Journal of Cell Science 113, 1651-1659 (2000) 1651 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0712 COMMENTARY The nuclear pore complex: mediator of translocation between nucleus and cytoplasm T. D. Allen1, J. M. Cronshaw1, S. Bagley1, E. Kiseleva2 and M. W. Goldberg1 1CRC Structural Cell Biology Group, Paterson Institute, Christie Hospital, Manchester, M20 4BX, UK 2Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090, Russia Published on WWW 18 April 2000 SUMMARY The enclosure of nuclear contents in eukaryotes means that receptors and adapters, and the molecules (and their cells require sites in the boundary that mediate exchange regulators) that underpin the transport mechanisms. Over of material between nucleus and cytoplasm. These sites, the past few years there has been an increasing interest in termed nuclear pore complexes (NPCs), number 100-200 in the pore complex: structural studies have been followed by yeast, a few thousand in mammalian cells and ~50 million elucidation of the biochemical aspects of nuclear import, in the giant nuclei of amphibian oocytes. NPCs are large and subsequent investigations into nuclear export. The (125 MDa) macromolecular complexes that comprise current challenge is to understand the interactions between 50-100 different proteins in vertebrates. In spite of their the structural elements of the pore complex and the size and complex structure, NPCs undergo complete mechanisms that drive the physical processes of breakdown and reformation at cell division. Transport translocation through it. through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport Movies available on-line: of complexes up to several megadaltons in molecular mass. http://www.biologists.com/JCS/movies/jcs0712.html Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for Key words: Nuclear pore complex (NPC), Nucleocytoplasmic different cargoes, and their transport signals, transport transport, NPC structure, NPC function, NPC dynamics INTRODUCTION nucleoporins, including the sequencing of all 30 nucleoporins found in isolated yeast NPCs has been published recently (Rout The vertebrate nuclear pore complex (NPC) is one of the et al., 2000). Fontoura et al. (1999) have initiated similar largest types of macromolecular complex in the cell: it has an approaches for the 50 nucleoporins isolated from rat liver estimated molecular mass of 125 MDa in vertebrates (Reichelt NPCs. The next major task is a full structural characterisation et al., 1990), and comprises 50-100 proteins (Fontoura et al., of the nucleoporins, their exact localisation within the overall 1999) termed nucleoporins. Yeast pore complexes are smaller, NPC structure and definition of the specific roles of these being made up of ~30 nucleoporins and having a molecular proteins in transport (Fig. 1). mass of ~66 MDa (Rout and Blobel, 1993; Rout et al., 2000). Amongst the variety of nucleoporins that make up the pores, several complexes have been identified, such as the p62 STRUCTURE OF THE NPC complex in vertebrates, which consists of p62, p58 and p54 in Xenopus (Finlay, 1991) as well as p45 in the p62 complexes Yeast and vertebrate NPCs display an overall similarity of core from rat (Guan et al., 1995). Several nucleoporin complexes structural elements at the electron microscopic level (Yang et have been described in yeast (see Ohno et al., 1998); a recently al., 1998), and equivalent yeast and vertebrate nucleoporins described example, the Nup84 complex (Rappsilber et al., share sequence similarity of ~25%. The increased size of the 2000), has also been structurally characterised as a triskelion vertebrate pore is most likely to reflect an increase in that might form an elemental building block of the pore complexity consistent with metazoan evolution, but the basic structure. Excellent accounts dealing with the nucleoporins structural similarities between the NPCs of yeast and higher themselves can be found in other recent reviews (Ohno et al., eukaryotes indicate that they probably share common 1998; Stoffler et al., 1999a). A full characterisation of the yeast translocation mechanisms. Extensive localisation studies of 1652 T. D. Allen and others yeast NPCs by immunoelectron microscopy have shown a peripheral structures of the NPC. Transport (both import and symmetrical distribution of potential transport-associated export) is then completed by a series of cargo–complex-NPC docking sites running in a continuous sequence through the interactions over a distance of up to 200 nm, required for pore structure (see Stoffler et al., 1999a; Rout et al., 2000). complete passage between nucleus and cytoplasm (Ohno et al., The overall structure of the vertebrate nuclear pore has been 1998). known for some time (Akey, 1989; Hinshaw et al., 1992; Goldberg and Allen, 1993, 1996; Panté and Aebi, 1996; Goldberg et al., 1999; Kiseleva et al., 1998). The presence of NUCLEAR TRANSPORT, SOLUBLE FACTORS AND a central plug or transporter (Akey and Radermacher, 1993; Ran Goldberg and Allen, 1996) as a fundamental element of pore structure remains controversial: an alternative view suggests Molecules destined for nuclear import (Fig. 3) or export (Fig. that central elements are merely cargo caught ‘in transit’ during 4) can be thought of as cargoes, and include proteins, various specimen preparation. Our own view is that a regular and types of RNA and RNPs. For recent comprehensive reviews, consistent structure is visualised in all well-prepared NPCs, see Nakielny and Dreyfuss (1999) and Görlich and Kutay either rapidly frozen (Akey, 1989) or chemically fixed, and can (1999). Crucial to the process of transport are the import be exposed by proteolysis (Goldberg and Allen, 1993, 1996). receptors (Görlich et al., 1995) such as importin α and importin This view is also supported by the direct visualisation of the β, the earliest-identified members of a family comprising at transporter as a central NPC structure through which mRNP least seven members (Mattaj and Engelmeier, 1998). In yeast fibres emerge into the cytoplasm during mRNA export in the import receptors are termed karyopherins. Export receptors Chironomus salivary glands (Kiseleva et al., 1998). Studies of (exportins) recognise nuclear-export signals (NESs) as specific the passage of gold particles through the centre of the pore sequences in the same way as nuclear-localisation signals complex by transmission EM indicate a central channel (NLSs) are recognised by importins. Further molecules (through the transporter) that has a maximum diameter of 26 necessary for transport include the small GTPase Ran and nm (Feldherr and Akin, 1993, 1994a,b). In the absence of any NTF2 (nuclear-transport factor 2). central structure in the pore complex, a considerably larger In a typical example of import, the NLS of nucleoplasmin central orifice (perhaps up to 45 nm) would be expected. is recognised by importin α. Importin α becomes complexed Our current view is that the transporter is suspended in the to importin β, and the cargo plus importin α and importin β is centre of the spoke-ring complex, which occupies most of the translocated through the NPC into the nucleus, where it is space delineated by the pore membrane (the area of membrane that joins the inner and outer nuclear envelopes). The radial arms of the spoke complex penetrate the pore membrane and project into the lumen of the nuclear envelope. Sandwiching the spoke- ring complex are the cytoplasmic coaxial ring and the nucleoplasmic coaxial ring, which anchor and support the cytoplasmic filaments and nuclear baskets, respectively (Fig. 2A,B). The cytoplasmic coaxial ring has at least three levels of substructure. It contains a ‘star ring’ in direct apposition to the outer NE membrane; the star ring itself is covered by a thin ring, to which further elements are added to form the entire ring, and this supports the attachment of eight cytoplasmic filaments (Fig. 2B). The cytoplasmic coaxial ring is joined to the entrance of the transporter by eight internal filaments, which are arranged rather like the hub and spokes of a wagon wheel. These substructures were originally identified by FEISEM after various fracturing and proteolytic treatments (Goldberg et al., 1996) and subsequently confirmed in successive stages of NPC assembly in vitro (Goldberg et al., 1997). Although 95% of the NPC’s in isolated oocyte envelopes show typical eightfold radial symmetry, a small proportion of examples of nine- and seven-fold symmetry are seen, often adjacent to one another; this might indicate local disruption of a self-assembly process. This is particularly evident in the basket structures, in which the increase in diameter required to accommodate nine rather than eight structural elements is clearly apparent (Goldberg and Allen, 1993). Nuclear transport is initiated by interaction of transport cargo with the Fig. 1. Functions of the nuclear pore complex. The nuclear pore complex 1653 dissociated from the importins by GTP-bound Ran. Ran-GTP rapid isolation and fixation of oocyte nuclear envelopes binds to importin β and recycles it back to the cytoplasm, revealed a much longer filamentous organisation (Goldberg where GTP hydrolysis of Ran is stimulated by the GTPase and Allen, 1996); this is also apparent in situ in transmission activating Ran-GAP; this releases importin β for the next round electron microscopy (TEM) when gold-labelled transport of import. The new Ran-GDP is taken back into the nucleus complexes are bound to the filaments (Richardson et al., 1988; by NTF2 (Fig. 5), where RCC1 stimulates exchange of Ran- Rutherford et al., 1997). There have also been suggestions that bound GDP for GTP, which effectively converts Ran-GDP elements of the cytoskeleton make direct contact with these back to Ran-GTP. Meanwhile importin α and the cargo have separated in the nucleus, and importin α is recycled to the A cytoplasm complexed to CAS (the product of the cellular apoptosis-susceptibility gene) and Ran-GTP (Kutay et al., 1997).