger than the assumed functional NPC diameter of 26 nm can be transported into the karyoplasm. The physiological relevance of this finding was supported by the observation that intact nucleocapsids of human hepatitis B virus with diameters of 32 and 36 nm are able to cross the nuclear pore without disassembly.

Ran/TC4 is a small nuclear G protein that forms a complex with the chromatin-bound guanine nucleotide release factor RCC1 (ref. 2). Loss of RCC1 causes defects in cell cycle progression, RNA export and nuclear protein import. Some of these can be suppressed by overexpression of Ran/TC4 (ref. 1), suggesting that Ran/TC4 functions downstream of RCC1. We have searched for proteins that bind Ran/TC4 by using a two-hybrid screen, and here we report the identification of RanBP2, a novel protein of 3,224 residues. This giant protein comprises an amino-terminal 700-residue leucine-rich region, four RanBP1-homologous (refs 9, 10) domains, eight zinc-finger motifs similar to those of NUP153 (refs 11, 12), and a carboxy terminus with high homology to cyclophilin. The molecule contains the XFXFG pentapeptide motif characteristic of nuclear pore complex (NPC) proteins, and immunolocalization suggests that RanBP2 is a constituent of the NPC. The fact that NLS-mediated nuclear import can be inhibited by an antibody directed against RanBP2 supports a functional role in protein import through the NPC.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.Transport through nuclear pores requires concerted action between the structural components of the nuclear pore complex (NPC) and the soluble transport factors that bind to the transport substrates and shuttle between the nuclear and cytoplasmic compartments (for reviews, see references 2 and 31). Substantial progress toward an understanding of nuclear protein import has been achieved in the past few years, but very little is known about how RNA is exported from the nucleus into the cytoplasm. Among the factors required for nuclear protein import are the classical nuclear localization signalreceptor complex, consisting of importin/karyopherin ␣ and ␤, the small GTPase Ran, and several Ran-binding proteins, as well as repeat sequences containing nucleoporins (for reviews, see references 5 and 11). Recently, additional routes of import into the nucleus were discovered, suggesting that major classes of transport substrates use different import pathways. Transportin and Kap123p were identified as novel transport factors that bind directly to their transport substrates, hnRNP protein A1 and ribosomal protein L25, respectively (30, 38). Transportin and Kap123p belong to a growing family of importin ␤-like proteins which have a Ran GTP-binding domain in their amino-terminal portions (5, 10). Recently, Mtr10p, which is also a member of this protein family, was shown to be the importin for yeast Np13p (34,41). An essential role for Ran in energydependent nuclear protein import has been firmly established, but how Ran and the many Ran activity-modulating proteins participate in the actual translocation process is still controversial.The Ran system is also involved in transport from the nucleus (9,14,18,36). It has been firmly established that nuclear export sequences (NES), first identified in viral proteins such as human immunodeficiency virus Rev and protein kinase inhibitor, mediate the exit of proteins from the nucleus (for a review, see reference 8). For the Rev protein, which is an RNA-binding protein with a specificity for unspliced or partially spliced viral transcripts, viral mRNA is coexported through association with Rev (3). Initially, it was found that Rev NES interact with Rip (6,47), which resembles repeat sequence-containing nucleoporins and accordingly was suggested to be a NES receptor at the nuclear pores. Recen...