The NPC is Largely Symmetrical and Constructed of Surprisingly Few Distinct Proteins
We present the results of an oversampled analysis that has identified the complement of yeast nups, and set them within the structural context of the NPC. In addition to previously characterized nups, our work has allowed the classification of Nup60p/Yar002p, Pom34p/Ylr018p, Cdc31p, Rip1p/Nup42p, Nup192p/Yjl039p, Gle1p, and Gle2p as nups. It is possible that our nup definition and subsequent classification system may exclude some bona fide constituents; nonetheless, the number so excluded is likely small, and we can, therefore, set an upper limit of ~30 distinct NPC components. This indicates a surprisingly simple composition for such a massive structure (e.g., given ~75 different proteins in a ribosome), and is significantly lower than previous estimates (Rout and Wente 1994
). We found three factors that explain this.
First, most nups were found on both the nucleoplasmic and cytoplasmic sides of the NPC, equidistant from the midplane of the NPC running parallel to the NE, and were estimated to be present in two or four copies per spoke (16 or 32 copies/NPC). Thus, the NPC appears to be mainly composed of 16 copies of a nup subcomplex, 8 copies on each side of the NPC midplane. This agrees with the high degree of twofold symmetry shown by the three-dimensional map of the isolated yeast NPC (Yang et al. 1998
), and indeed our positional plot for the nups superimposes well around a mask derived from a central vertical section from this map (). Complications from the biased and gene duplicate nups do not change this overall picture. A relatively minor degree of asymmetry is introduced by placing proteins unequally on either the nucleoplasmic or cytoplasmic face of this symmetrical superstructure, which could contribute to the observed minor morphological asymmetry of the yeast NPC (Rout and Blobel 1993
; Yang et al. 1998
). Second, economy of composition is also achieved by the NPC sharing proteins with other cellular components, such as the COPII component Sec13p, and the spindle pole components Ndc1p and (as shown here) Cdc31p. Third, the high NPC mass is also a result of the large average molecular weight (~100 kD) of yeast nups. Thus only 30 such proteins, each present in 16 copies, would produce a structure of ~50 MD. This combination of a limited number of large nups, present in multiple copies in a highly symmetric structure, can thus completely account for the mass of the NPC.
A Brownian Affinity Gating Model for Nucleocytoplasmic Transport
Constrained by the above observations, a coherent model of nucleocytoplasmic transport must explain the following: (a) NLS or NES signal–mediated gating at the NPC, (b) unidirectional (vectorial) transport across the NPC, (c) recycling to allow unlimited rounds of transport, and (d) the energy requirements of these processes. We propose that Brownian motion (diffusion) accounts for translocation while vectoriality is a combined effort of both the asymmetric nups and the asymmetric distribution of soluble transport factors. Two gating mechanisms remain possible. One involves a dilatory gate. However, given the number and disposition of FG nups, we propose another mechanism that does not require conventional mechanical gating.
Two characteristics of the NPC could account for gating (). On one hand, the NPC contains a narrow central tube, ringed at each end by a dense array of filamentous FG nups (). This confined channel (which may be further occluded by the filamentous FG nups) presents a significant barrier to the passive diffusion of macromolecules (Paine et al. 1975
; Feldherr and Akin 1997
). Thus, macromolecules that do not bind to nups are less likely to diffuse across the NPC and are largely barred from passage across the NE ( I). On the other hand, macromolecules that bind to nups (specifically cargo-carrying transport factors binding to FG nups) increase their residence time at the entrance of the central tube, and so their diffusion across the NPC is greatly facilitated ( II). In a sense, the FG nups provide an attraction force for transport factors that could counter the repulsion force from diffusive exclusion. Given a sufficiently high release rate, rapid and reversible binding would promote a fast diffusional exchange of transport factors among the symmetrically disposed docking sites and so between the two faces of the NPC. By restricting the number of diffusional degrees of freedom (e.g., along the filaments), the FG nups may further facilitate the passage of transport factors across the NPC. In effect, the apparent size of the diffusion channel would become very much larger for proteins that can bind the FG nups than for those proteins unable to bind. This difference provides a virtual gate, in which the apparent size of the NPC increases for a cargo only while its signal-mediated translocation is active, but which does not necessarily involve a physical dilation of the NPC (Blobel 1995
Figure 10 The Brownian affinity gate model. (I) The narrow bore of the central tube ensures that macromolecules that do not bind to nups find in hard to diffuse across the NPC, and are thus largely excluded; the diffusive movement of the filamentous nups (more ...)
Our model is clearly a simplified one. In particular, some aspects of gating still seem to require the existence of a mechanical gate at the central tube. However, we suggest an alternative that does not require such large-scale concerted motions of the central tube. Thus, we propose that diffusive movement of the tethered filamentous FG nups may contribute to the discrimination between nonbinding and binding macromolecules. Brownian motion would drive the rapid motion of these filaments; small molecules could readily slip past the flailing filaments, while large molecules would tend to be deflected away from the channel by frequent impacts. Such entropic exclusion has been demonstrated (Brown and Hoh 1997
). The dilation of the central channel and nuclear basket observed for the largest transport substrates (Akey 1990
; Kiseleva et al. 1996
) would then be a consequence, rather than cause, of substrate translocation.
For unidirectional transport across the NPC, translocating transport factors need cues to determine which side of the NPC they are on. Vectorial cues arise from two potential sources, the asymmetry of the FG nups and the asymmetric distribution of Ran-GTP (Mattaj and Englmeier 1998
). Transport is then concluded by a vectorial step.
Consider a cargo imported by its kap into the nucleoplasm (). The cargo alone cannot easily pass through the diffusion barrier of the NPC. In the cytoplasm, the kap is exposed only to Ran-GDP, allowing it to acquire the cargo. This cargo–kap complex can now bind the NPC ( I), passing through the now effectively open Brownian gate and diffusing across the NPC by binding symmetric FG nups. This allows the cargo–kap complex access to both faces of the NPC ( II). We propose that in this state, the cargo–kap complex has its highest affinity for the one-sided FG nups on the nuclear face. At the nuclear face, the kap moves preferentially away from the symmetric region of the nuclear pore to these higher affinity binding sites, preventing its exchange back to the cytoplasmic face. In the absence of any other influence this high affinity interaction would be stable, and the cargo–kap complex would arrest here, bound to the FG nups at the nuclear extremity of the NPC ( III). This situation is seen in the case of kap mutants that cannot bind Ran (Gorlich et al. 1996
). However, the peripheral localization of these nups exposes their bound kap complex to the nucleoplasmic milieu and in particular, to Ran-GTP. The binding of Ran-GTP to the kap induces cargo release and undocking from the NPC, which causes the cargo and kap to be liberated into the nucleoplasm ( IV). Once nucleoplasmic, the cargo can no longer bind the NPC, and the Brownian gate, in effect, shuts.
Diagram of NPC illustrating a model for nucleocytoplasmic transport. See Discussion for details.
Export would be similar ( I–IV). An exporting kap binds both Ran-GTP and its export cargo in the nucleoplasm. After passing through the symmetric FG nups, the cargo-Ran-kap complex is preferentially drawn to the high affinity binding sites, this time on the NPC cytoplasmic face. In isolation, this complex would be stable, as has been observed (Floer and Blobel 1999
; Kehlenbach et al. 1999
). However, the kap-bound Ran-GTP is now at the cytoplasmic periphery of the NPC, exposed to the cytoplasmic RanGAP, which induces the hydrolysis of the GTP on Ran. This in turn, causes undocking of the kap complex from the cytoplasmic binding sites and the release of both Ran and the cargo from the kap. It can readily be seen how recycling of the export kap back to the nucleoplasm would be akin to cargoless import, and likewise how recycling of an importing kap akin to cargoless export. In this fashion, the transport cycles are completed and the system restored, powered by the hydrolysis of GTP.
Numerous mechanistic refinements can be added. For instance, as exporting and importing kaps differ only in which Ran-bound state they load and dispose of cargo, it is possible that some kaps may carry material in both directions. The various transport factors apparently favor different FG nups (Aitchison et al. 1996
; Rout et al. 1997
; Marelli et al. 1998
), and may also differ as to which vectorial cues (asymmetric FG nups versus soluble factors) they emphasize; some may only require symmetric FG nups and Ran. Such techniques for segregating transport factors into alternative pathways would serve to decrease congestion at the NPC, and furthermore provide opportunities for differential regulation by altering the affinity for a particular docking site to its particular preferred class of transport factor (Saavedra et al. 1997
; Marelli et al. 1998
). Furthermore, some transport factors may not require Ran at all, gaining directional cues and energy from alternative sources. For example, NPC-associated, ATP-driven RNA helicases (Snay-Hodge et al. 1998
; Tseng et al. 1998
), plus the exchange of RNA-binding proteins at the NPC, might reel mRNPs across the NPC in a process that is largely independent of Ran-mediated GTP hydrolysis. We also expect that in higher eukaryotes other mechanisms, like incorporating binding sites for Ran and its cofactors within the NPC, have been called into play to improve the efficiency and regulation of transport.