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Nucleocytoplasmic transport: Diffusion channel or phase transition? Gwénaël Rabut and Jan Ellenberg
How exactly large molecules translocate through nuclear pores has been mysterious for a long time. Recent kinetic measurements of transport rates through the pore have led to a novel translocation model that elegantly combines selectivity with very high transport rates. Address: Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D–69117 Heidelberg, Germany. E-mail:
[email protected] Current Biology 2001, 11:R551–R554 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Communication between the nucleus and the cytosol of a eukaryotic cell is achieved by a unique molecular machine: the nuclear pore complex (NPC), a large protein assembly that spans the double membrane of the nuclear envelope, forming an aqueous channel. All nuclear proteins and all cytoplasmic RNAs have to traffic through NPCs at least once, and many do so more often, generating an enormous mass flow. Since the initial discovery that NPCs mediate nucleocytoplasmic traffic [1], enormous progress has been made in identifying and understanding the factors that mediate this process [2,3]. But we still do not understand the mechanism of translocation through the channel itself. This question is vital, because the limited number of NPCs per nucleus have to achieve a large flux while maintaining selectivity of transport, two goals that seem intuitively contradictory. Recent work has started to address the translocation mechanism by analyzing the kinetics of the translocation event [4,5], or by inference from ultrastructural observations [6]. Spectacularly, Ribbeck and Görlich [5] found single NPCs of permeabilized cells mediate close to 1000 translocation events per second. Here, we review the current models of NPC translocation in the light of the recent findings on transport kinetics. General properties of nucleocytoplasmic transport
The overall structure of the NPC is conserved among all eukaryotes [7,8]. For the purpose of translocation, we can simplify the vertebrate NPC to a flat cylinder with an outer diameter of ~120 nm and a length of ~40 nm (Figure 1). Filaments emanate from the rims of the cylinder and form distinct structures on the nuclear and cytoplasmic side (Figure 1). The 30–50 different proteins that make up the NPC, referred to as nucleoporins, frequently contain repeats of the hydrophobic amino acid phenylalanine paired with glycine (FG). There are two modes of
passage through the NPC. Small molecules move through nuclear pores rapidly and efficiently, and without selectivity, by free diffusion. For objects larger than about 30 kDa, diffusion is inefficient and translocation has to be facilitated and can thus be selective. In most cases, this is achieved by the formation of a complex between the translocating species and a transport receptor that specifically interacts with the NPC. Many transport receptors are known to interact with FG repeats, making FG nucleoporins likely key players in the translocation process [9,10]. Notably, both modes of passage occur through a single channel inside the NPC [11,12]. Molecules transported by facilitated translocation can move against a gradient of chemical activity. Sustained transport is an energy-consuming task, and it was believed for a long time that translocation itself is the active process. However, it has become clear more recently that a single round of transport does not require energy [13,14]. As a consequence, it appears that translocation of transport complexes occurs solely by diffusion inside the pore. The energy requirement for sustained transport lies in maintaining a chemical gradient of the small GTPase Ran across the nuclear envelope, with a high concentration of RanGTP in the nucleus and a high concentration of RanGDP in the cytosol. Nucleocytoplasmic transport uses this gradient for directionality, by moving RanGTP along the gradient to the cytoplasm either in antiport (import) or symport (export) mode. In the cytoplasm, RanGTP is immediately hydrolyzed to RanGDP, which equilibrates efficiently between cytoplasm and nucleus. To sustain traffic then, the nuclear RanGTP pool must be replenished constantly, consuming GTP [2,3]. Thus, the translocation step itself has neither to be active nor vectorial to achieve nucleocytoplasmic transport. Translocation models
What then are our current models to explain the molecular mechanism of facilitated translocation through the NPC? The key problem is to understand how the interactions of transport complexes with NPC components can enable their efficient yet selective translocation. Affinity gradient of binding sites
One hypothesis for translocation has been that transport complexes diffuse inside NPCs in a stepwise manner, hopping along a path of increasing affinity [10,15]. This would make the translocation process itself directional. BenEfraim and Gerace [16] recently reported some evidence in support of this hypothesis, from experiments using a solidphase binding assay with recombinant proteins. They
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Figure 1 Schematic illustration of Brownian affinity gate and selective phase translocation models. (a) In the Brownian affinity gate model, translocation occurs through a narrow (~5 nm diameter) aqueous channel. A hypothetical import substrate (green, comparable in size to 97 kDa Importin β) interacting with FG repeat nucleoporins on cytoplasmic filaments is concentrated at the entrance of the channel by constant binding and dissociation (dotted arrows). This increases its probability of entering the channel and being translocated (solid arrows). By contrast, inert molecules (red, comparable in size to a globular 70 kDa protein) that diffuse randomly (solid arrows) are unlikely to enter the channel and are thus excluded from translocation. (b) In the selective phase model, FG repeat nucleoporins (thin black lines) form a meshwork linked by hydrophobic interactions (dark green spots) that acts as a sieve and selective phase. A hypothetical large import substrate (blue) is shown in the inset below. If such large molecules/complexes can recruit transport receptors or have hydrophobic surface properties (light green), they can dissolve into the FG repeat mesh and be translocated. Inert hydrophilic molecules above the mesh size
(a)
(b) Cytosol
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would be excluded, as in (a) (not shown). NPC components — nuclear membrane, black; spoke ring complex, light gray; nuclear and
determined that an import substrate binds to FG nucleoporins of the NPC cytoplasmic filaments, central channel and nuclear filaments with apparent dissociation constants ranging from ~200 nM, through ~100 nM to ~10 nM, respectively. It is not clear, however, how the binding affinities measured on individual nucleoporins in vitro reflect those in the intact NPC in vivo. High-affinity interactions during translocation would slow down transport, as they saturate at low concentrations, and would thus reduce the flux through NPCs. Moreover, the affinities measured for nucleoporins of the central channel were similar, and we currently do not have any indication that FG nucleoporins are asymmetrically distributed inside the pore [6], a prerequisite for the gradient model. Brownian affinity gate
Rout et al. [6] recently proposed a translocation model that does not rely on high-affinity interactions (Figure 1a). In a comprehensive study, these authors identified virtually all yeast nucleoporins, and localized their protein A-tagged variants by preembedding electron microscopy. Most FG nucleoporins were found to be symmetrically localized surrounding the central channel. This distribution, and the assumption that the NPC diffusion channel has a narrow functional diameter [11], are the basis for the Brownian affinity gate translocation model. In this model, gating is achieved by the narrow channel entrance itself, which could potentially be obstructed further by the ‘flailing’ of the NPC filaments as a result of their Brownian motion.
cytoplasmic filaments, dark gray — as well as translocated molecules are approximately drawn to scale. Scale bar: 20 nm.
Cellular macromolecules of a significant size compared to the channel diameter would be extremely unlikely to enter the channel via the random walk of diffusing particles (Figure 1a, red particles). Substrates capable of binding to FG repeat nucleoporins of the filaments, however, would increase their residency time at the entrance of the channel, greatly increasing their probability of entering the channel and freely diffusing through it (Figure 1a, green particles). This model is attractive, because it accounts for the selectivity of the NPC for proteins that can interact with the FG nucleoporins surrounding the channel. Such a selectivity mechanism would maintain large fluxes, as the process of translocation is simply based on fast aqueous diffusion and does not require high-affinity interactions as obligatory steps. This model does not, however, account for the translocation of the large particles, up to 36 nm diameter, that have been shown to pass through the NPC efficiently [17,18]. Selective hydrophobic phase
In their recent study, Ribbeck and Görlich [5] monitored the influx kinetics of recombinant fluorescent import substrates into permeabilized cell nuclei supplied with an excess of transport factors. Uniquely in this assay, translocation depended solely on substrate concentration — analogous to classical enzyme kinetics — because competition from other substrates and limiting transport receptors was excluded. A single NPC was found to translocate up to 1000 substrate molecules per second, corresponding to a
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mass flow of 100 MDa per NPC per second! This is a 250 times higher translocation rate than previously estimated from measurements in cell extracts [4], and 40 times higher than estimates from measurements in intact cells [19]. Translocation was found to saturate partially at high substrate concentration, consistent with a KM of only ~4 µM. Thus, only low-affinity interactions are required for translocation. Ribbeck and Görlich calculate that facilitated translocation through the NPC is almost as effective as free diffusion through an aqueous channel of 40 nm in diameter and length. To explain the surprisingly high, but nevertheless selective, flux on the basis of low-affinity interactions, Ribbeck and Görlich [5] propose the selective phase translocation model. Abundant FG nucleoporins inside the central channel would interact mutually via their hydrophobic repeats, forming a flexible meshwork of nucleoporins (Figure 1b). For hydrophilic molecules, such a meshwork would act as a sieve, allowing only the passage of molecules smaller than the mesh size. For large molecules, the mesh would effectively form a hydrophobic phase, excluding them unless they are able to interact with FG repeats. Such molecules would be soluble in the hydrophobic phase and thus efficiently translocated (Figure 1b, blue particles). The selective phase model is especially elegant as it accounts for high fluxes and selectivity, as well as the translocation of large particles. The dissolution of such particles in the mesh would result in an apparent dilation of the pore, often observed by electron microscopy [20]. However, dilation would be the consequence of the interaction with the FG repeats and not an active gating process. Testing the models
The Brownian affinity gate and the selective phase model both predict that molecules that directly interact with FG repeat nucleoporins can diffuse through NPCs. Both assume a uniform translocation mechanism, which contradicts the distinct nucleoporin requirements for different transport pathways known from many biochemical and genetic studies [21–24]. However, almost all the pathway-specific effects have been observed by interfering with peripherally distributed nucleoporins, especially components of the filaments, and are therefore compatible with a homogenous channel. Nevertheless, the pathwayspecific data do suggest that there are substrate-specific interactions, rather than just generic FG repeat affinities before and/or after translocation. One way that the two models might be distinguished is their different predictions for other aspects of nucleocytoplasmic transport. According to the Brownian affinity gate model, translocation efficiency should be proportional to the concentration of the substrate at the channel entrance.
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This explains why many transport substrates accumulate at the nuclear envelope, which has classically been interpreted as a docking step. Data on cells lacking nucleoporin Nup98 also seem to support this prediction. NPCs from these cells lack nucleoporins of the cytoplasmic extensions and show reduced nuclear import and docking of transport complexes [25]. However, facilitated translocation of some substrates can occur without a detectable accumulation at the NPCs, which therefore does not seem to be an absolute prerequisite for translocation [5]. The selective phase model predicts that interference with hydrophobic interactions between FG repeats should compromise the ability of the NPC to act as a permeability barrier. Indeed, preliminary data obtained by Görlich and Ribbeck indicate that the small amphipathic compound hexanediol can abolish NPC selectivity. In hexanedioltreated cells, maltose binding protein, normally excluded from the nucleus, can pass through the NPC without restriction (D. Görlich, K. Ribbeck, personal communication). This effect is specific, as it can be prevented by wheat germ agglutinin, a lectin known to extensively bind and crosslink FG-repeat nucleoporins. Despite its elegance, the selective phase model is most likely an oversimplification. Specifically, even a wider hydrophobic channel would exclude large randomly diffusing substrates, and thus benefit from Brownian-gate-type low-affinity binding sites at its entrance. Furthermore, peripheral binding sites could also include interactions biased towards specific substrates. Future experiments on the kinetics of translocation with model substrates and manipulated NPCs that lack defined components will undoubtedly refine the current translocation models. Importantly, the recent quantitative approaches [4,5] have made translocation accessible to kinetic modeling [26], which will help to shed light on the in situ affinities of substrate–nucleoporin interactions. Ultimately, kinetic measurements should also be carried out in living cells to determine mass fluxes occurring in vivo and exclude artifacts of permeabilized cells. Acknowledgements The authors would like to thank Dirk Görlich for sharing unpublished results and Elisa Izaurralde and Iain Mattaj for critical reading of the manuscript. G.R. was supported by an EMBL International PhD Programme fellowship.
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